FARNESYL DIBENZODIAZEPINONE AND PROCESSES FOR ITS PRODUCTION

Abstract

a novel farnesylated dibenzodiazepinone, named ECO-04601, its pharmaceutically acceptable salts and derivatives, and to methods for obtaining such compounds. One method of obtaining the ECO-04601 compound is by cultivation of a novel strain of Micromonospora sp., 046-ECO11; another method involves expression of biosynthetic pathway genes in transformed host cells. The present invention further relates to Micromonospora sp. strain 046-ECO11, to the use of and its pharmaceutically acceptable salts and derivatives as pharmaceuticals, in particular to their use as inhibitors of cancer cell growth, bacterial cell growth, mammalian lipoxygenase, and to pharmaceutical compositions comprising ECO-04601 or a pharmaceutically acceptable salt or derivative thereof. Finally, the invention relates to novel polynucleotide sequences and their encoded proteins, which are involved in the biosynthesis of ECO-04601.

Description

RELATED APPLICATIONS

[0001]This Application is a divisional of U.S. Utility application Ser. No. 11/511,586, filed Aug. 28, 2006, which is a divisional of U.S. Utility application Ser. No. 10/762,107, filed Jan. 21, 2004, now issued as U.S. Pat. No. 7,101,872, and which claims priority to each of U.S. Provisional Application 60/441,126, filed Jan. 21, 2003; U.S. Provisional Application 60/492,997, filed Aug. 7, 2003; and U.S. Provisional Application 60/518,286, filed Nov. 10, 2003. The entire teachings of each of the above-noted applications are incorporated herein by reference.

FIELD OF THE INVENTION

[0002]This invention relates to a novel farnesylated dibenzodiazepinone, named ECO-04601, its pharmaceutically acceptable salts and derivatives, and to methods for obtaining the compound. One method of obtaining the compound is by cultivation of a novel strain of Micromonospora sp., i.e., 046-ECO11 or [S01]046; another method involves expression of biosynthetic pathway genes in transformed host cells. The present invention further relates to Micromonospora sp. strains 046-ECO11 and [S01]046, to the use of ECO-04601 and its pharmaceutically acceptable salts and derivatives as pharmaceuticals, in particular to their use as inhibitors of cancer cell growth, bacterial cell growth, mammalian lipoxygenase, and for treating acute and chronic inflammation, and to pharmaceutical compositions comprising ECO-04601 or a pharmaceutically acceptable salt or derivative thereof. Finally, the invention relates to novel polynucleotide sequences and their encoded proteins, which are involved in the biosynthesis of ECO-04601.

BACKGROUND OF THE INVENTION

[0003]The euactinomycetes are a subset of a large and complex group of Gram-positive bacteria known as actinomycetes. Over the past few decades these organisms, which are abundant in soil, have generated significant commercial and scientific interest as a result of the large number of therapeutically useful compounds, particularly antibiotics, produced as secondary metabolites. The intensive search for strains able to produce new antibiotics has led to the identification of hundreds of new species.

[0004]Many of the euactinomycetes, particularly Streptomyces and the closely related Saccharopolyspora genera, have been extensively studied. Both of these genera produce a notable diversity of biologically active metabolites. Because of the commercial significance of these compounds, much is known about the genetics and physiology of these organisms.

[0005]Another representative genus of euactinomycetes, Micromonospora, has also generated commercial interest. For example, U.S. Pat. No. 5,541,181 (Ohkuma et al.) discloses a dibenzodiazepinone compound, specifically 5-farnesyl-4,7,9-trihydroxy-dibenzodiazepin-11-one (named "BU-4664L"), produced by a known euactinomycetes strain, Micromonospora sp. M990-6 (ATCC 55378). The Ohkurma et al. patent reports that BU-4664L and its chemically synthesized di- and tri-alkoxy and acyloxy derivatives possess anti-inflammatory and anti-tumor cell activities.

[0006]Although many biologically active compounds have been identified from bacteria, there remains the need to obtain novel naturally occurring compounds with enhanced properties. Current methods of obtaining such compounds include screening of natural isolates and chemical modification of existing compounds, both of which are costly and time consuming. Current screening methods are based on general biological properties of the compound, which require prior knowledge of the structure of the molecules. Methods for chemically modifying known active compounds exist, but still suffer from practical limitations as to the type of compounds obtainable.

[0007]Thus, there exists a considerable need to obtain pharmaceutically active compounds in a cost-effective manner and with high yield. The present invention solves these problems by providing a novel strain of Micromonospora capable of producing a potent new therapeutic compound, as well as reagents (e.g., polynucleotides, vectors comprising the polynucleotides and host cells comprising the vectors) and methods to generate novel compounds by de novo biosynthesis rather than by chemical synthesis.

SUMMARY OF THE INVENTION

[0008]In one aspect, the invention relates to a compound of the formula

##STR00001##

(Formula II) or a pharmaceutically acceptable salt thereof.

[0009]In another aspect, the invention relates to a pharmaceutical composition comprising a compound of the formula

##STR00002##

or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.

[0010]In a further aspect, the invention relates to a class of compounds represented by Formula I:

##STR00003##

wherein, [0011]W¹, W² and W³ is each independently selected from

##STR00004##

[0011]or

[0012]the chain from the tricycle may terminate at W³, W² or W¹ with W³, W² or W¹ respectively being either --CH═O or --CH₂OH;

[0013]A is selected from --NH--, --NCH₂R¹, --NC(O)R¹;

[0014]R¹ is selected from C1-6 alkyl, C2-6 alkene, aryl or heteroaryl;

[0015]R², R³, and R⁴ is each independently selected from H, R⁵, --C(O)R⁶

[0016]R⁵ is each independently selected from C_1-6 alkyl, C_2-7 alkalene, aryl or heteroaryl;

[0017]R⁶ is each independently selected from H, C_1-6 alkyl, C_2-7 alkalene, aryl or heteroaryl; or a pharmaceutically acceptable salt thereof.

[0018]In one embodiment, A is NH.

[0019]In another embodiment, A is --NCH₂R¹.

[0020]In another embodiment, A is --NC(O)R¹.

[0021]In another embodiment, R² is H.

[0022]In another embodiment, R³ is H.

[0023]In another embodiment, R⁴ is H.

[0024]In another embodiment, R², R³ and R⁴ are each H.

[0025]In another embodiment, R², R³ and R⁴ are each H, and W¹ is --CH═CH--.

[0026]In another embodiment, R², R³ and R⁴ are each H, and W² is --CH═CH--.

[0027]In another embodiment, R², R³ and R⁴ are each H, and W³ is --CH═CH--.

[0028]In another embodiment, A is NH and R², R³ and R⁴ are each H.

[0029]In another embodiment, A is NH, each of W¹, W², and W³ is --CH═CH--.

[0030]The invention further encompasses a compound selected from the group consisting of:

##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012##

[0031]In one embodiment, the invention relates to compositions of the compounds of Formula I together with a pharmaceutically acceptable carrier.

[0032]The invention further encompasses a farnesyl dibenzodiazepinone obtained by a method comprising: a) cultivating Micromonospora sp. strain [S01]046, wherein the cultivation is performed under aerobic conditions in a nutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms; and b) isolating a farnesyl dibenzodiazepinone from the bacteria cultivated in step (a). In one embodiment the farnesyl dibenzodiazapinone is the compound of Formula II.

[0033]In one embodiment, the farnesyl dibenzodiazepinone generates NMR spectra essentially as shown in FIGS. 3, 4, 5, 6 and 7. In another embodiment, the farnesyl dibenzodiazepinone generates an ¹H NMR spectrum of FIG. 3.

[0034]The invention further encompasses a process for making a farnesyl dibenzodiazapinone compound, comprising cultivation of Micromonospora sp. strain 046-ECO11, in a nutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms, and isolation and purification of the compound.

[0035]The invention further encompasses a process for making a farnesyl dibenzodiazepinone compound comprising cultivation of Micromonospora sp. strain [S01]046 in a nutrient medium comprising at least one source of carbon atoms and at least one source of nitrogen atoms, and isolation and purification of the compound.

[0036]In one embodiment, the cultivation occurs under aerobic conditions.

[0037]In another embodiment, the carbon atom and nitrogen atom sources are chosen from the components shown in Table 16.

[0038]In another embodiment, the cultivation is carried out at a temperature ranging from 18° C. to 40° C. In a further embodiment, the temperature range is 18° C. to 29° C.

[0039]In another embodiment, the cultivation is carried out at a pH ranging from 6 to 9.

[0040]The invention further encompasses the Micromonospora sp. having IDAC Accession No. 231203-01.

[0041]The invention further encompasses a method of inhibiting the growth of a cancer cell, the method comprising contacting the cancer cell with a compound of Formula I, such that growth of the cancer cell is inhibited.

[0042]In one embodiment, the compound is ECO-04601.

[0043]The invention further encompasses a method of inhibiting the growth of a cancer cell in a mammal, the method comprising administering a compound of Formula I to a mammal comprising a cancer cell, such that growth of the cancer cell is inhibited in the mammal.

[0044]In one embodiment, the compound is ECO-04601.

[0045]The invention further encompasses a method of treating a pre-cancerous or cancerous condition in a mammal, comprising the step of administering to the mammal a therapeutically effective amount of a compound of Formula I, such that a pre-cancerous or cancerous condition is treated.

[0046]In one embodiment, the compound is ECO-04601.

[0047]The invention further encompasses a method of treating a bacterial infection in a mammal, comprising administering a therapeutically effective amount of a compound of Formula I to a mammal having a bacterial infection, such that the bacterial infection is treated.

[0048]In one embodiment, the compound is ECO-04601.

[0049]The invention further encompasses a method of reducing inflammation in a mammal, comprising administering to a mammal having inflammation a therapeutically effective amount of a compound of Formula I, such that the inflammation is reduced.

[0050]In one embodiment, the compound is ECO-04601.

[0051]The invention further encompasses an isolated polynucleotide comprising one or more of SEQ ID NOs. 1, 64 and 73, wherein the polynucleotide encodes a polypeptide that participates in a biosynthetic pathway for a farnesyl dibenzodiazepinone.

[0052]The invention further encompasses an isolated polynucleotide comprising SEQ ID NOs. 1, 64 and 73, wherein the polynucleotide encodes a polypeptide that participates in a biosynthetic pathway for a farnesyl dibenzodiazepinone.

[0053]The invention further encompasses an isolated polynucleotide that encodes a polypeptide selected from the group consisting of SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88.

[0054]In one embodiment, the isolated polynucleotide comprising SEQ ID No. 1 encodes a polypeptide selected from the group consisting of SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 58, 60 and 62.

[0055]In another embodiment, the isolated polynucleotide comprising SEQ ID No. 64 encodes a polypeptide selected from the group consisting of SEQ ID NOS: 65, 67, 69 and 71.

[0056]In another embodiment, the isolated polynucleotide comprising SEQ ID No. 73, encodes a polypeptide selected from the group consisting of SEQ ID NOS: 74, 76, 78, 80, 82 84, 86 and 88.

[0057]The invention further encompasses an isolated polypeptide of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 or 88.

[0058]In one embodiment, the polypeptide participates in a biosynthetic pathway for a farnesyl dibenzodiazepinone.

[0059]The invention further encompasses an expression vector comprising one or more of the polynucleotides described herein.

[0060]The invention further encompasses a recombinant prokaryotic organism comprising one or more such expression vectors.

[0061]In one embodiment, the organism is an actinomycete.

[0062]In another embodiment, the organism requires the expression vector to synthesize a farnesyl dibenzodiazepinone. That is, the organism is deficient in the ability to synthesize a farnesyl dibenzodiazepinone before transformation with a polynucleotide as described herein.

[0063]The invention further encompasses a method of making a farnesyl dibenzodiazepinone de novo in a prokaryote, comprising the steps of: (a) providing a prokaryote that is incapable of synthesizing a farnesyl dibenzodiazepinone; (b) transforming the prokaryote with an expression vector as described herein; and (c) culturing the prokaryote; wherein the culturing results in the synthesis of a farnesyl dibenzodiazepinone in the prokaryote.

[0064]In one embodiment, the prokaryote is an actinomycete.

[0065]In another embodiment, the vector expresses a polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 or 88.

BRIEF DESCRIPTION OF THE FIGURES

[0066]FIG. 1 shows the mass of ECO-04601 determined by electrospray mass spectrometry to be 462.6.

[0067]FIG. 2 shows the absorption spectrum of purified ECO-04601 with a UVmax at 230 nm and a shoulder at 290 nm.

[0068]FIG. 3 shows proton NMR data for the compound dissolved in MeOH-d₄.

[0069]FIG. 4 shows multidimensional pulse sequences gDQCOSY.

[0070]FIG. 5 shows multidimensional pulse sequences gHSQC.

[0071]FIG. 6 shows multidimensional pulse sequences gHMBC.

[0072]FIG. 7 shows multidimensional pulse sequences NOESY.

[0073]FIG. 8 shows the in vitro anti-inflammatory activity of ECO-04601. Graph shows percent inhibition of 5-lipoxygenase activity plotted against the Log μM concentration of ECO-04601 and NDGA. Graph shows the EC₅₀ of ECO-04601 to be 0.93 μM.

[0074]FIG. 9 shows inhibition of tumor growth resulting from administration of 10 to 30 mg/kg of ECO-04601 to glioblastoma-bearing mice beginning one day after tumor cell inoculation.

[0075]FIG. 10 shows inhibition of tumor growth resulting from administration of 20-30 mg/kg of ECO-04601 to glioblastoma-bearing mice beginning ten days after tumor cell inoculation.

[0076]FIG. 11 shows micrographs of tumor sections from mice bearing glioblastoma tumors and treated with saline or ECO-04601. The cell density of tumor treated with ECO-04601 appears decreased and nuclei from ECO-04601-treated tumor cells are larger and pynotic suggesting a cytotoxic effect.

[0077]FIG. 12 shows the biosynthetic locus of ECO-04601, isolated from Micromonospora sp. strain 046-ECO11, including the positions of cosmids 046KM and 046KQ.

[0078]FIG. 13 shows a schematic diagram of the biosynthetic pathway for the production of the farnesyl-diphosphate group of ECO-04601 with biosynthetic enzymes indicated by their ORF number and family designation.

[0079]FIG. 14 shows a schematic diagram of the biosynthetic pathway for the production of (a) 3-hydroxy-anthranilate-adenylate, and (b) 2-amino-6-hydroxy-[1,4]benzoquinone components as specified by ORFs present in the locus encoding ECO-04601. Biosynthetic enzymes are indicated by their ORF number and family designation.

[0080]FIG. 15 shows a schematic diagram of the biosynthetic pathway for the assembly of the ECO-04601 precursors, farnesyl-diphosphate, 3-hydroxy-anthranilate-adenylate and 2-amino-6-hydroxy-[1,4]benzoquinone. Biosynthetic enzymes are indicated by their ORF number and family designation.

[0081]FIG. 16 shows a sequence listing table indicating the SEQ ID NO. and function for each of the open reading frames (ORFs) of the 046D biosynthetic locus and the corresponding gene product.

[0082]FIG. 17 shows results of the fatty acid analysis of Micromonospora sp. strain 046ECO11 (Accession No. IDAC 070303-01). Analysis was conducted using gas chromatography on fatty acid methyl esters (FAME).

[0083]FIG. 18 illustrates the 16 S ribosomal RNA analysis of Micromonospora sp. strain 046ECO11 (Accession No. IDAC 070303-01). Alignment of 16 S ribosomal RNA sequences demonstrates the phylogenetic relatedness of Micromonospora sp. strain 046ECO11 (indicated as MID352 ECOPIA#1 con) to Micromonospora chalcea.

[0084]FIG. 19 shows the complete ¹H and ¹3C NMR assignments for ECO-04601 when measured in MeOH-d4.

DETAILED DESCRIPTION OF THE INVENTION

[0085]The present invention relates to a novel farnesyl dibenzodiazepinone, referred to herein as "ECO-04601," which was isolated from novel strains of actinomycetes, Micromonospora sp. strain 046-ECO11 and strain [S01]046. These microorganisms were analysed using gas chromatography as Fatty acid methyl esters (FAME) (FIG. 17) 6 S ribosomal RNA determination (FIG. 18) and were found to belong to the genus of Micromonospora. These organisms were deposited on Mar. 7, 2003, and Dec. 23, 2003, respectively, with the International Depository Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2, under Accession Nos. IDAC 070303-01 and IDAC 231203-01, respectively.

[0086]The invention further relates to pharmaceutically acceptable salts and derivatives of ECO-04601, and to methods for obtaining such compounds. One method of obtaining the compound is by cultivating Micromonospora sp. strain 046-ECO11, or a mutant or a variant thereof, under suitable Micromonospora culture conditions, preferably using the fermentation protocol described hereinbelow.

[0087]The invention also relates to a method for producing novel polyketide compounds, namely farnesyl dibenzodiazepinones, by selectively altering the genetic information of an organism. The present invention further provides isolated and purified polynucleotides that encode farnesyl dibenzodiazepinone domains, i.e., polypeptides from farnesyl dibenzodiazepinone-producing microorganisms, fragments thereof, vectors containing those polynucleotides, and host cells transformed with those vectors. These polynucleotides, fragments thereof, and vectors comprising the polynucleotides can be used as reagents in the above described method. Portions of the polynucleotide sequences disclosed herein are also useful as primers for the amplification of DNA or as probes to identify related domains from other farnesyl dibenzodiazepinone producing microorganisms.

[0088]The present invention also relates to pharmaceutical compositions comprising ECO-04601 and its pharmaceutically acceptable salts and derivatives. ECO-04601 is useful as a pharmaceutical, in particular for use as an inhibitor of cancer cell growth, bacterial cell growth, and mammalian lipoxygenase. The invention also relates to novel polynucleotide sequences and their encoded proteins, which are involved in the biosynthesis of ECO-04601.

[0089]The following detailed description discloses how to make and use ECO-04601 and compositions containing this compound to inhibit microbial growth and/or specific disease pathways.

[0090]Accordingly, certain aspects of the present invention relate to pharmaceutical compositions comprising the farnesylated dibenzodiazepinone compounds of the present invention together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit bacterial growth, and methods of using the pharmaceutical compositions to treat diseases, including cancer, and chronic and acute inflammation.

I. Definitions

[0091]For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below.

[0092]As used herein, the term "farnesyl dibenzodiazepinone" refers to a class of dibenzodiazepinone compounds containing a farnesyl moiety. The term includes, but is not limited to, the exemplified compound of the present invention, 10-farnesyl-4,6,8-trihydroxy-dibenzodiazepin-11-one, which is referred to herein as "ECO-04601." As used herein, the term "farnesyl dibenzodiazepinone" includes compounds of this class that can be used as intermediates in chemical syntheses. As used herein, the term "alkyl" refers to linear or branched hydrocarbon groups. Examples of alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, pentyl, hexyl, heptyl, cyclopentyl, cyclohexyl, cyclohexymethyl, and the like. Alkyl may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, oxo, guanidino and formyl.

[0093]The term "alkenyl" refers to linear, branched or cyclic hydrocarbon groups containing at least one carbon-carbon double bond. Examples of alkenyl groups include, without limitation, vinyl, 1-propen-2-yl, 1-buten-4-yl, 2-buten-4-yl, 1-penten-5-yl and the like. Alkenyl may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, formyl, oxo and guanidino. The double bond portion(s) of the unsaturated hydrocarbon chain may be either in the cis or trans configuration.

[0094]The terms "cycloalkyl" and "cycloalkyl ring" refer to a saturated or partially unsaturated carbocyclic ring in a single or fused carbocyclic ring system having from three to fifteen ring members. Examples of cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl. Cycloalkyl may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.

[0095]The terms "heterocyclyl" and "heterocyclic" refer to a saturated or partially unsaturated ring containing one to four hetero atoms or hetero groups selected from O, N, NH, NRx, PO2, S, SO or SO₂ in a single or fused heterocyclic ring system having from three to fifteen ring members. Examples of a heterocyclyl or heterocyclic ring include, without limitation, morpholinyl, piperidinyl, and pyrrolidinyl. Heterocyclyl, heterocyclic or heterocyclyl ring may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, oxo, thiocarbonyl, imino, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.

[0096]The term "amino acid" refers to any natural amino acid, all natural amino acids are well known to a person skilled in the art.

[0097]The term "halo" refers to a halogen atom, e.g., bromine, chlorine, fluorine and iodine.

[0098]The terms "aryl" and "aryl ring" refer to aromatic groups in a single or fused ring system, having from five to fifteen ring members. Examples of aryl include, without limitation, phenyl, naphthyl, biphenyl, terphenyl. Aryl may optionally be substituted with one or more substituent group selected from acyl, amino, acylamino, acyloxy, azido, alkylthio, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.

[0099]The terms "heteroaryl" and "heteroaryl ring" refer to aromatic groups in a single or fused ring system, having from five to fifteen ring members and containing at least one hetero atom such as O, N, S, SO and SO₂. Examples of heteroaryl groups include, without limitation, pyridinyl, thiazolyl, thiadiazoyl, isoquinolinyl, pyrazolyl, oxazolyl, oxadiazoyl, triazolyl, and pyrrolyl groups. Heteroaryl groups may optionally be substituted with one or more substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, thiocarbonyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, and formyl.

[0100]The terms "aralkyl" and "heteroaralkyl" refer to an aryl group or a heteroaryl group, respectively bonded directly through an alkyl group, such as benzyl. Aralkyl and heteroaralkyl may be optionally substituted as the aryl and heteroaryl groups.

[0101]Similarly, the terms "aralkenyl" and "heteroaralkenyl" refer to an aryl group or a heteroaryl group, respectively bonded directly through an alkene group, such as benzyl. Aralkenyl and heteroaralkenyl may be optionally substituted as the aryl and heteroaryl groups.

[0102]The compounds of the present invention can possess one or more asymmetric carbon atoms and can exist as optical isomers forming mixtures of racemic or non-racemic compounds. The compounds of the present invention are useful as single isomers or as a mixture of stereochemical isomeric forms. Diastereoisomers, i.e., nonsuperimposable stereochemical isomers, can be separated by conventional means such as chromatography, distillation, crystallization or sublimation. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes.

[0103]The invention encompasses isolated or purified compounds. An "isolated" or "purified" compound refers to a compound which represents at least 10%, 20%, 50%, 80% or 90% of the compound of the present invention present in a mixture, provided that the mixture comprising the compound of the invention has demonstrable (i.e. statistically significant) biological activity including antibacterial, cytostatic, cytotoxic, antiinflammatory or enzyme inhibitory action when tested in conventional biological assays known to a person skilled in the art.

[0104]The terms "farnesyl dibenzodiazepinone-producing microorganism" and "producer of farnesyl dibenzodiazepinone," as used herein, refer to a microorganism that carries genetic information necessary to produce a farnesyl dibenzodiazepinone compound, whether or not the organism naturally produces the compound. The terms apply equally to organisms in which the genetic information to produce the farnesyl dibenzodiazepinone compound is found in the organism as it exists in its natural environment, and to organisms in which the genetic information is introduced by recombinant techniques.

[0105]Specific organisms contemplated herein include, without limitation, organisms of the family Micromonosporaceae, of which preferred genera include Micromonospora, Actinoplanes and Dactylosporangium; the family Streptomycetaceae, of which preferred genera include Streptomyces and Kitasatospora; the family Pseudonocardiaceae, of which preferred genera are Amycolatopsis and Saccharopolyspora; and the family Actinosynnemataceae, of which preferred genera include Saccharothrix and Actinosynnema; however the terms are intended to encompass all organisms containing genetic information necessary to produce a farnesyl dibenzodiazepinone compound. A preferred producer of a farnesyl dibenzodiazepinone compound includes microbial strain 046-ECO11, a deposit of which was made on Mar. 7, 2003, with the International Depository Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E, 3R2, under Accession No. IDAC 070303-01.

[0106]The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as, where applicable, intervening regions (introns) between individual coding segments (exons).

[0107]The terms "gene locus, "gene cluster," and "biosynthetic locus" refer to a group of genes or variants thereof involved in the biosynthesis of a farnesyl benzodiazepinone compound. The biosynthetic locus in strain 046-ECO11 that directs the production of ECO-04601 is often referred to herein, in both the written description and Figures, as "046D." Genetic modification of gene locus, gene cluster or biosynthetic locus refers to any genetic recombinant techniques known in the art including mutagenesis, inactivation, or replacement of nucleic acids that can be applied to generate variants of ECO-04601.

[0108]A DNA or nucleotide "coding sequence" or "sequence encoding" a particular polypeptide or protein, is a DNA sequence which is transcribed and translated into a polypeptide or protein when placed under the control of an appropriate regulatory sequence.

[0109]"Oligonucleotide" refers to a nucleic acid, generally of at least 10, preferably 15 and more preferably at least 20 nucleotides in length, preferably no more than 100 nucleotides in length, that are hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA or other nucleic acid of interest.

[0110]A promoter sequence is "operably linked to" a coding sequence recognized by RNA polymerase which initiates transcription at the promoter and transcribes the coding sequence into mRNA.

[0111]The term "replicon" as used herein means any genetic element, such as a plasmid, cosmid, chromosome or virus, that behaves as an autonomous unit of polynucleotide replication within a cell. A "expression vector" or "vector" is a replicon in which another polynucleotide fragment is attached, such as to bring about the replication and/or expression of the attached fragment. "Plasmids" are designated herein by a lower case "p" preceded or followed by capital letters and/or numbers. The starting plasmids disclosed herein are commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accordance with published procedures. In addition, equivalent plasmids to those described herein are known in the art and will be apparent to the skilled artisan.

[0112]The terms "express" and "expression" means allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product" such as a protein. The expression product itself, e.g. the resulting protein, may also be said to be "expressed" by the cell. An expression product can be characterized as intracellular, extracellular or secreted.

[0113]"Digestion" of DNA refers to enzymatic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinary skilled artisan. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37° C. are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the gel electrophoresis may be performed to isolate the desired fragment.

[0114]The term "isolated" as used herein means that the material is removed from its original environment (e.g. the natural environment where the material is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, which is separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that the vector or composition is not part of the natural environment.

[0115]The term "restriction fragment" as used herein refers to any linear DNA generated by the action of one or more restriction enzymes.

[0116]The term "transformation" means the introduction of a foreign gene, foreign nucleic acid, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a "cloned" or "foreign" gene or sequence, may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been "transformed" and is a "transformant" or a "clone" or "recombinant". The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.

[0117]The terms "recombinant polynucleotide" and "recombinant polypeptide" as used herein mean a polynucleotide or polypeptide which by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide or polypeptide with which it is associated in nature and/or is linked to a polynucleotide or polypeptide other than that to which it is linked in nature.

[0118]The term "host cell" as used herein, refer to both prokaryotic and eukaryotic cells which are used as recipients of the recombinant polynucleotides and vectors provided herein. In one embodiment, the host cell is a prokaryote.

[0119]The terms "open reading frame" and "ORF" as used herein refers to a region of a polynucleotide sequence which encodes a polypeptide; this region may represent a portion of a coding sequence or a total coding sequence.

[0120]As used herein and as known in the art, the term "identity" is the relationship between two or more polynucleotide sequences, as determined by comparing the sequences. Identity also means the degree of sequence relatedness between polynucleotide sequences, as determined by the match between strings of such sequences. Identity can be readily calculated (see, e.g., Computation Molecular Biology, Lesk, A. M., eds., Oxford University Press, New York (1998), and Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993), both of which are incorporated by reference herein). While there exist a number of methods to measure identity between two polynucleotide sequences, the term is well known to skilled artisans (see, e.g., Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); and Sequence Analysis Primer, Gribskov., M. and Devereux, J., eds., M. Stockton Press, New York (1991)). Methods commonly employed to determine identity between sequences include, for example, those disclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math. (1988) 48:1073. "Substantially identical," as used herein, means there is a very high degree of homology (preferably 100% sequence identity) between subject polynucleotide sequences. However, polynucleotides having greater than 90%, or 95% sequence identity may be used in the present invention, and thus sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence can be tolerated.

[0121]As used herein, the term "treatment" refers to the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disorder, e.g., a disease or condition, a symptom of disease, or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of disease, or the predisposition toward disease.

[0122]As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a farnesyl dibenzodiazepinone and a pharmaceutically acceptable carrier. As used herein, "pharmacologically effective amount," "therapeutically effective amount" or simply "effective amount" refers to that amount of a farnesyl dibenzodiazepinone effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.

[0123]The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

[0124]The term "pharmaceutically acceptable salt" refers to both acid addition salts and base addition salts. The nature of the salt is not critical, provided that it is pharmaceutically acceptable. Exemplary acid addition salts include, without limitation, hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulphuric, phosphoric, formic, acetic, citric, tartaric, succinic, oxalic, malic, glutamic, propionic, glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, malonic, galactaric, galacturonic acid and the like. Suitable pharmaceutically acceptable base addition salts include, without limitation, metallic salts made from aluminium, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, procaine and the like. Additional examples of pharmaceutically acceptable salts are listed in Journal of Pharmaceutical Sciences (1977) 66:2. All of these salts may be prepared by conventional means from a farnesyl dibenzodiazepinone by treating the compound with the appropriate acid or base.

II. Farnesylated Dibenzodiazepinone Compounds

[0125]In one aspect, the invention relates to a novel farnesyl dibenzodiazepinone, referred to herein as "ECO-04601" and having the chemical structure represented by the following formula:

##STR00013##

[0126]ECO-04601 may be described as a new dibenzodiazepinone having a 10-farnesyl substituent located on the nitrogen atom in the 10 position of the dibenzodiazepine ring (i.e., the amide nitrogen in the diazepinone ring), and three phenolic hydroxy substituents in the 4,6 and 8 positions of the dibenzodiazepinone ring. ECO-04601 may be characterized by any one or more of its physicochemical and spectral properties given below, such as its mass, UV, and NMR spectroscopic data. Mass was determined by electrospray mass spectrometry to be 462.6 (FIG. 1); UV=230 nm with a shoulder at 290 nm (FIG. 2). NMR data were collected using MeOH-d4, including proton (FIG. 3), and multidimensional pulse sequences gDQCOSY (FIG. 4), gHSQC (FIG. 5), gHMBC (FIG. 6), and NOESY (FIG. 7).

[0127]In another aspect, the invention relates to a novel class of farnesyl dibenzodiazepinone compounds represented by Formula I:

##STR00014##

wherein, [0128]W¹, W² and W³ is each independently selected from

##STR00015##

[0128]or

[0129]the chain from the tricycle may terminate at W³, W² or W¹ with W³, W² or W¹ respectively being either --CH═O or --CH₂OH;

[0130]A is selected from --NH--, --NCH₂R¹, --NC(O)R¹;

[0131]R¹ is selected from C1-6 alkyl, C2-6 alkene, aryl or heteroaryl;

[0132]R², R³, and R⁴ is each independently selected from H, R⁵, --C(O)R⁶

[0133]R⁵ is each independently selected from C_1-6 alkyl, C_2-7 alkalene, aryl or heteroaryl;

[0134]R⁶ is each independently selected from H, C_1-6 alkyl, C_2-7 alkalene, aryl or heteroaryl; or a pharmaceutically acceptable salt thereof.

[0135]In other embodiments, the invention provides compounds of Formula I, wherein A is selected from the group consisting of NH, NCH2R1, and NC(O)R1; wherein R2 is H; R3 is H; and R4 is H. In another embodiment, R2, R3 and R4 are each H; and all other groups are as previously defined. In a further embodiment, R2, R3 and R4 are each H; and W1 is --CH═CH-- and all other groups are as previously defined. In a further embodiment, R2, R3 and R4 are each H, and W2 is --CH═CH-- and all other groups are as previously defined. In a further embodiment, R2, R3 and R4 are each H; and W3 is --CH═CH--; and all other groups are as previously defined. In a further embodiment, A is NH; R2, R3 and R4 are each H; and all other groups are as previously defined. In a further embodiment, A is NH; each of W1, W2, and W3 is --CH ═CH--; and all other groups are as previously defined. The invention encompasses all pharmaceutically acceptable salts of the foregoing compounds.

[0136]The following are exemplary compounds of the invention:

##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024##

[0137]Certain embodiments expressly exclude one or more of the compounds of Formula I. In one embodiment, the compound of Formula II is excluded.

[0138]The compounds of this invention may be formulated into pharmaceutical compositions comprised of compounds of Formula I in combination with a pharmaceutical acceptable carrier, as discussed in Section V below.

III. Method of Making a Farnesyl Dibenzodiazepinone by Fermentation

[0139]In one embodiment, ECO-04601 is obtained by cultivating a novel strain of Micromonospora, namely Micromonospora sp. strain 046-ECO11. Strain 046-ECO11 was deposited on Mar. 7, 2003, with the International Depositary Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2, under Accession No. 070303-01. The deposit of the strain was made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure. The deposited strains will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposited strains are provided merely as convenience to those skilled in the art and are not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. §112.

[0140]It is to be understood that the present invention is not limited to use of the particular strain 046-ECO11. Rather, the present invention contemplates the use of other ECO-04601 producing organisms, such as mutants or variants of 046-ECO11 that can be derived from this organism by known means such as X-ray irradiation, ultraviolet irradiation, treatment with nitrogen mustard, phage exposure, antibiotic selection and the like; or through the use of recombinant genetic engineering techniques, as described in Section IV below.

[0141]The farnesyl dibenzodiazepinone compounds of the present invention may be biosynthesized by various microorganisms. Microorganisms that may synthesize the compounds of the present invention include but are not limited to bacteria of the order Actinomycetales, also referred to as actinomycetes. Non-limiting examples of members belonging to the genera of Actinomycetes include Nocardia, Geodermatophilus, Actinoplanes, Micromonospora, Nocardioides, Saccharothrix, Amycolatopsis, Kutzneria, Saccharomonospora, Saccharopolyspora, Kitasatospora, Streptomyces, Microbispora, Streptosporangium, and Actinomadura. The taxonomy of actinomycetes is complex and reference is made to Goodfellow, Suprageneric Classification of Actinomycetes (1989); Bergey's Manual of Systematic Bacteriology, Vol. 4 (Williams and Wilkins, Baltimore, pp. 2322-2339); and to Embley and Stackebrandt, "The molecular phylogeny and systematics of the actinomycetes," Annu. Rev. Microbiol. (1994) 48:257-289, each of which is hereby incorporated by reference in its entirety, for genera that may synthesize the compounds of the invention.

[0142]Farnesyl dibenzodiazepinone-producing microorganisms are cultivated in culture medium containing known nutritional sources for actinomycetes. Such media having assimilable sources of carbon, nitrogen, plus optional inorganic salts and other known growth factors at a pH of about 6 to about 9. Suitable media include, without limitation, the growth media provided in Table 16. Microorganisms are cultivated at incubation temperatures of about 18° C. to about 40° C. for about 3 to about 40 days.

[0143]The culture media inoculated with the farnesyl dibenzodiazepinone-producing microorganisms may be aerated by incubating the inoculated culture media with agitation, for example, shaking on a rotary shaker, or a shaking water bath. Aeration may also be achieved by the injection of air, oxygen or an appropriate gaseous mixture to the inoculated culture media during incubation. Following cultivation, the farnesyl dibenzodiazepinone compounds can be extracted and isolated from the cultivated culture media by techniques known to a skilled person in the art and/or disclosed herein, including for example centrifugation, chromatography, adsorption, filtration. For example, the cultivated culture media can be mixed with a suitable organic solvent such as n-butanol, n-butyl acetate or 4-methyl-2-pentanone, the organic layer can be separated for example, by centrifugation followed by the removal of the solvent, by evaporation to dryness or by evaporation to dryness under vacuum. The resulting residue can optionally be reconstituted with for example water, ethanol, ethyl acetate, methanol or a mixture thereof, and re-extracted with a suitable organic solvent such as hexane, carbon tetrachloride, methylene chloride or a mixture thereof. Following removal of the solvent, the compounds may be further purified by the use of standard techniques, such as chromatography.

[0144]The farnesyl dibenzodiapezinones biosynthesized by microorganisms may optionally be subjected to random and/or directed chemical modifications to form compounds that are derivatives or structural analogs. Such derivatives or structural analogs having similar functional activities are within the scope of the present invention. Farnesyl dibenzodiapezinone compounds may optionally be modified using methods known in the art and described herein.

IV. Method of Making a Farnesyl Dibenzodiazepinone by Recombinant Technology

[0145]In another embodiment, the present invention relates to nucleic acid molecules that encode proteins useful in the production of farnesyl benzodiazepinones. Specifically, the present invention provides recombinant DNA vectors and nucleic acid molecules that encode all or part of the biosynthetic locus in strain 046-ECO11, which directs the production of ECO-04601, and is referred to herein as "046D." The invention further includes genetic modification of 046D using conventional genetic recombinant techniques, such as mutagenesis, inactivation, or replacement of nucleic acids, to produce chemical variants of ECO-04601.

[0146]The invention thus provides a method for making a farnesyl benzodiazepinone compound using a transformed host cell comprising a recombinant DNA vector that encodes one or more of the polypeptides of the present invention, and culturing the host cell under conditions such that farnesyl benzodiazepinone is produced. The host cell is a prokaryote. In one embodiment, the host cell is an actinomycete. In another embodiment, the host cell is a Streptomyces host cell.

[0147]The invention provides recombinant nucleic acids that produce a variety of farnesyl dibenzodiazepinone compounds that cannot be readily synthesized by chemical methodology alone. The invention allows direct manipulation of 046D biosynthetic locus via genetic engineering of the enzymes involved in the biosynthesis of a farnesyl benzodiazepinone according to the invention. The 046A biosynthetic locus is described in Example 11.

Recombinant DNA Vectors

[0148]Vectors of the invention typically comprise the DNA of a transmissible agent, into which foreign DNA is inserted. A common way to insert one segment of DNA into another segment of DNA involves the use of specific enzymes called restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites. A "cassette" refers to a DNA coding sequence or segment of DNA that codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. Generally, a nucleic acid molecule that encodes a protein useful in the production of a farnesyl benzodiazepinone is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a prokaryote e.g. actinomycte, by transformation (see below). A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a "DNA construct". A common type of vector is a "plasmid" which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can be readily introduced into a suitable host cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme. In one embodiment of the invention, the coding DNA encodes for polypeptides of SEQ ID NOs. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 or 89 that are required for the biosynthesis of a farnesyl benzodiazepinone.

[0149]Promoter DNA of a recombinant vector is a DNA sequence that initiates, regulates, or otherwise mediates or controls the expression of the coding DNA. Promoter DNA and coding may be from the same or different organisms. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes. Vector constructs may be produced using conventional molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0150]Examples of promoters that function in actinomycetes, e.g. Streptomyces, are taught in U.S. Pat. Nos. 5,830,695 and 5,466,590. Another example of a transcription promoter useful in Actinomycetes expression vectors is tipA, a promoter inducible by the antibiotic thiostrepton [c.f. Murakami, T., et al., (1989), J. Bacteriol., 171,1459].

Transformation of Actinomycetes

[0151]A suitable transformation method for use with an actinomycete comprises forming the actinomycete culture into spheroplasts using lysozyme. A buffer solution containing recombinant DNA vectors and polyethylene glycol is then added, in order to introduce the vector into the host cells, by using either of the methods of Thompson or Keiser [c. f. Thompson, C. J., et al., (1982), J. Bacteriol., 151, 668-677 or Keiser, T. et al. (2000), "Practical Streptomyces Genetics", The John Innes Foundation, Norwich], for example. A thiostrepton-resistance gene is frequently used as a selective marker in the transformation plasmid [c.f. Hopwood, D. A., et al., (1987), "Methods in Enzymology" 153, 116, Academic Press, New York], but the present invention is not limited thereto. Additional methods for the transformation of actinomycetes are taught in U.S. Pat. No. 5,393,665.

Assay for Farnesyl Dibenzodiazepinone or Biosynthetic Intermediates

[0152]Actinomycetes defective in farnesyl dibenzodiazepinone biosynthesis are transformed with one or more expression vectors encoding one or more proteins in the farnesyl benzodiazepinone biosynthetic pathway, thus restoring farnesyl benzodiazepinone biosynthesis by genetic complementation of the specific defect.

[0153]The presence or absence of farnesyl dibenzodiazepinone or intermediates in the biosynthetic pathway (see FIGS. 13, 14 and 15) in a recombinant actinomycete can be determined using methodologies that are well known to persons of skill in the art. For example, ethyl acetate extracts of fermentation media used for the culture of a recombinant actinomycete are processed as described in Example 2 and fractions containing farnesyl dibenzodiazepinone or intermediates detected by TLC on commercial Kieselgel 60F₂₅₄ plates. Farnesyl dibenzodiazepinone and intermediate compounds are visualized by inspection of dried plates under UV light or by spraying the plates with a spray containing vanillin (0.75%) and concentrated sulfuric acid (1.5%, v/v) in ethanol and subsequently heating the plate. The exact identity of the compounds separated by TLC is then determined using gas chromatography-mass spectroscopy. Methods of mass spectroscopy are taught in the published U.S. Patent Application No. US2003/0052268.

Mutagenesis

[0154]The invention allows direct manipulation of 046D biosynthetic locus via genetic engineering of the enzymes involved in the biosynthesis of a farnesyl benzodiazepinone according to the invention.

[0155]A number of methods are known in the art that permit the random as well as targeted mutation of the DNA sequences of the invention (see for example, Ausubel et. al. Short Protocols in Molecular Biology (1995) 3rd Ed. John Wiley & Sons, Inc.). In addition, there are a number of commercially available kits for site-directed mutagenesis, including both conventional and PCR-based methods. Examples include the EXSITE® PCR-Based Site-directed Mutagenesis Kit available from Stratagene (Catalog No. 200502) and the QUIKCHANGE® Site-directed mutagenesis Kit from Stratagene (Catalog No. 200518), and the CHAMELEON® double-stranded Site-directed mutagenesis kit, also from Stratagene (Catalog No. 200509).

[0156]In addition the nucleotides of the invention may be generated by insertional mutation or truncation (N-terminal, internal or C-terminal) according to methodology known to a person skilled in the art.

[0157]Older methods of site-directed mutagenesis known in the art rely on sub-cloning of the sequence to be mutated into a vector, such as an M13 bacteriophage vector, that allows the isolation of single-stranded DNA template. In these methods, one anneals a mutagenic primer (i.e., a primer capable of annealing to the site to be mutated but bearing one or more mismatched nucleotides at the site to be mutated) to the single-stranded template and then polymerizes the complement of the template starting from the 3' end of the mutagenic primer. The resulting duplexes are then transformed into host bacteria and plaques are screened for the desired mutation.

[0158]More recently, site-directed mutagenesis has employed PCR methodologies, which have the advantage of not requiring a single-stranded template. In addition, methods have been developed that do not require sub-cloning. Several issues must be considered when PCR-based site-directed mutagenesis is performed. First, in these methods it is desirable to reduce the number of PCR cycles to prevent expansion of undesired mutations introduced by the polymerase. Second, a selection must be employed in order to reduce the number of non-mutated parental molecules persisting in the reaction. Third, an extended-length PCR method is preferred in order to allow the use of a single PCR primer set. And fourth, because of the non-template-dependent terminal extension activity of some thermostable polymerases it is often necessary to incorporate an end-polishing step into the procedure prior to blunt-end ligation of the PCR-generated mutant product.

[0159]The protocol described below accommodates these considerations through the following steps. First, the template concentration used is approximately 1000-fold higher than that used in conventional PCR reactions, allowing a reduction in the number of cycles from 25-30 down to 5-10 without dramatically reducing product yield. Second, the restriction endonuclease Dpn I (recognition target sequence: 5-Gm6ATC-3, where the A residue is methylated) is used to select against parental DNA, since most common strains of E. coli Dam methylate their DNA at the sequence 5-GATC-3. Third, Taq Extender is used in the PCR mix in order to increase the proportion of long (i.e., full plasmid length) PCR products. Finally, Pfu DNA polymerase is used to polish the ends of the PCR product prior to intramolecular ligation using T4 DNA ligase.

[0160]A non-limiting example for the isolation of mutant polynucleotides is described in detail as follows:

[0161]Plasmid template DNA (approximately 0.5 pmole) is added to a PCR cocktail containing: 1× mutagenesis buffer (20 mM Tris HCl, pH 7.5; 8 mM MgCl2; 40 g/ml BSA); 12-20 pmole of each primer (one of skill in the art may design a mutagenic primer as necessary, giving consideration to those factors such as base composition, primer length and intended buffer salt concentrations that affect the annealing characteristics of oligonucleotide primers; one primer must contain the desired mutation, and one (the same or the other) must contain a 5' phosphate to facilitate later ligation), 250 M each dNTP, 2.5 U Taq DNA polymerase, and 2.5 U of Taq Extender (Available from Stratagene; See Nielson et al. (1994) Strategies 7: 27, and U.S. Pat. No. 5,556,772). Primers can be prepared using the triester method of Matteucci et al., 1981, J. Am. Chem. Soc. 103:3185-3191, incorporated herein by reference. Alternatively automated synthesis may be preferred, for example, on a Biosearch 8700 DNA Synthesizer using cyanoethyl phosphoramidite chemistry.

[0162]The PCR cycling is performed as follows: 1 cycle of 4 min at 94° C., 2 min at 50° C. and 2 min at 72° C.; followed by 5-10 cycles of 1 min at 94° C., 2 min at 54° C. and 1 min at 72° C. The parental template DNA and the linear, PCR-generated DNA incorporating the mutagenic primer are treated with DpnI (10 U) and Pfu DNA polymerase (2.5 U). This results in the DpnI digestion of the in vivo methylated parental template and hybrid DNA and the removal, by Pfu DNA polymerase, of the non-template-directed Taq DNA polymerase-extended base(s) on the linear PCR product. The reaction is incubated at 37° C. for 30 min and then transferred to 72° C. for an additional 30 min. Mutagenesis buffer (115 ul of 1×) containing 0.5 mM ATP is added to the DpnI-digested, Pfu DNA polymerase-polished PCR products. The solution is mixed and 10 ul are removed to a new microfuge tube and T4 DNA ligase (2-4 U) is added. The ligation is incubated for greater than 60 min at 37° C. Finally, the treated solution is transformed into competent E. coli according to standard methods.

[0163]Methods of random mutagenesis, which will result in a panel of mutants bearing one or more randomly situated mutations, exist in the art. Such a panel of mutants may then be screened for those exhibiting reduced uracil detection activity relative to the wild-type polymerase (e.g., by measuring the incorporation of 10 nmoles of dNTPs into polymeric form in 30 minutes in the presence of 200 M dUTP and at the optimal temperature for a given DNA polymerase). An example of a method for random mutagenesis is the so-called "error-prone PCR method". As the name implies, the method amplifies a given sequence under conditions in which the DNA polymerase does not support high fidelity incorporation. The conditions encouraging error-prone incorporation for different DNA polymerases vary, however one skilled in the art may determine such conditions for a given enzyme. A key variable for many DNA polymerases in the fidelity of amplification is, for example, the type and concentration of divalent metal ion in the buffer. The use of manganese ion and/or variation of the magnesium or manganese ion concentration may therefore be applied to influence the error rate of the polymerase.

[0164]Genes for desired mutant polypeptides generated by mutagenesis may be sequenced to identify the sites and number of mutations. For those mutants comprising more than one mutation, the effect of a given mutation may be evaluated by introduction of the identified mutation to the wild-type gene by site-directed mutagenesis in isolation from the other mutations borne by the particular mutant. Screening assays of the single mutant thus produced will then allow the determination of the effect of that mutation alone.

V. Genes and Proteins for the Production of ECO-04601

[0165]As discussed in more detail below, the isolated, purified or enriched nucleic acids of one of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 may be used to prepare one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88, respectively, or fragments comprising at least 50, 75, 100, 200, 300, 500 or more consecutive amino acids of one of the polypeptides of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88.

[0166]Accordingly, another aspect of the present invention is an isolated, purified or enriched nucleic acid which encodes one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200, 300 or more consecutive amino acids of one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88. The coding sequences of these nucleic acids may be identical to one of the coding sequences of one of the nucleic acids of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 or a fragment thereof, or may be different coding sequences which encode one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200, 300 consecutive amino acids of one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 as a result of the redundancy or degeneracy of the genetic code. The genetic code is well known to those of skill in the art and can be obtained, for example, from Stryer, Biochemistry, 3^rd edition, W.H. Freeman & Co., New York.

[0167]The isolated, purified or enriched nucleic acid which encodes one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 may include, but is not limited to: (1) only the coding sequences of one of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89; (2) the coding sequences of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 and additional coding sequences, such as leader sequences or proprotein; and (3) the coding sequences of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 and non-coding sequences, such as non-coding sequences 5' and/or 3' of the coding sequence. Thus, as used herein, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide that includes only coding sequence for the polypeptide as well as a polynucleotide that includes additional coding and/or non-coding sequence.

[0168]The invention relates to polynucleotides based on SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 but having polynucleotide changes that are "silent", for example changes which do not alter the amino acid sequence encoded by the polynucleotides of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89. The invention also relates to polynucleotides which have nucleotide changes which result in amino acid substitutions, additions, deletions, fusions and truncations of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88. Such nucleotide changes may be introduced using techniques such as site directed mutagenesis, random chemical mutagenesis, exonuclease III deletion, and other recombinant DNA techniques.

[0169]The isolated, purified or enriched nucleic acids of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89, the sequences complementary thereto, or a fragment comprising at least 100, 150, 200, 300, 400 or more consecutive bases of one of the sequence of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89, or the sequences complementary thereto may be used as probes to identify and isolate DNAs encoding the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 respectively. In such procedures, a genomic DNA library is constructed from a sample microorganism or a sample containing a microorganism capable of producing a farnesyl dibenzodiazepinone. The genomic DNA library is then contacted with a probe comprising a coding sequence or a fragment of the coding sequence, encoding one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88, or a fragment thereof under conditions which permit the probe to specifically hybridize to sequences complementary thereto. In a preferred embodiment, the probe is an oligonucleotide of about 10 to about 30 nucleotides in length designed based on a nucleic acid of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89. Genomic DNA clones which hybridize to the probe are then detected and isolated. Procedures for preparing and identifying DNA clones of interest are disclosed in Ausubel et al., Current Protocols in Molecular Biology, John Wiley 503 Sons, Inc. 1997; and Sambrook et al., Molecular Cloning: A Laboratory Manual 2d Ed., Cold Spring Harbor Laboratory Press, 1989. In another embodiment, the probe is a restriction fragment or a PCR amplified nucleic acid derived from SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89.

[0170]The isolated, purified or enriched nucleic acids of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89, the sequences complementary thereto, or a fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400 or 500 consecutive bases of one of the sequences of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 or the sequences complementary thereto may be used as probes to identify and isolate related nucleic acids. In some embodiments, the related nucleic acids may be genomic DNAs (or cDNAs) from potential farnesyl dibenzodiazepinone producers. In such procedures, a nucleic acid sample containing nucleic acids from a potential farnesyl dibenzodiazepinone producer is contacted with the probe under conditions that permit the probe to specifically hybridize to related sequences. The nucleic acid sample may be a genomic DNA (or cDNA) library from the potential farnesyl dibenzodiazepinone-producer. Hybridization of the probe to nucleic acids is then detected using any of the methods described above.

[0171]Hybridization may be carried out under conditions of low stringency, moderate stringency or high stringency. As an example of nucleic acid hybridization, a polymer membrane containing immobilized denatured nucleic acids is first prehybridized for 30 minutes at 45° C. in a solution consisting of 0.9 M NaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mM Na₂EDTA, 0.5% SDS, 10×Denhardt's, and 0.5 mg/ml polyriboadenylic acid. Approximately 2×10⁷ cpm (specific activity 4-9×10⁸ cpm/ug) of ³²P end-labeled oligonucleotide probe are then added to the solution. After 12-16 hours of incubation, the membrane is washed for 30 minutes at room temperature in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh 1×SET at Tm-10° C. for the oligonucleotide probe where Tm is the melting temperature. The membrane is then exposed to autoradiographic film for detection of hybridization signals.

[0172]By varying the stringency of the hybridization conditions used to identify nucleic acids, such as genomic DNAs or cDNAs, which hybridize to the detectable probe, nucleic acids having different levels of homology to the probe can be identified and isolated. Stringency may be varied by conducting the hybridization at varying temperatures below the melting temperatures of the probes. The melting temperature of the probe may be calculated using the following formulas:

[0173]For oligonucleotide probes between 14 and 70 nucleotides in length the melting temperature (Tm) in degrees Celcius may be calculated using the formula: T_m=81.5+16.6(log [Na.sup.+])+0.41 (fraction G+C)-(600/N) where N is the length of the oligonucleotide.

[0174]If the hybridization is carried out in a solution containing formamide, the melting temperature may be calculated using the equation Tm=81.5+16.6(log [Na+])+0.41 (fraction G+C)-(0.63% formamide)-(600/N) where N is the length of the probe.

[0175]Prehybridization may be carried out in 6×SSC, 5×Denhardt's reagent, 0.5% SDS, 0.1 mg/ml denatured fragmented salmon sperm DNA or 6×SSC, 5×Denhardt's reagent, 0.5% SDS, 0.1 mg/ml denatured fragmented salmon sperm DNA, 50% formamide. The composition of the SSC and Denhardt's solutions are listed in Sambrook et al., supra.

[0176]Hybridization is conducted by adding the detectable probe to the hybridization solutions listed above. Where the probe comprises double stranded DNA, it is denatured by incubating at elevated temperatures and quickly cooling before addition to the hybridization solution. It may also be desirable to similarly denature single stranded probes to eliminate or diminish formation of secondary structures or oligomerization. The filter is contacted with the hybridization solution for a sufficient period of time to allow the probe to hybridize to cDNAs or genomic DNAs containing sequences complementary thereto or homologous thereto. For probes over 200 nucleotides in length, the hybridization may be carried out at 15-25° C. below the Tm. For shorter probes, such as oligonucleotide probes, the hybridization may be conducted at 5-10° C. below the Tm. Preferably, the hybridization is conducted in 6×SSC, for shorter probes. Preferably, the hybridization is conducted in 50% formamide containing solutions, for longer probes. All the foregoing hybridizations would be considered to be examples of hybridization performed under conditions of high stringency.

[0177]Following hybridization, the filter is washed for at least 15 minutes in 2×SSC, 0.1% SDS at room temperature or higher, depending on the desired stringency. The filter is then washed with 0.1×SSC, 0.5% SDS at room temperature (again) for 30 minutes to 1 hour. Nucleic acids which have hybridized to the probe are identified by conventional autoradiography and non-radioactive detection methods.

[0178]The above procedure may be modified to identify nucleic acids having decreasing levels of homology to the probe sequence. For example, to obtain nucleic acids of decreasing homology to the detectable probe, less stringent conditions may be used. For example, the hybridization temperature may be decreased in increments of 5° C. from 68° C. to 42° C. in a hybridization buffer having a Na+ concentration of approximately 1M. Following hybridization, the filter may be washed with 2×SSC, 0.5% SDS at the temperature of hybridization. These conditions are considered to be "moderate stringency" conditions above 50° C. and "low stringency" conditions below 50° C. A specific example of "moderate stringency" hybridization conditions is when the above hybridization is conducted at 55° C. A specific example of "low stringency" hybridization conditions is when the above hybridization is conducted at 45° C.

[0179]Alternatively, the hybridization may be carried out in buffers, such as 6×SSC, containing formamide at a temperature of 42° C. In this case, the concentration of formamide in the hybridization buffer may be reduced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe. Following hybridization, the filter may be washed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered to be "moderate stringency" conditions above 25% formamide and "low stringency" conditions below 25% formamide. A specific example of "moderate stringency" hybridization conditions is when the above hybridization is conducted at 30% formamide. A specific example of "low stringency" hybridization conditions is when the above hybridization is conducted at 10% formamide. Nucleic acids which have hybridized to the probe are identified by conventional autoradiography and non-radioactive detection methods.

[0180]The preceding methods may be used to isolate nucleic acids having at least 97%, at least 95%, at least 90%, at least 85%, at least 80%, or at least 70% sequence identity to a nucleic acid sequence selected from the group consisting of the sequences of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89. The isolated nucleic acid may have a coding sequence that is a naturally occurring allelic variant of one of the coding sequences described herein. Such allelic variant may have a substitution, deletion or addition of one or more nucleotides when compared to the nucleic acids of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89, or the sequences complementary thereto.

[0181]Additionally, the above procedures may be used to isolate nucleic acids which encode polypeptides having at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, or at least 70% identity to a polypeptide having the sequence of one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200, 300 consecutive amino acids thereof.

[0182]Another aspect of the present invention is an isolated or purified polypeptide comprising the sequence of one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof. As discussed herein, such polypeptides may be obtained by inserting a nucleic acid encoding the polypeptide into a vector such that the coding sequence is operably linked to a sequence capable of driving the expression of the encoded polypeptide in a suitable host cell. For example, the expression vector may comprise a promoter, a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for modulating expression levels, an origin of replication and a selectable marker.

[0183]Promoters suitable for expressing the polypeptide or fragment thereof in bacteria include the E. coli lac or trp promoters, the lad promoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter, the lambda P_R promoter, the lambda P_L promoter, promoters from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), and the acid phosphatase promoter. Fungal promoters include the α factor promoter. Eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, heat shock promoters, the early and late SV40 promoter, LTRs from retroviruses, and the mouse metallothionein-I promoter. Other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses may also be used.

[0184]Mammalian expression vectors may also comprise an origin of replication, any necessary ribosome binding sites, a polyadenylation site, splice donors and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. In some embodiments, DNA sequences derived from the SV40 splice and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

[0185]Vectors for expressing the polypeptide or fragment thereof in eukaryotic cells may also contain enhancers to increase expression levels. Enhancers are cis-acting elements of DNA, usually from about 10 to about 300 bp in length that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancers.

[0186]In addition, the expression vectors preferably contain one or more selectable marker genes to permit selection of host cells containing the vector. Examples of selectable markers that may be used include genes encoding dihydrofolate reductase or genes conferring neomycin resistance for eukaryotic cell culture, genes conferring tetracycline or ampicillin resistance in E. coli, and the S. cerevisiae TRP1 gene.

[0187]The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is ligated to the desired position in the vector following digestion of the insert and the vector with appropriate restriction endonucleases. Alternatively, appropriate restriction enzyme sites can be engineered into a DNA sequence by PCR. A variety of cloning techniques are disclosed in Ausbel et al. Current Protocols in Molecular Biology, John Wiley 503 Sons, Inc. 1997 and Sambrook et al., Molecular Cloning: A Laboratory Manual 2d Ed., Cold Spring Harbour Laboratory Press, 1989. Such procedures and others are deemed to be within the scope of those skilled in the art.

[0188]The vector may be, for example, in the form of a plasmid, a viral particle, or a phage. Other vectors include derivatives of chromosomal, nonchromosomal and synthetic DNA sequences, viruses, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. A variety of cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989).

[0189]Particular bacterial vectors which may be used include the commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), pGEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9 (Qiagen), pD10, phiX174, pBluescript® II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However, any other vector may be used as long as it is replicable and stable in the host cell.

[0190]The host cell may be any of the host cells familiar to those skilled in the art, including prokaryotic cells or eukaryotic cells. As representative examples of appropriate hosts, there may be mentioned: bacteria cells, such as E. coli, Streptomyces lividans, Streptomyces griseofuscus, Streptomyces ambofaciens, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, Bacillus, and Staphylococcus, fungal cells, such as yeast, insect cells such as Drosophila S2 and Spodoptera Sf9, animal cells such as CHO, COS or Bowes melanoma, and adenoviruses. The selection of an appropriate host is within the abilities of those skilled in the art.

[0191]The vector may be introduced into the host cells using any of a variety of techniques, including electroporation transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Where appropriate, the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter may be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells may be cultured for an additional period to allow them to produce the desired polypeptide or fragment thereof.

[0192]Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells employed for expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art. The expressed polypeptide or fragment thereof can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be employed for final purification steps.

[0193]Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts (described by Gluzman, Cell, 23:175 (1981)), and other cell lines capable of expressing proteins from a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK cell lines. The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Polypeptides of the invention may or may not also include an initial methionine amino acid residue.

[0194]Alternatively, the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof can be synthetically produced by conventional peptide synthesizers. In other embodiments, fragments or portions of the polynucleotides may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides.

[0195]Cell-free translation systems can also be employed to produce one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof using mRNAs transcribed from a DNA construct comprising a promoter operably linked to a nucleic acid encoding the polypeptide or fragment thereof. In some embodiments, the DNA construct may be linearized prior to conducting an in vitro transcription reaction. The transcribed mRNA is then incubated with an appropriate cell-free translation extract, such as a rabbit reticulocyte extract, to produce the desired polypeptide or fragment thereof.

[0196]The present invention also relates to variants of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof. The term "variant" includes derivatives or analogs of these polypeptides. In particular, the variants may differ in amino acid sequence from the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination.

[0197]The variants may be naturally occurring or created in vitro. In particular, such variants may be created using genetic engineering techniques such as site directed mutagenesis, random chemical mutagenesis, exonuclease III deletion procedures, and standard cloning techniques. Alternatively, such variants, fragments, analogs, or derivatives may be created using chemical synthesis or modification procedures.

[0198]Other methods of making variants are also familiar to those skilled in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids that encode polypeptides having characteristics which enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. Preferably, these nucleotide differences result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates.

[0199]The variants of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 may be variants in which one or more of the amino acid residues of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code.

[0200]Conservative substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the following replacements: replacements of an aliphatic amino acid such as Ala, Val, Leu and IIe with another aliphatic amino acid; replacement of a Ser with a Thr or vice versa; replacement of an acidic residue such as Asp or Glu with another acidic residue; replacement of a residue bearing an amide group, such as Asn or Gln, with another residue bearing an amide group; exchange of a basic residue such as Lys or Arg with another basic residue; and replacement of an aromatic residue such as Phe or Tyr with another aromatic residue.

[0201]Other variants are those in which one or more of the amino acid residues of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 include a substituent group. Still other variants are those in which the polypeptide is associated with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol). Additional variants are those in which additional amino acids are fused to the polypeptide, such as leader sequence, a secretory sequence, a proprotein sequence or a sequence that facilitates purification, enrichment, or stabilization of the polypeptide.

[0202]In some embodiments, the fragments, derivatives and analogs retain the same biological function or activity as the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88. In other embodiments, the fragment, derivative or analogue includes a fused heterologous sequence that facilitates purification, enrichment, detection, stabilization or secretion of the polypeptide that can be enzymatically cleaved, in whole or in part, away from the fragment, derivative or analogue.

[0203]Another aspect of the present invention are polypeptides or fragments thereof which have at least 70%, at least 80%, at least 85%, at least 90%, or more than 95% identity to one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or a fragment comprising at least 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof. It will be appreciated that amino acid "substantially identity" includes conservative substitutions such as those described above.

[0204]The polypeptides or fragments having homology to one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or a fragment comprising at least 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof may be obtained by isolating the nucleic acids encoding them using the techniques described above.

[0205]Alternatively, the homologous polypeptides or fragments may be obtained through biochemical enrichment or purification procedures. The sequence of potentially homologous polypeptides or fragments may be determined by proteolytic digestion, gel electrophoresis and/or microsequencing. The sequence of the prospective homologous polypeptide or fragment can be compared to one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or a fragment comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof.

[0206]The polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments, derivatives or analogs thereof comprising at least 40, 50, 75, 100, 150, 200 or 300 consecutive amino acids thereof invention may be used in a variety of applications. For example, the polypeptides or fragments, derivatives or analogs thereof may be used to catalyze biochemical reactions as described elsewhere in the specification.

[0207](a)

VI. Pharmaceutical Compositions Comprising Farnesyl Dibenzodiazepinones

[0208]In another embodiment, the invention relates to a pharmaceutical composition comprising a farnesyl dibenzodiazepinone, as described in the preceding section, and a pharmaceutically acceptable carrier, as described below. The pharmaceutical composition comprising the farnesyl dibenzodiazepinone is useful for treating a variety of diseases and disorders, including cancer, inflammation and bacterial infections.

[0209]The compounds of the present invention, or pharmaceutically acceptable salts thereof, can be formulated for oral, intravenous, intramuscular, subcutaneous, topical or parenteral administration for the therapeutic or prophylactic treatment of diseases, particularly bacterial infections, acute and chronic inflammation and cancer. For oral or parental administration, compounds of the present invention can be mixed with conventional pharmaceutical carriers and excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers and the like. The compositions comprising a compound of this present invention will contain from about 0.1% to about 99.9%, about 1% to about 98%, about 5% to about 95%, about 10% to about 80% or about 15% to about 60% by weight of the active compound.

[0210]The pharmaceutical preparations disclosed herein are prepared in accordance with standard procedures and are administered at dosages that are selected to reduce, prevent, or eliminate bacterial infection, cancer or inflammation. (See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.; and Goodman and Gilman, Pharmaceutical Basis of Therapeutics, Pergamon Press, New York, N.Y., the contents of which are incorporated herein by reference, for a general description of the methods for administering various antimicrobial agents for human therapy). The compositions of the present invention can be delivered using controlled (e.g., capsules) or sustained release delivery systems (e.g., bioerodable matrices). Exemplary delayed release delivery systems for drug delivery that are suitable for administration of the compositions of the invention (preferably of Formula I) are described in U.S. Pat. Nos. 4,452,775 (issued to Kent), 5,239,660 (issued to Leonard), 3,854,480 (issued to Zaffaroni).

[0211]The pharmaceutically acceptable compositions of the present invention comprise one or more compounds of the present invention in association with one or more non-toxic, pharmaceutically acceptable carriers and/or diluents and/or adjuvants and/or excipients, collectively referred to herein as "carrier" materials, and if desired other active ingredients. The compositions may contain common carriers and excipients, such as corn starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid. The compositions may contain crosarmellose sodium, microcrystalline cellulose, sodium starch glycolate and alginic acid.

[0212]Tablet binders that can be included are acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (Providone), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose.

[0213]Lubricants that can be used include magnesium stearate or other metallic stearates, stearic acid, silicon fluid, talc, waxes, oils and colloidal silica.

[0214]Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring or the like can also be used. It may also be desirable to add a coloring agent to make the dosage form more aesthetic in appearance or to help identify the product comprising a compound of the present invention.

[0215]For oral use, solid formulations such as tablets and capsules are particularly useful. Sustained released or enterically coated preparations may also be devised. For pediatric and geriatric applications, suspension, syrups and chewable tablets are especially suitable. For oral administration, the pharmaceutical compositions are in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a therapeutically-effective amount of the active ingredient. Examples of such dosage units are tablets and capsules. For therapeutic purposes, the tablets and capsules which can contain, in addition to the active ingredient, conventional carriers such as binding agents, for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth; fillers, for example, calcium phosphate, glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, for example, magnesium stearate, polyethylene glycol, silica or talc: disintegrants, for example, potato starch, flavoring or coloring agents, or acceptable wetting agents. Oral liquid preparations generally are in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs and may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous agents, preservatives, coloring agents and flavoring agents. Examples of additives for liquid preparations include acacia, almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.

[0216]For intravenous (iv) use, compounds of the present invention can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion. Intravenous fluids include, without limitation, physiological saline or Ringer's solution.

[0217]Formulations for parental administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions or suspensions can be prepared from sterile powders or granules having one or more of the carriers mentioned for use in the formulations for oral administration. The compounds can be dissolved in polyethylene glycol, propylene glycol, ethanol, corn oil, benzyl alcohol, sodium chloride, and/or various buffers.

[0218]For intramuscular preparations, a sterile formulation of compounds of the present invention or suitable soluble salts forming the compound, can be dissolved and administered in a pharmaceutical diluent such as Water-for-Injection (WFI), physiological saline or 5% glucose. A suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl oleate.

[0219]For topical use the compounds of present invention can also be prepared in suitable forms to be applied to the skin, or mucus membranes of the nose and throat, and can take the form of creams, ointments, liquid sprays or inhalants, lozenges, or throat paints. Such topical formulations further can include chemical compounds such as dimethylsulfoxide (DMSO) to facilitate surface penetration of the active ingredient.

[0220]For application to the eyes or ears, the compounds of the present invention can be presented in liquid or semi-liquid form formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints or powders.

[0221]For rectal administration the compounds of the present invention can be administered in the form of suppositories admixed with conventional carriers such as cocoa butter, wax or other glyceride.

[0222]Alternatively, the compound of the present invention can be in powder form for reconstitution in the appropriate pharmaceutically acceptable carrier at the time of delivery. In another embodiment, the unit dosage form of the compound can be a solution of the compound or a salt thereof in a suitable diluent in sterile, hermetically sealed ampoules.

[0223]The amount of the compound of the present invention in a unit dosage comprises a therapeutically-effective amount of at least one active compound of the present invention which may vary depending on the recipient subject, route and frequency of administration. A recipient subject refers to a plant, a cell culture or an animal such as an ovine or a mammal including a human.

[0224]According to this aspect of the present invention, the novel compositions disclosed herein are placed in a pharmaceutically acceptable carrier and are delivered to a recipient subject (including a human subject) in accordance with known methods of drug delivery. In general, the methods of the invention for delivering the compositions of the invention in vivo utilize art-recognized protocols for delivering the agent with the only substantial procedural modification being the substitution of the compounds of the present invention for the drugs in the art-recognized protocols.

[0225]Likewise, the methods for using the claimed composition for treating cells in culture, for example, to eliminate or reduce the level of bacterial contamination of a cell culture, utilize art-recognized protocols for treating cell cultures with antibacterial agent(s) with the only substantial procedural modification being the substitution of the compounds of the present invention for the agents used in the art-recognized protocols.

[0226]The compounds of the present invention provide a method for treating bacterial infections, pre-cancerous or cancerous conditions, and acute or chronic inflammatory disease. As used herein, the term "unit dosage" refers to a quantity of a therapeutically effective amount of a compound of the present invention that elicits a desired therapeutic response. As used herein, the phrase "therapeutically effective amount" means an amount of a compound of the present invention that prevents the onset, alleviates the symptoms, or stops the progression of a bacterial infection, inflammatory condition, or pre-cancerous or cancerous condition. The term "treating" is defined as administering, to a subject, a therapeutically effective amount of at least one compound of the present invention, both to prevent the occurrence of a bacterial infection, inflammation or pre-cancer or cancer condition, or to control or eliminate a bacterial infection, inflammation or pre-cancer or cancer condition. The term "desired therapeutic response" refers to treating a recipient subject with a compound of the present invention such that a bacterial or inflammatory condition or pre-cancer or cancer condition is reversed, arrested or prevented in a recipient subject.

[0227]The compounds of the present invention can be administered as a single daily dose or in multiple doses per day. The treatment regime may require administration over extended periods of time, e.g., for several days or for from two to four weeks. The amount per administered dose or the total amount administered will depend on such factors as the nature and severity of the disease condition, the age and general health of the recipient subject, the tolerance of the recipient subject to the compound and the type of the bacterial infection, inflammatory disorder, or type of cancer.

[0228]A compound according to this invention may also be administered in the diet or feed of a patient or animal. The diet for animals can be normal foodstuffs to which the compound can be added or it can be added to a premix.

[0229]The compounds of the present invention may be taken in combination, together or separately with any known clinically approved antibiotic, inflammation or anti-cancer agent to treat a recipient subject in need of such treatment.

VII. Method of Inhibiting Tumor Growth

[0230]In another embodiment, the present invention relates to a method of inhibiting tumor growth. Compounds as described herein can possess antitumor activity. The compounds are effective against mammalian tumor cells such as leukemia cells, melanoma cells, breast carcinoma cells, lung carcinoma cells, pancreatic carcinoma cells, ovarian carcinoma cells, renal carcinoma cells, colon carcinoma cells prostate carcinoma cells and glioma cells. The antitumor method of the invention results in inhibition of tumor cells. The term "inhibition", when used in conjunction with the antitumor method refers to suppression, killing, stasis, or destruction of tumor cells. The antitumor method preferably results in prevention, reduction or elimination of invasive activity and related metastasis of tumor cells. The term "effective amount" when used in conjunction with the antitumor cell method refers to the amount of the compound sufficient to result in the inhibition of mammalian tumor cells.

[0231]The inhibition of mammalian tumor growth according to this method can be monitored in several ways. First, tumor cells grown in vitro can be treated with the compound and monitored for growth or death relative to the same cells cultured in the absence of the compound. A cessation of growth or a slowing of the growth rate (i.e., the doubling rate), e.g., by 10% or more, is indicative of tumor cell inhibition. Alternatively, tumor cell inhibition can be monitored by administering the compound to an animal model of the tumor of interest. Examples of experimental animal tumor models are known in the art and described in the examples herein. A cessation of tumor growth (i.e., no further increase in size) or a reduction in tumor size (i.e., tumor volume) or cell number (e.g., at least a 10% decrease in either) in animals treated with a compound as described herein relative to tumors in control animals not treated with the compound is indicative of tumor growth inhibition.

[0232]To monitor the efficacy of tumor treatment in a human, tumor size or tumor cell titer is measured before and after initiation of the treatment, and treatment is considered effective if either the tumor size or titer ceases further growth, or if the tumor is reduced in size or titer, e.g., by at least 10% or more (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100%, that is, the absence of the tumor). Methods of determining the size or cell titer of a tumor in vivo vary with the type of tumor, and include, for example, various imaging techniques well known to those in the medical imaging or oncology fields (MRI, CAT, PET, etc.), as well as histological techniques and flow cytometry.

[0233]For the antitumor method of the invention, a typical effective dose of the compounds given orally or parenterally would be from about 5 to about 100 mg/kg of body weight of the subject with a daily dose ranging from about 15 to about 300 mg/kg of body weight of the subject.

VIII. Method of Inhibiting Lipoxygenase

[0234]In another embodiment, the present invention also provides for a method of treating diseased states, in particular inflammation, caused by the 5-lipoxygenase system and/or by the synthesis of the Leukotrienes C₄, D₄, E₄ and F₄ as well as Leukotriene B₄ in mammals, especially in human subjects. This method comprises administering to a subject an effective amount of ECO-04601. Compound ECO-04601 may be used alone or in combination with other anti-inflammatory compounds to treat or prevent disease states related to inflammation including pulmonary conditions, inflammation, cardiovascular conditions, central nervous system conditions or skin conditions. More specific diseases include gastritis; erosive esophagitis; inflammatory bowel disease; ethanol-induced hemorrhagic erosions; hepatic ischemia; ischemic neuronal injury; noxious agent induced damage or necrosis of hepatic, pancreatic, renal, neuronal or myocardial tissue; liver parenchymal damage caused by hepatoxic agents such as CCl₄ and D-galactosamine; ischemic renal failure; disease-induced hepatic damage; trauma- or stress-induced cell damage; asthma; multiple sclerosis; ischemic reperfusion; edema; rheumatoid arthritis; viral encephalitis; bacterial pneumonia; neurodegeneration; Alzheimer's disease and glycerol-induced renal failure.

[0235]For the method of the invention related to the 5-lipoxygenase system and/or the biosynthesis of Leukotrienes, a typical effective unit dose of ECO-04601 given orally or parenterally would be from about 5 to about 100 mg/kg of body weight of the subject with a daily dose ranging from about 15 to about 300 mg/kg of body weight of the subject.

[0236]The inhibition of lipoxygenase enzymes is monitored using methods well known in the art and as described in the examples herein. A decrease in enzyme activity by at least 10%, relative to the activity in the absence of a compound as described herein is indicative of effective inhibition of lipoxygenase activity.

[0237]Farnesyl dibenzodiazepinone compounds useful according to the invention can be used to reduce or prevent inflammation. Among the hallmarks of local acute inflammation are heat, redness, swelling, pain and loss of function. These changes are induced largely by changes in vascular flow and caliber, changes in vascular permeability and leukocyte exudation (Robbins et al., "Pathologic Basis of Disease", 6^th Ed., W.B. Saunders Co., Philadelphia, Pa.). Anti-inflammatory therapy performed using compounds useful according to the invention can be monitored for success by tracking any of these changes. For example, a decrease in swelling (e.g., at least 10% decrease following treatment) or reported pain (e.g., a sustained decrease of 1 point or more on a 1-10 scale reported by the patient, with 10 being the worst pain experienced in association with this disorder prior to treatment, and 0 being no pain) can be used to indicate successful treatment.

[0238]Other measurable hallmarks of inflammation include leukocyte infiltration and inflammatory cytokine levels. These hallmarks can be monitored by biopsy of the affected tissue. A decrease of 10% or more in leukocyte infiltration in fixed, stained tissue relative to infiltration in similar tissue prior to treatment can be used to indicate successful treatment, as can a decrease of 10% or more in the level of any given inflammatory cytokine, relative to the level before treatment. Those skilled in the art can readily assay for inflammatory cytokine levels in tissue, blood, or other fluid samples. Alternatively, the level of systemic indicators of inflammation such as C reactive protein levels and erythrocyte sedimentation rate can be monitored. Each of these has established normal ranges in medicine, and treatment is considered successful if one or more of such indicators goes from outside the normal range to inside the normal range after the initiation of treatment.

IX. Method of Inhibiting Bacterial Growth

[0239]In another embodiment, the present invention relates to a method for treating bacterial infection in a mammalian subject in need thereof, comprising the step of administering to the mammal a therapeutically effective amount of compound ECO-04601, a compound as described herein, or a pharmaceutically acceptable derivative or prodrug thereof.

[0240]According to another embodiment, the invention provides a method of decreasing bacterial quantity in a biological sample. This method comprises the step of contacting the biological sample with a compound ECO-04601, a compound as described herein, or a pharmaceutically acceptable derivative or prodrug thereof. This method is effective if the number of bacteria decreases by at least 10%, and preferably more, e.g., 25%, 50%, 75% or even 100% after contacting the biological sample with compound ECO-04601, a compound as described herein, or a pharmaceutically acceptable derivative or prodrug thereof.

[0241]These pharmaceutical compositions effective to treat or prevent a bacterial infection which comprise ECO-04601, a compound as described herein, or a pharmaceutically acceptable derivative or prodrug thereof in an amount sufficient to measurably decrease bacterial quantity, and a pharmaceutically acceptable carrier, are another embodiment of the present invention. The term "measurably decrease bacterial quantity", as used herein means a measurable change in the number of bacteria between a sample containing the inhibitor and a sample not containing the inhibitor.

[0242]Agents which increase the susceptibility of bacterial organisms to antibiotics are known. For example, U.S. Pat. No. 5,523,288, U.S. Pat. No. 5,783,561 and U.S. Pat. No. 6,140,306 describe methods of using bactericidal/permeability-increasing protein (BPI) for increasing antibiotic susceptibility of gram-positive and gram-negative bacteria. Agents that increase the permeability of the outer membrane of bacterial organisms have been described by Vaara, M. in Microbiological Reviews (1992) pp. 395-411, and the sensitization of gram-negative bacteria has been described by Tsubery, H., et al, in J. Med. Chem. (2000) pp. 3085-3092.

[0243]For the method of the invention related to treatment of subjects with a bacterial infection, a typical effective unit dose of ECO-04601, a compound described herein or a pharmaceutically acceptable derivative or prodrug thereof given orally or parenterally would be from about 5 to about 100 mg/kg of body weight of the subject with a daily dose ranging from about 15 to about 300 mg/kg of body weight of the subject.

[0244]Another preferred embodiment of this invention relates to a method, as described above, of treating a bacterial infection in a mammal in need thereof, but further comprising the step of administering to the mammal an agent which increases the susceptibility of bacterial organisms to antibiotics.

[0245]According to another preferred embodiment, the invention provides a method, as described above, of decreasing bacterial quantity in a biological sample, but further comprising the step of contacting the biological sample with an agent which increases the susceptibility of bacterial organisms to antibiotics.

[0246]Methods of decreasing bacterial quantity are effective if the number of bacteria decreases at least 10%, and preferably more, e.g., 25%, 50%, 75% or even 100% after contacting the biological sample with compound ECO-04601, a compound as described herein, or a pharmaceutically acceptable derivative or prodrug thereof.

[0247]The pharmaceutical compositions and methods of this invention will be useful generally for controlling bacterial infections in vivo. Examples of bacterial organisms that may be controlled by the compositions and methods of this invention include, but are not limited to the following organisms: Streptococcus pneumoniae, Streptococcus pyrogenes, Enterococcus fecalis, Enterococcus faecium, Klebsiella pneumoniae, Enterobacter spp., Proteus spp., Pseudomonas aeruginosa, E. coli, Serratia marcesens, Staphylococcus aureus, Coagulase negative Staphylococcus, Haemophilus infuenzae, Bacillus anthracis, Mycoplasma pneumoniae, and Staphylococcus epidermidis. The compositions and methods will therefore be useful for controlling, treating or reducing the advancement, severity or effects of nosocomial or non-nosocomial infections. Examples of nosocomial uses include, but are not limited to, urinary tract infections, pneumonia, surgical wound infections, bacteremia and therapy for febrile neutropenic patients. Examples of non-nosocomial uses include but are not limited to urinary tract infections, pneumonia, prostatitis, skin and soft tissue infections and intra-abdominal infections.

[0248]In addition to the compounds of this invention, pharmaceutically acceptable derivatives or prodrugs of the compounds of this invention may also be employed in compositions to treat or prevent the above-identified disorders.

[0249]A "pharmaceutically acceptable derivative or prodrug" means any pharmaceutically acceptable salt, ester, salt of an ester or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention or an inhibitorily active metabolite or residue thereof. Particularly favored derivatives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species.

[0250]Pharmaceutically acceptable prodrugs of the compounds of this invention include, without limitation, esters, amino acid esters, phosphate esters, metal salts and sulfonate esters.

[0251]Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, IC₅₀ and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of significant figures and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set in the examples, Tables and Figures are reported as precisely as possible. Any numerical values may inherently contain certain errors resulting from variations in experiments, testing measurements, statistical analyses and such.

[0252]Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES

Example 1

Preparation of Production Culture

[0253]Unless otherwise noted, all reagents were purchased from Sigma Chemical Co. (St. Louis, Mo.), (Aldrich). Micromonospora spp. (deposit accession number IDAC 070303-01) was maintained on agar plates of ISP2 agar (Difco Laboratories, Detroit, Mich.). An inoculum for the production phase was prepared by transferring the surface growth of the Micromonospora spp. from the agar plates to 125-mL flasks containing 25 mL of sterile medium comprised of 24 g potato dextrin, 3 g beef extract, 5 g Bacto-casitone, 5 g glucose, 5 g yeast extract, and 4 g CaCO₃ made up to one liter with distilled water (pH 7.0). The culture was incubated at about 28° C. for approximately 60 hours on a rotary shaker set at 250 rpm. Following incubation, 10 mL of culture was transferred to a 2 L baffled flask containing 500 mL of sterile production medium containing 20 g/L potato dextrin, 20 g/L glycerol, 10 g/L Fish meal, 5 g/L Bacto-peptone, 2 g/L CaCO₃, and 2 g/L (NH₄)₂SO₄, pH 7.0. Fermentation broth was prepared by incubating the production culture at 28° C. in a rotary shaker set at 250 rpm for one week.

Example 2

Isolation

[0254]500 mL ethyl acetate was added to 500 mL of fermentation broth prepared as described in Example 1 above. The mixture was agitated for 30 minutes on an orbital shaker at 200 rpm to create an emulsion. The phases were separated by centrifugation and decantation. Between 4 and 5 g of anhydrous MgSO₄ was added to the organic phase, which was then filtered and the solvents removed in vacuo.

[0255]An ethyl acetate extract from 2 L fermentation was mixed with HP-20 resin (100 mL; Mitsubishi Casei Corp., Tokyo, Japan) in water (300 mL). Ethyl acetate was removed in vacuo, the resin was filtered on a Buchner funnel and the filtrate was discarded. The adsorbed HP-20 resin was then washed successively with 2×125 mL of 50% acetonitrile in water, 2×125 mL of 75% acetonitrile in water and 2×125 mL of acetonitrile.

[0256]Fractions containing the compound of Formula II were evaporated to dryness and 100 mg was digested in the 5 mL of the upper phase of a mixture prepared from chloroform, cyclohexane, methanol, and water in the ratios, by volume, of 5:2:10:5. The sample was subjected to centrifugal partition chromatography using a High Speed Countercurrent (HSCC) system (Kromaton Technologies, Angers, France) fitted with a 200 mL cartridge and prepacked with the upper phase of this two-phase system. The HSCC was run with the lower phase mobile and the compound of Formula II was eluted at approximately one-half column volume. Fractions were collected and the compound of Formula II was detected by TLC of aliquots of the fractions on commercial Kieselgel 60F₂₅₄ plates. Compound could be visualized by inspection of dried plates under UV light or by spraying the plates with a spray containing vanillin (0.75%) and concentrated sulfuric acid (1.5%, v/v) in ethanol and subsequently heating the plate. Fractions contained substantially pure compound of Formula II, although highly colored. A buff-colored sample could be obtained by chromatography on HPLC as follows.

[0257]6 mg of sample was dissolved in acetonitrile and injected onto a preparative HPLC column (XTerra ODS (10 μm), 19×150 mm, Waters Co., Milford, Mass.), with a 9 mL/min flow rate and UV peak detection at 300 nm. The column was eluted with acetonitrile/buffer (20 mM of NH₄HCO₃) according to the following gradient shown in Table 1

TABLE-US-00001 TABLE 1 Time (min) Water (%) Acetonitrile (%) 0 70 30 10 5 95 15 5 95 20 70 30

[0258]Fractions containing the compound of Formula II eluted at approximately 11:0 min and were combined, concentrated and lyophilized to give a yield of 3.8 mg compound.

[0259]Alternative Protocol 1

[0260]The compound of Formula II was also isolated using the following alternative protocol. At the end of the incubation period, the fermentation broth from the baffled flasks of Example 1 was centrifuged and the supernatant decanted from the pellet containing the bacterial mycelia. 100 mL of 100% MeOH was added to the mycelial pellet and the sample was stirred for 10 minutes and centrifuged for 15 minutes. The methanolic supernatant was decanted and saved. 100 mL of acetone was then added to the mycelial pellet and stirred for 10 minutes then centrifuged for 15 minutes. The acetonic supernatant was decanted and combined with the methanolic supernatant. Finally, 100 mL of 20% MeOH/H₂O was added to the mycelial pellet, stirred for 10 minutes and centrifuged for 15 minutes. The supernatant was combined with the acetonic and methanolic supernatants.

[0261]The combined supernatant was added to 400 ml of HP-20 resin in 1000 mL of water and the organics were removed in vacuo. The resulting slurry was filtered on a Buchner funnel and the filtrate was discarded. Adsorbed HP-20 resin was washed successively with 2×500 mL of 50% MeOH/H₂O, 2×500 mL of 75% MeOH/H₂O and 2×500 mL of MeOH.

[0262]The individual washes were collected separately and analyzed by TLC as described above. Those fractions containing the compound of Formula II were evaporated to near dryness and lyophilized. The lyophilizate was dissolved in methanol and injected onto a preparative HPLC column (Xterra ODS (10 μm), 19×150 mm, Waters Co., Milford, Mass.) with a flow rate of 9 mL/min and peak detection at 300 nm.

[0263]The column was eluted with acetonitrile/buffer (5 mM of NH₄HCO₃) according to gradient shown in Table 2.

TABLE-US-00002 TABLE 2 Time (min) Buffer (%) Acetonitrile (%) 0 95 5 15 45 55 20 5 95 30 5 95 35 95 5

[0264]Fractions containing the compound of Formula II were combined, concentrated and lyophilized to yield about 33.7 mg of compound.

[0265]Alternative Protocol 2

[0266]10 liters of the whole broth from Example 1 are extracted twice with equal volumes of ethyl acetate and the two extracts are combined and concentrated to dryness. The dried extract is weighed, and for every gram of dry extract, 100 mL of MeOH--H₂O (2:1 v/v) and 100 mL of hexane is added. The mixture is swirled gently but well to achieve dissolution. The two layers are separated and the aqueous layer is washed with 100 mL of hexane. The two hexane layers are combined and the combined hexane solution is washed with 100 mL methanol:water (2:1, v/v). The two methanol:water layers are combined and treated with 200 mL of EtOAc and 400 mL of water. The layers are separated and the aqueous layer is extracted twice more with 200 mL portions of EtOAc. The EtOAc layers are combined and concentrated. The residue obtained will be suitable for final purification, either by HSCC or by HPLC as described above. This extraction process achieves a ten-fold purification when compared with the extraction protocol used above.

Example 3

Elucidation of the Structure of Compound of Formula II

[0267]The structure of the compound of Formula II was derived from spectroscopic data, including mass, UV, and NMR spectroscopy. Mass was determined by electrospray mass spectrometry to be 462.6 (FIG. 1), UVmax 230 nm with a shoulder at 290 nm (FIG. 2). NMR data were collected dissolved in MeOH-d₄ including proton (FIG. 3), and multidimensional pulse sequences gDQCOSY (FIG. 4), gHSQC (FIG. 5), gHMBC (FIG. 6), and NOESY (FIG. 7).

[0268]A number of cross peaks in the 2D spectra of ECO-04601 are key in the structural determination. For example, the farnesyl chain is placed on the amide nitrogen by a strong cross peak between the proton signal of the terminal methylene of that chain at 4.52 ppm and the amide carbonyl carbon at 170 ppm in the gHMBC experiment. This conclusion is confirmed by a cross peak in the NOESY spectrum between the same methylene signals at 4.52 ppm and the aromatic proton signal at 6.25 ppm from one of the two protons of the tetra substituted benzenoid ring.

[0269]Based on the mass, UV and NMR spectroscopy data, the structure of the compound was determined to be the structure of Formula II.

Example 4

Antibacterial Activity (Minimal Inhibitory Concentration Determination)

[0270]Minimal Inhibitory Concentration (MIC) is defined as the lowest concentration of drug that inhibits more than 99% of the bacterial population. The MIC determination of ECO-04601 against bacteria strains (Bacillus subtilis--ATCC 23857; Micrococcus luteus --ATCC 9341) was performed using broth microdilution assay (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Fifth Edition. NCCLS document M7-A5 (ISBN 1-56238-394-9). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pa. 19087-1898 USA.).

Test compound preparation: The test article ECO-04601 is prepared as 100× stock solutions in DMSO, with concentrations ranging from 3.2 mg/ml to 0.0625 mg/ml (a two-fold dilution series over 10 points). The first dilution (3.2 mg/ml) was prepared by resuspending 0.5 mg of each test article in 156.25 μl of DMSO. The stock is then serially diluted by two-fold decrement to obtain the desired concentration range.Inoculum preparation: From an overnight culture in Mueller Hinton (MH) broth, cell density for each indicator strain (Bacillus subtilis; Micrococcus luteus) was adjusted to 0.5 Mc Farland units in 0.85% saline, then further diluted 1/100 in appropriate assay medium (˜1×10⁶ cells/ml).MIC determination: The 100×ECO-04601 solutions was diluted 50 times in MH broth and dispensed in a 96 well plate, one test concentration per column of wells, 10 columns in total. The 11^th column of wells contained MH broth with 1% DMSO, the 12^th column of wells contained 100 μl of broth alone. 50 μl of the final cell dilution of each indicator strain was added to each corresponding well of the microplate containing 50 μl of diluted drug or media alone. Assay plates were incubated at 35° C. for 24 hrs.The results of the MIC for the compound of ECO-04601, shown in Table 3, demonstrate a range of antibacterial effects:

TABLE-US-00003 TABLE 3 Indicator strain MIC (μg/mL) Bacillus subtilis ATCC 23857 12.5 Micrococcus luteus ATCC 9341 6.25

Example 5

Anticancer Activity In Vitro Against Human and Animal Tumor Cell Lines from Various Tissues

[0271]Culture conditions: The cell lines listed in Table 4 were used to characterize the cytotoxicity of ECO-04601 against human and animal tumor cell lines. These cell lines were shown to be free of mycoplasma infection and were maintained on the appropriate media (Table 4) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin, under 5% CO₂ at 37° C. Cells were passaged twice to three times per week. Viability was examined by staining with 0.25% trypan blue and only flasks where cell viability was >95% were used for this study.Cell lines amplification and plating: Tumor cells were seeded (1-3×10³ cells per 100 μL) in 96-wells flat bottom microtiter plates and incubated at 37° C. and 5% CO₂ for 16 hrs before treatment in drug-free medium supplemented with 10% serum.Evaluation of inhibitory activity on cell proliferation: Cells were incubated for 96 hrs with 6 log₁₀-fold concentrations of the test substance starting at 10 μg/ml (20 μM). The test substance stock solution (5 mg/mL) was initially diluted at 1/70 fold in medium supplemented with serum. Other concentrations were then obtained from 1/10 fold successive dilutions in the same supplemented medium. Cell survival was evaluated 96 h later by replacing the culture media with 150 μL fresh medium containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4. Next, 50 μL of 2.5 mg/mL of 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in phosphate buffer solution, pH 7.4, was added. After 3-4 h of incubation at 37° C., the medium and soluble MTT was removed, and 200 μL of dimethylsulfoxide was added to dissolve the precipitate of reduced MTT followed by addition of 25 μL glycine buffer (0.1 M glycine plus 0.1 M NaCl, pH 10.5). The absorbance was determined at 570 nm with a microplate reader. Results were expressed as the concentration of drug which inhibits 50% of the cell growth (IC₅₀). The IC₅₀ values shown in Table 4 demonstrated a pharmacologically relevant cytotoxic activity of ECO-04601 against a variety of tumor types such as leukemias, melanomas, pancreatic and breast carcinomas.

TABLE-US-00004 TABLE 4 Cell Culture IC₅₀ lines Type Origin Source medium (x10^-5 M) K562 Leukemia Human ATCC RPMI 1640 8.6 myelogeneous P388 Leukemia Mouse ATCC RPMI 1640 10.9 I83 Leukemia Human ATCC RPMI 1640 2.7 B16 (F10) Melanoma Mouse ATCC RPMI 1640 11.4 SK-MEL 28 Melanoma Human ATCC RPMI 1640 14.0 SK-MEL Melanoma Human ATCC RPMI 1640 14.3 28VEGF (expressing VEGF) SK-MEL-1 Melanoma Human ATCC EMEM 1% 14.1 non- essential amino acid 1% Sodium puryvate Panc 96 Pancreatic Human ATCC RPMI 1% 12.5 carcinoma Sodium puryvate Panc Pancreatic Human ATCC RPMI 1% 14.2 10.05 carcinoma Sodium puryvate Insulin MCF-7 Breast Human ATCC RPMI 1640 9.7 adeno- carcinoma

Example 6

Anticancer Activity In Vitro Against Various Human Tumor Cell Lines from the U.S. National Cancer Institute Panel

[0272]A study measuring the in vitro antitumor activity of ECO-04601 was performed by the National Cancer Institute (National Institutes of Health, Bethesda, Md., USA) against panel of human cancer cell lines in order to determine the ECO-04601 concentrations needed to obtain a 50% inhibition of cell proliferation (GI₅₀). The operation of this unique screen utilizes 50 different human tumor cell lines, representing leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney.

Culture conditions and plating: The human tumor cell lines of the cancer-screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells were inoculated into 96 well microtiter plates in 100 μL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines (Table 5). After cell inoculation, the microtiter plates were incubated at 37° C., 5% CO₂, 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs. After 24 h, two plates of each cell line were fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz).

[0273]Evaluation of inhibitory activity on cell proliferation: ECO-04601 was provided as a lyophilized powder with an estimated purity of 90+%. The compound was stored at -20° C. until day of use. ECO-04601 was solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 μg/mL gentamicin. Additional four, 10-fold or 1/2 log serial dilutions were made to provide a total of five drug concentrations plus control. Aliquots of 100 μl of these different drug dilutions were added to the appropriate microtiter wells already containing 100 μl of medium, resulting in the required final drug concentrations (8.0×10^-5 M to 8.0×10^-9 M).

[0274]Following drug addition, the plates were incubated for an additional 48 h at 37° C., 5% CO₂, 95% air, and 100% relative humidity. For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 μl of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4° C. Supernatants were discarded, and the plates were washed five times with tap water and air-dried. Sulforhodamine B (SRB) solution (100 μl) at 0.4% (w/v) in 1% acetic acid was added to each well, and plates were incubated for 10 minutes at room temperature. After staining, unbound dye was removed by washing five times with 1% acetic acid and the plates were air-dried. Bound stain was subsequently solubilized with 10 mM trizma base, and the absorbance was read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology was the same except that the assay was terminated by fixing settled cells at the bottom of the wells by gently adding 50 μl of 80% TCA (final concentration, 16% TCA).

[0275]The growth inhibitory activity of ECO-04601 was measured by NCl utilizing the GI₅₀ value, rather than the classical IC₅₀ value. The GI₅₀ value emphasizes the correction for the cell count at time zero and, using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], GI₅₀ is calculated as [(Ti-Tz)/(C-Tz)]×100=-50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation.

Result: ECO-04601 shows a significant antitumor activity against several types of tumor as revealed by the NCl screening. Results of the screen are shown in Table 5, and more detailed results of activity against gliomas are shown in Example 7 (Table 6).

TABLE-US-00005 TABLE 5 Inoculation Density (number of Cell Line Name Type Origin cells/well) GI₅₀ (×10^-6 M) CCRF-CEM Leukemia Human 40,000 1.08 K-562 Leukemia Human 5,000 1.43 RPMI-8226 Leukemia Human 20,000 3.15 A549/ATCC Non-Small Cell Lung Human 7,500 9.10 EKVX Non-Small Cell Lung Human 20,000 0.23 HOP-62 Non-Small Cell Lung Human 10,000 8.29 NCl-H226 Non-Small Cell Lung Human 20,000 2.00 NCl-H23 Non-Small Cell Lung Human 20,000 2.02 NCl-H460 Non-Small Cell Lung Human 7,500 13.60 NCl-H522 Non-Small Cell Lung Human 20,000 3.44 COLO 205 Colon Human 15,000 12.70 HCT-116 Colon Human 5,000 2.92 HCT-15 Colon Human 10,000 9.73 HT29 Colon Human 5,000 20.70 SW-620 Colon Human 10,000 2.72 SF-268 CNS Human 15,000 4.94 SF-295 CNS Human 10,000 12.70 SF-539 CNS Human 15,000 0.0075 SNB-19 CNS Human 15,000 2.90 SNB-75 CNS Human 20,000 7.71 U251 CNS Human 7,500 2.19 LOX IMVI Melanoma Human 7,500 4.53 M14 Melanoma Human 15,000 4.57 SK-MEL-2 Melanoma Human 20,000 25.0 SK-MEL-28 Melanoma Human 10,000 11.6 SK-MEL-5 Melanoma Human 10,000 7.80 UACC-257 Melanoma Human 20,000 2.31 UACC-62 Melanoma Human 10,000 1.55 IGR-OV1 Ovarian Human 10,000 3.11 OVCAR-3 Ovarian Human 10,000 13.50 OVCAR-4 Ovarian Human 15,000 9.67 OVCAR-5 Ovarian Human 20,000 2.81 OVCAR-8 Ovarian Human 10,000 2.65 SK-OV-3 Ovarian Human 20,000 4.00 786-0 Renal Human 10,000 6.99 A498 Renal Human 25,000 22.30 ACHN Renal Human 10,000 3.10 CAKI-1 Renal Human 10,000 15.20 RXF 393 Renal Human 15,000 7.71 SN12C Renal Human 15,000 3.85 UO-31 Renal Human 15,000 19.70 DU-145 Prostate Human 10,000 3.56 MCF7 Breast Human 10,000 10.10 NCI/ADR-RES Breast Human 15,000 18.30 MDA-MB-231/ATCC Breast Human 20,000 2.72 HS 578T Breast Human 20,000 2.76 MDA-MB-435 Breast Human 15,000 15.30 BT-549 Breast Human 20,000 0.11 T-47D Breast Human 20,000 0.77

[0276]The results indicate that ECO-04601 was effective against most of the human tumor cell lines that have been assayed in the NCl screening panel suggesting a broad anticancer activity against several types of human cancer.

Example 7

In Vitro Antiproliferative Study Against a Panel of Glioma Cell Lines

[0277]The anticancer activity of ECO-04601 was evaluated using a panel of glioma cancer cell lines shown in Table 6, and the 50% inhibition of cell proliferation (IC₅₀) was determined.

Culture conditions: The cell lines listed in Table 6 were shown to be free of mycoplasma infection and were maintained on DMEM medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin, under 5% CO₂ at 37° C. Cells were passaged once a week. Prior to use the cells were detached from the culture flask by treating with trypsin for five to ten minutes. The cells were counted with a Neubauer glass slide and viability assessed by 0.25% trypan blue exclusion. Only flasks with >95% cell viability, were used in the study.Cell lines amplification and plating: Cells, 5×10³ cells per well in 100 μL drug-free medium supplemented with 10% serum, were plated in 96-well flat bottom microtiter plates and incubated at 37° C. for 48 hrs before treatment.Evaluation of inhibitory activity on cell proliferation: Cells (in triplicate wells) were incubated 96 hrs with medium containing different concentrations of ECO-04601, starting at 5.0 μg/ml (10 μM). The compound was used in a solution of 1% DMSO in D-MEM or RPMI media (or other equivalent media). The concentrations of ECO-04601 were as follows: 10 μM (5.0 μg/ml), 1 μM (0.50 μg/ml), 0.5 μM (0.25 μg/ml), 0.1 μM (0.050 μg/ml), 0.5 μM (0.025 μg/ml), 0.01 μM (0.0050 μg/ml), 0.001 μM (0.00050 μg/ml). Negative controls were cells treated with vehicle alone (1% DMSO in culture medium). Positive controls were cells treated with 4 to 6 increasing concentrations of cisplatin (CDDP) (data not shown). The optical density was measured before incubation (time 0) and following 96 hrs of incubation with test compound in order to measure the growth rate of each cell line.

[0278]At the end of the cell treatment, cell culture media was replaced with 150 μl of fresh medium containing 10 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4. Then 50 μl of 2.5 mg/ml of 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide in PBS pH 7.4, were added to each well and the culture plates incubated for 4 hrs at 37° C. The resulting supernatant was removed and formazan crystals were dissolved with 200 μl of DMSO followed by 25 μl of glycine buffer (0.1 M glycine plus 0.1 M NaCl, pH 10.5). The optical density was read in each well using a single wavelength spectrophotometer plate reader at 570 nm. Results were expressed as the concentration of drug, which inhibits 50% of the cell growth (IC₅₀). Each of the cell lines was tested in at least 3 independent experiments.

[0279]Results shown in Table 6 confirmed the activity of ECO-04601 against different brain cancer cell lines including gliosarcoma, which is the most malignant form of type IV glioblastoma multiform. Gliosarcomas are a mixture of glial and endothelial cells and are resistant to any chemotherapy.

TABLE-US-00006 TABLE 6 Cell IC₅₀ lines Type Origin Source (x 10^-6 M) 9L Gliosarcoma Rat ATCC 6.82 ± 2.90 GHD Astrocytoma Human ATCC 6.29 ± 2.98 U 373 Astrocytoma Human ATCC 3.83 ± 1.37 GL26 Glioblastoma Human ATCC 8.93 ± 1.10 C6 Glioblastoma Rat ATCC 4.28 ± 2.82 DN Oligodendro- Human ATCC 3.26 ± 0.93 glioma GHA Oligodendro- Human ATCC 1.78 ± 0.84 glioma

Example 8

Effect on the Enzymatic Activity of Human Lipoxygenase (5-LO)

[0280]5-Lipoxygenase catalyzes the oxidative metabolism of arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE), the initial reaction leading to formation of leukotrienes. Eicosanoids derived from arachidonic acid by the action of lipoxygenases or cycloxygenases have been found to be involved in acute and chronic inflammatory diseases (i.e. asthma, multiple sclerosis, rheumatoid arthritis, ischemia, edema) as well in neurodegeneration (Alzheimer disease), aging and various steps of carcinogenesis, including tumor promotion, progression and metastasis.

[0281]The aim of this study was to determine whether ECO-04601, is able to block the formation of leukotrienes by inhibiting the enzymatic activity of human 5-LO. Methods employed are based on Carter et al (1991) J. Pharmacol. Exp. Ther. 256(3):929-937, and Safayhi (2000), Planta Medica 66:110-113 which are incorporated herein in their entirety by reference.

Experimental Design Human peripheral blood mononuclear cells (PMNs) were isolated through a Ficoll-Paque density gradient. PMNs were stimulated by addition A23187 (30 μM final concentration). Stimulated PMNs were adjusted to a density of 5×10⁶ cells/mL in HBBS medium and incubated with the vehicle control (DMSO), ECO-04601 (at final concentrations of 0.1, 0.5, 1, 2.5, 5 and 10 μM) and NDGA as positive control (at final concentrations of 3, 1, 0.3, 0.1 and 0.03 μM) for 15 minutes at 37° C. Following incubation, samples were neutralized with NaOH and centrifuged. Leukotriene B4 content was measured in the supernatant using an Enzyme Immunosorbant Assay (EIA) assay.Results: Results shown in FIG. 8 demonstrated that ECO-04601 inhibited the activity of human 5-LO with an apparent IC₅₀=0.93 μM (versus 0.1 μM for the positive control NDGA) and therefore displays anti-inflammatory properties.

Example 9

In Vivo Efficacy in a Glioma Model

[0282]The aim of this study was to test whether ECO-04601 administered by i.p. route prevents or delays tumor growth in C6 glioblastoma cell-bearing mice, and to determine an effective dosage regimen.

Animals: A total of 60 six-week-old female mice (Mus musculus nude mice), ranging between 18 to 25 g in weight, were observed for 7 days before treatment. Animal experiments were performed according to ethical guidelines of animal experimentation (Charte du comite d'ethique du CNRS, juillet 2003) and the English guidelines for the welfare of animals in experimental neoplasia (WORKMAN, P., TWENTYMAN, P., BALKWILL, F., et al. (1998). United Kingdom Coordinating Committee on Cancer Research (UKCCCR) Guidelines for the welfare of animals in experimental neoplasia(Second Edition, July 1997; British Journal of Cancer 77:1-10). Any dead or apparently sick mice were promptly removed and replaced with healthy mice. Sick mice were euthanized upon removal from the cage. Animals were maintained in rooms under controlled conditions of temperature (23±2° C.), humidity (45±5%), photoperiodicity (12 hrs light/12 hrs dark) and air exchange. Animals were housed in polycarbonate cages (5/single cage) that were equipped to provide food and water. Animal bedding consisted of sterile wood shavings that were replaced every other day. Food was provided ad libitum, being placed in the metal lid on the top of the cage. Autoclaved tap water was provided ad libitum. Water bottles were equipped with rubber stoppers and sipper tubes. Water bottles were cleaned, sterilized and replaced once a week. Two different numbers engraved on two earrings identified the animals. Each cage was labelled with a specific code.Tumor Cell Line: The C6 cell line was cloned from a rat glial tumor induced by N-nitrosomethyurea (NMU) by Premont et al. (Premont J, Benda P, Jard S., [3H] norepinephrine binding by rat glial cells in culture. Lack of correlation between binding and adenylate cyclase activation. Biochim Biophys Acta. 1975 Feb. 13; 381(2):368-76.) after series of alternate culture and animal passages.

[0283]Cells were grown as adherent monolayers at 37° C. in a humidified atmosphere (5% CO₂, 95% air). The culture medium was DMEM supplemented with 2 mM L-glutamine and 10% fetal bovine serum. For experimental use, tumor cells were detached from the culture flask by a 10 min treatment with trypsin-versen. The cells were counted in a hemocytometer and their viability assessed by 0.25% trypan blue exclusion.

Preparation of the Test Article: for the Test Article, the Following Procedure was Followed for reconstitution (performed immediately preceding injection). The vehicle consisted of a mixture of benzyl alcohol (1.5%), ethanol (8.5%), propylene glycol (27%), PEG 400 (27%), dimethylacetamide (6%) and water (30%). The vehicle solution was first vortexed in order to obtain a homogeneous liquid. 0.6 mL of the vortexed vehicle solution was added to each vial containing the test article (ECO-04601). Vials were mixed thoroughly by vortexing for 1 minute and inverted and shaken vigorously. Vials were mixed again prior to injection into each animal.Animal Inoculation with tumor cells: Experiment started at day 0 (D₀). On D₀, mice received a superficial intramuscular injection of C6 tumor cells (5×10⁵ cells) in 0.1 mL of DMEM complete medium into the upper right posterior leg.

Treatment Regimen and Results

[0284]In a first series of experiments, treatment started 24 hrs following inoculation of C6 cells. On the day of the treatment, each mouse was slowly injected with 100 μL of test or control articles by i.p. route. For all groups, treatment was performed until the tumor volume of the saline-treated mice (group 1) reached approximately 3 cm³ (around day 16). Mice of group 1 were treated daily with a saline isosmotic solution for 16 days. Mice of group 2 were treated daily with the vehicle solution for 16 days. Mice of group 3 were treated daily with 10 mg/kg of ECO-04601 for 16 days. Mice of group 3 were treated every two days with 30 mg/kg of ECO-04601 and received 8 treatments. Mice of group 5 were treated every three days with 30 mg/kg of ECO-04601 and received 6 treatments. Measurement of tumor volume started as soon as tumors became palpable (>100 mm³; around day 11 post-inoculation) and was evaluated every second day until the end of the treatment using callipers. As shown in Table 7 and FIG. 9, the mean value of the tumor volume of all ECO-04601 treated groups (6 mice/group) was significantly reduced as demonstrated by the one-way analysis of variance (Anova) test followed by the non-parametric Dunnett's multiple comparison test comparing treated groups to the saline group. An asterisk in the P value column of Table 7 indicates a statistically significant value, while "ns" signifies not significant.

TABLE-US-00007 TABLE 7 Tumor volume Treatment (mm³) % P Treatment regimen (mean ± SEM) Inhibition value Saline Q1 × 16 3,004.1 ± 249.64 -- -- Vehicle Q1 × 16 2,162.0 ± 350.0 28.0% >0.05 ns solution ECO-04601 Q1 × 16 1,220.4 ± 283.46 59.4% <0.01* (10 mg/kg) ECO-04601 Q2 × 8 1,236.9 ± 233.99 58.8% <0.01* (30 mg/kg) ECO-04601 Q3 × 6 1,184.1 ± 221.45 60.6% <0.01* (30 mg/kg)

[0285]In a second series of experiments, treatment started at day 10 following inoculation of C6 cells when tumors became palpable (around 100 to 200 mm³). Treatment was repeated daily for 5 consecutive days. On the day of the treatment, each mouse was slowly injected with 100 μL of ECO-04601 by i.p. route. Mice of group 1 were treated daily with saline isosmotic solution. Mice of group 2 were treated daily with the vehicle solution. Mice of group 3 were treated daily with 20 mg/kg of ECO-04601. Mice of group 4 were treated daily with 30 mg/kg of ECO-04601. Mice were treated until the tumor volume of the saline-treated control mice (group 1) reached around 4 cm³. Tumor volume was measured every second day until the end of the treatment using callipers. As shown in Table 8 and FIG. 10, the mean value of the tumor volume of all treated groups (6 mice/group) was significantly reduced as demonstrated by the one-way analysis of variance (Anova) test followed by the non-parametric Dunnett's multiple comparison test comparing treated groups to the saline group. An asterisk in the P value column of Table 8 indicates a statistically significant value, while "ns" signifies not statistically significant.

[0286]Histological analysis of tumor sections showed pronounced morphological changes between ECO-04601-treated tumors and control groups. In tumors treated with ECO-04601 (20-30 mg/kg), cell density was decreased and the nuclei of remaining tumor cells appeared larger and pycnotic while no such changes were observed for vehicle-treated mice (FIG. 11).

TABLE-US-00008 TABLE 8 Tumor volume Treatment (mm³) % P Treatment regimen (mean ± SEM) Inhibition value Saline Q1 × 5 4,363.1 ± 614.31 -- -- Vehicle solution Q1 × 5 3,205.0 ± 632.37 26.5% >0.05 ns ECO-04601 Q1 × 5 1,721.5 ± 374.79 60.5% <0.01* (20 mg/kg) ECO-04601 Q1 × 5 1,131.6 ± 525.21 74.1% <0.01* (30 mg/kg)

Example 10

Generation of Variants of Eco-04601 According to the Invention

[0287]Variants of the ECO-04601 molecule, for example those identified herein as Formulae III-LIX, can be generated by standard organic chemistry approaches. General principles of organic chemistry required for making and manipulating the compounds described herein, including functional moieties, reactivity and common protocols are described, for example, in "Advanced Organic Chemistry," 3^rd Edition by Jerry March (1985) which is incorporated herein by reference in its entirety. In addition, it will be appreciated by one of ordinary skill in the art that the synthetic methods described herein may use a variety of protecting groups, whether or not they are explicitly described. A "protecting group" as used herein means a moiety used to block one or more functional moieties such as reactive groups including oxygen, sulfur or nitrogen, so that a reaction can be carried out selectively at another reactive site in a polyfunctional compound. General principles for the use of protective groups, their applicability to specific functional groups and their uses are described for example in T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3^rd Edition, John Wiley & Sons, New York (1999).

Scheme 1: Epoxide variants

[0288]The epoxide compounds of the present invention (e.g., compounds according to exemplary Formulae VII-XIV) are made from the compound of Formula II (ECO-04601) by treatment with any of a number of epoxidizing reagents such as perbenzoic acid, monoperphthalic acid or more preferably by m-chloroperbenzoic acid in an inert solvent such as tetrahydrofuran (THF) dichloromethane or 1,2-dichloroethane. It will be appreciated by one of ordinary skill in the art that slightly greater than one molecule equivalent of epoxidizing agent will result in the maximal yield of mono-epoxides, and that the reagent, solvent, concentration and temperature of the reaction will dictate the ratio of specific mono-epoxides formed. It will also be appreciated that the mono-epoxides will be enantiomeric mixtures, and that the di-epoxides and the tri-epoxide can be prepared as diastereomers and that the conditions of the reaction will determine the ratios of the products. One skilled in the art will appreciate that under most conditions of reactions the product will be a mixture of all possible epoxides and that these may be separated by standard methods of chromatography. Exemplary approaches to the generation of mono-, di-, and tri-epoxides are provided below. [0289]A) Mono-Epoxides of the Formulae VII, VIII, and IX by Epoxidation of the Compound of Formula II:

##STR00025##

[0290]To a solution of the compound of Formula II dissolved in tetrahydrofuran (THF) is added 1.1 equivalents of meta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0° C. for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of Sephadex LH-20 to isolate a mixture of predominantly the compounds of Formulae VII, VIII and IX, contaminated with some unchanged starting material and some di- and tri-epoxides. The compounds of Formulae VII, VIII and XIX are separated and purified by HPLC using the system described in Example 2 for the purification of the compound of Formulae II. In a typical experiment yields of 15% to 25% are obtained for each of the compounds of Formulae VII, VIII and IX. [0291]B) Synthesis of Compounds of Formulae X, XI, and XII by Di-Epoxidation of Compound of Formula II:

##STR00026##

[0292]To a solution of the compound of Formula II dissolved in tetrahydrofuran (THF) is added 2.3 equivalents of meta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0° C. for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of Sephadex LH-20 to isolate a mixture of predominantly the compounds of Formulae X, XI and XII, contaminated with traces of unchanged starting material and some mono- and tri-epoxides. The Compounds of Formulae X, XI and XII are separated and purified by HPLC using the system described in Example 2 for the purification of the compound of Formulae II. In a typical experiment, yields of 15% to 20% are obtained for each of the compounds of Formulae X, XI and XII. [0293]C) Synthesis of Compound of Formula XIII by Tri-Epoxidation of Compound of Formula II:

##STR00027##

[0294]To a solution of the compound of Formula II, dissolved in tetrahydrofuran (THF), is added 3.5 equivalents of meta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0° C. for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of Sephadex LH-20 to isolate the compound of Formula XIII as a mixture of diasteriomers in a yield of 80+%.

##STR00028##

[0295]To a solution of Compound of Formula II dissolved in tetrahydrofuran (THF) is added 1.2 equivalents of acetic anhydride and a few drops of triethylamine. The reaction mixture allowed to stand at room temperature for 1-2 hours and then evaporated to dryness under reduced pressure to obtain the Compound of Formula III in an essentially pure form in an almost quantitative yield

##STR00029##

[0296]To a solution of Compound of Formula II dissolved in terachloroethylene is added 1.2 equivalents of the appropriate alkyl bromide (benzyl bromide for the compound of formula IV or ethyl bromide for the Compound of Formula V). The reaction mixture the reaction mixture is heated under reflux for 1-2 hours and then evaporated to dryness under reduced pressure to obtain the Compound of Formula IV or the Compound of Formula V respectively, in an essentially pure form in an almost quantitative yield.

##STR00030##

[0297]A solution of the Compound of Formula II (462 mg) in ethanol (200 ml) with palladium on charcoal (25 mg of 5%) is shaken in an hydrogenation apparatus in an atmosphere of hydrogen. The uptake of hydrogen by the reaction is measured carefully and at the point where one millimole of hydrogen has been consumed, shaking is stopped, the vessel is rapidly evacuated and the atmosphere is replaced with nitrogen. The catalyst is removed by filtration and the filtrate is concentrated to obtain a crude mixture of the Compounds of Formulae XL, XLI and XLII contaminated by unreacted starting material and minor amounts of over reduced products. The desired products may be separated and purified by HPLC or HSCC chromatography using the systems as described in Example 2 above, to obtain approximately 100 mg of each of the Compounds of Formulae XL, XLI and XLII.

##STR00031##

[0298]A solution of the Compound of Formula II (462 mg) in ethanol (200 ml) with palladium on charcoal (25 mg of 5%) is shaken in an hydrogenation apparatus in an atmosphere of hydrogen. The uptake of hydrogen by the reaction is measured carefully and at the point where two millimoles of hydrogen has been consumed, shaking is stopped, the vessel is rapidly evacuated and the atmosphere is replaced with nitrogen. The catalyst is removed by filtration and the filtrate is concentrated to obtain a crude mixture of the Compounds of Formulae XLIII, XLIV and XLV contaminated by trace amounts unreacted starting material and minor amounts of under and over reduced products. The desired products may be separated and purified by HPLC or HSCC chromatography using the systems as described in Example 2 above, to obtain approximately 100 mg of each of the Compounds of Formulae XLIII, XLIV and XLV.

##STR00032##

[0299]A solution of the Compound of Formula II (462 mg) in ethanol (200 ml) with palladium on charcoal (25 mg of 5%) is shaken in an hydrogenation apparatus in an atmosphere of hydrogen. The uptake of hydrogen by the reaction is measured carefully and at the point where three millimoles of hydrogen has been consumed, shaking is stopped, the vessel is rapidly evacuated and the atmosphere is replaced with nitrogen. The catalyst is removed by filtration and the filtrate is concentrated to obtain an essentially pure sample of the Compound of Formula XLVI

##STR00033##

[0300]A solution of the Compound of Formula II (100 mg) in acetic anhydride (5 ml) is treated with pyridine (250 ul). The reaction mixture is allowed to stand overnight at room temperature and is then diluted with toluene (100 ml). The toluene solution is washed well with aqueous 5% sodium bicarbonate solutions, then with water and is finally concentrated under reduced pressure to give an essentially pure sample of the Compound of Formula VI in almost quantitative yield.

##STR00034##

[0301]A solution of the Compound of Formula VII (100 mg) in tetrahydrofuran (50 ml) is treated with 1N aqueous hydrochloric acid (5 ml). The reaction mixture is stirred overnight at room temperature and is then diluted with toluene (100 ml) and water (200 ml). The toluene layer is separated and the aqueous layer is extracted with a further 100 ml of toluene. The combined toluene layers are washed once more with water (50 ml) and the separated and dried under vacuum to give the vicinal glycol Compound of Formula LI.

##STR00035##

[0302]A solution of the Compound of Formula II (462 mg) in dry ethyl acetate (200 ml) in an ozonolysis apparatus is cooled to below -20° C. A stream of ozone-containing oxygen is passed into the solution from an ozone generator, which has been precalibrated such that the rate of ozone generation is known. To obtain predominantly the compound of Formula XLVII the passage of ozone is halted after 0.9 millimole have been generated. To obtain predominantly the compound of Formula XLIX the ozone passage is halted after 2 millimoles have been generated and to obtain the compound of Formula LI as the predominant product 3.3 millimoles of ozone are generated.

[0303]At the completion of the ozonolysis, the reaction mixture is transferred to an hydrogenation apparatus, 5% palladium on calcium carbonate catalyst (0.2 g) is added to the reaction mixture which is maintained at less than -20° C. and is hydrogenated. When hydrogen uptake is complete the hydrogen atmosphere is replaced with nitrogen and the reaction mixture is allowed to come to room temperature, filtered to remove catalyst and the filtrate is concentrated. The crude product may be purified by chromatography using either HPLC or HSCC with the systems as described in Example 2 to give, dependent on the amount of ozone used, Compounds of Formulae XLVII, XLIX and LI.

##STR00036##

[0304]A solution of the Compound of Formula XLVIII (50 mg) in isopropanol (5 ml) is cooled in an ice-salt bath and sodium borohydride (10 mg) is added and the mixture is stirred for 20 minutes. It is then diluted with water (20 ml) and extracted twice with toluene (10 ml portions) at ambient temperature. The combined toluene extracts are filtered and the filtrate is concentrated to give the Compound of Formula XLVII.

##STR00037##

[0305]To a solution of Compound of Formula XLII dissolved in tetrahydrofuran (THF) is added 1.1 equivalents of meta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0° C. for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of Sephadex LH-20 to isolate a mixture of predominantly the Compounds of Formulae XIV, and XV, contaminated with some unchanged starting material and some diepoxide. The Compounds of Formulae XIV and XV are separated and purified by HPLC or HSCC using one of the systems described in Example 2 for the purification of the Compound of Formulae II. In a typical experiment yields of 35% to 40% are obtained for each of the Compounds of Formulae XIV and XV.

##STR00038##

[0306]To a solution of Compound of Formula XL dissolved in tetrahydrofuran (THF) is added 2.2 equivalents of meta-chloroperbenzoic acid. The reaction is cooled in an ice bath and stirred at 0° C. for 1-2 hours. The reaction mixture is then evaporated to dryness, re-dissolved in methanol and subjected to liquid chromatography on a column of Sephadex LH-20 to isolate essentially pure Compound of Formulae XIX in good yield.

##STR00039##

[0307]To a solution of Compound of Formula II dissolved in toluene (9 parts) tetrahydrofuran (1 part), cooled in an ice-bath is added 1.1 equivalents of acetic anhydride and two drops of boron trifluoride etherate. The reaction is maintained cool in an ice bath and stirred at 0° C. for 1-2 hours. The reaction mixture is then poured into aqueous 5% sodium bicarbonate solution shaken and the toluene layer is removed. The aqueous layer is re-extracted with toluene and the combined toluene layers are concentrated to a mixture of predominantly the Compounds of Formulae XXVI, XXVII and XXVIII, contaminated with some unchanged starting material and some diacetates. The Compounds of Formulae XXVI, XXVII and XXVIII are separated and purified by HPLC or HSCC using one of the systems described in Example 2 for the purification of the Compound of Formulae II. In a typical experiment yields of 25% to 30% are obtained for each of the Compounds of Formulae XXVI, XXVII and XXVIII.

##STR00040##

[0308]A solution of the Compound of Formula II (1 g) in tetrahydrofuran 50 (ml) is titrated with exactly one equivalent of sodium methoxide, allowed to stand for 30 minutes at room temperature and then treated with 1.2 equivalents of dimethylsulphate. Heat the mixture under reflux for one hour, cool to room temperature and pour into a mixture of toluene (200 ml) and water (200 ml). The layers are separated and the aqueous layer is extracted once more with an equal portion of toluene. The combined toluene layers are washed once with 1N aqueous acetic acid and then concentrated to s crude product, which is predominantly a mixture of the Compounds of Formulae XXXIII, XXXIV and XXXV with some unchanged starting material and traces of over-methylated derivatives. The desired products may be separated and purified by HPLC or HSCC chromatography using the systems as described in Example 2 above, to obtain approximately 200 mg of each of the Compounds of Formulae XXXIII, XXXIV and XXXV.

Example 11

Genes and Proteins for the Production of Compounds of Formula

[0309]Micromonospora sp. strain 046-ECO11 is a representative microorganism useful in the production of the compound of the invention. Strain 046-ECO11 has been deposited with the International Depositary Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2 on Mar. 7, 2003 and was assigned IDAC accession no. 070303-01. The biosynthetic locus for the production of the compound of Formula II was identified in the genome of Micromonospora sp. strain 046-ECO11 using the genome scanning method described in U.S. Ser. No. 10/232,370, CA 2,352,451 and Zazopoulos et. al., Nature Biotechnol., 21, 187-190 (2003).

[0310]The biosynthetic locus spans approximately 52,400 base pairs of DNA and encodes 43 proteins. More than 10 kilobases of DNA sequence were analyzed on each side of the locus and these regions were deemed to contain primary genes or genes unrelated to the synthesis of the compound of Formula II. As illustrated in FIG. 12, the locus is contained within three sequences of contiguous base pairs, namely Contig 1 having the 36,602 contiguous base pairs of SEQ ID NO: 1 and comprising ORFs 1 to 31 (SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 and 63), Contig 2 having the 5,960 contiguous base pairs of SEQ ID NO: 64 and comprising ORFs 32 to 35 (SEQ ID NOS: 66, 68, 70 and 72), and Contig 3 having the 9,762 base pairs of SEQ ID NO: 73 and comprising ORFs 36 to 43 (SEQ ID NOS: 75, 77, 79, 81, 83, 85, 87 and 89). The order, relative position and orientation of the 43 open reading frames representing the proteins of the biosynthetic locus are illustrated schematically in FIG. 12. The top line in FIG. 12 provides a scale in base pairs. The gray bars depict the three DNA contigs (SEQ ID NOS: 1, 64 and 73) that cover the locus. The empty arrows represent the 43 open reading frames of this biosynthetic locus. The black arrows represent the two deposited cosmid clones covering the locus.

[0311]The biosynthetic locus will be further understood with reference to the sequence listing which provides contiguous nucleotide sequences and deduced amino acid sequences of the locus from Micromonospora sp. strain 046-ECO11. The contiguous nucleotide sequences are arranged such that, as found within the biosynthetic locus, Contig 1 (SEQ ID NO: 1) is adjacent to the 5' end of Contig 2 (SEQ ID NO: 64), which in turn is adjacent to Contig 3 (SEQ ID NO: 73). The ORFs illustrated in FIG. 12 and provided in the sequence listing represent open reading frames deduced from the nucleotide sequences of Contigs 1, 2 and 3 (SEQ ID NOS: 1, 64 and 73). Referring to the Sequence Listing, ORF 1 (SEQ ID NO: 3) is the polynucleotide drawn from residues 2139 to 424 of SEQ ID NO: 1, and SEQ ID NO: 2 represents that polypeptide deduced from SEQ ID NO: 3. ORF 2 (SEQ ID NO: 5) is the polynucleotide drawn from residues 2890 to 4959 of SEQ ID NO: 1, and SEQ ID NO: 4 represents the polypeptide deduced from SEQ ID NO: 5. ORF 3 (SEQ ID NO: 7) is the polynucleotide drawn from residues 7701 to 5014 of SEQ ID NO: 1, and SEQ ID NO: 6 represents the polypeptide deduced from SEQ ID NO: 7. ORF 4 (SEQ ID NO: 9) is the polynucleotide drawn from residues 8104 to 9192 of SEQ ID NO: 1, and SEQ ID NO: 8 represents the polypeptide deduced from SEQ ID NO: 9. ORF 5 (SEQ ID NO: 11) is the polynucleotide drawn from residues 9192 to 10256 of SEQ ID NO: 1, and SEQ ID NO: 10 represents the polypeptide deduced from SEQ ID NO: 11. ORF 6 (SEQ ID NO: 13) is the polynucleotide drawn from residues 10246 to 11286 of SEQ ID NO: 1, and SEQ ID NO: 12 represents the polypeptide deduced from SEQ ID NO: 13. ORF 7 (SEQ ID NO: 15) is the polynucleotide drawn from residues 11283 to 12392 of SEQ ID NO: 1, and SEQ ID NO: 14 represents the polypeptide deduced from SEQ ID NO: 15. ORF 8 (SEQ ID NO: 17) is the polynucleotide drawn from residues 12389 to 13471 of SEQ ID NO: 1, and SEQ ID NO: 16 represents the polypeptide deduced from SEQ ID NO: 17. ORF 9 (SEQ ID NO: 19) is the polynucleotide drawn from residues 13468 to 14523 of SEQ ID NO: 1, and SEQ ID NO: 18 represents the polypeptide deduced from SEQ ID NO: 19. ORF 10 (SEQ ID NO: 21) is the polynucleotide drawn from residues 14526 to 15701 of SEQ ID NO: 1, and SEQ ID NO: 20 represents the polypeptide deduced from SEQ ID NO: 21. ORF 11 (SEQ ID NO: 23) is the polynucleotide drawn from residues 15770 to 16642 of SEQ ID NO: 1, and SEQ ID NO: 22 represents the polypeptide deduced from SEQ ID NO: 23. ORF 12 (SEQ ID NO: 25) is the polynucleotide drawn from residues 16756 to 17868 of SEQ ID NO: 1, and SEQ ID NO: 24 represents the polypeptide deduced from SEQ ID NO: 25. ORF 13 (SEQ ID NO: 27) is the polynucleotide drawn from residues 17865 to 18527 of SEQ ID NO: 1, and SEQ ID NO: 26 represents the polypeptide deduced from SEQ ID NO: 27. ORF 14 (SEQ ID NO: 29) is the polynucleotide drawn from residues 18724 to 19119 of SEQ ID NO: 1, and SEQ ID NO: 28 represents the polypeptide deduced from SEQ ID NO: 29. ORF 15 (SEQ ID NO: 31) is the polynucleotide drawn from residues 19175 to 19639 of SEQ ID NO: 1, and SEQ ID NO: 30 represents the polypeptide deduced from SEQ ID NO: 31. ORF 16 (SEQ ID NO: 33) is the polynucleotide drawn from residues 19636 to 21621 of SEQ ID NO: 1, and SEQ ID NO: 32 represents the polypeptide deduced from SEQ ID NO: 33. ORF 17 (SEQ ID NO: 35) is the polynucleotide drawn from residues 21632 to 22021 of SEQ ID NO: 1, and SEQ ID NO: 34 represents the polypeptide deduced from SEQ ID NO: 35. ORF 18 (SEQ ID NO: 37) is the polynucleotide drawn from residues 22658 to 22122 of SEQ ID NO: 1, and SEQ ID NO: 36 represents the polypeptide deduced from SEQ ID NO: 37. ORF 19 (SEQ ID NO: 39) is the polynucleotide drawn from residues 24665 to 22680 of SEQ ID NO: 1, and SEQ ID NO: 38 represents the polypeptide deduced from SEQ ID NO: 39. ORF 20 (SEQ ID NO: 41) is the polynucleotide drawn from residues 24880 to 26163 of SEQ ID NO: 1, and SEQ ID NO: 40 represents the polypeptide deduced from SEQ ID NO: 41. ORF 21 (SEQ ID NO: 43) is the polynucleotide drawn from residues 26179 to 27003 of SEQ ID NO: 1, and SEQ ID NO: 42 represents the polypeptide deduced from SEQ ID NO: 43. ORF 22 (SEQ ID NO: 45) is the polynucleotide drawn from residues 27035 to 28138 of SEQ ID NO: 1, and SEQ ID NO: 44 represents the polypeptide deduced from SEQ ID NO: 45. ORF 23 (SEQ ID NO: 47) is the polynucleotide drawn from residues 28164 to 28925 of SEQ ID NO: 1, and SEQ ID NO: 46 represents the polypeptide deduced from SEQ ID NO: 47. ORF 24 (SEQ ID NO: 49) is the polynucleotide drawn from residues 28922 to 30238 of SEQ ID NO: 1, and SEQ ID NO: 48 represents the polypeptide deduced from SEQ ID NO: 49. ORF 25 (SEQ ID NO: 51) is the polynucleotide drawn from residues 30249 to 31439 of SEQ ID NO: 1, and SEQ ID NO: 50 represents the polypeptide deduced from SEQ ID NO: 51. ORF 26 (SEQ ID NO: 53) is the polynucleotide drawn from residues 31439 to 32224 of SEQ ID NO: 1, and SEQ ID NO: 52 represents the polypeptide deduced from SEQ ID NO: 53. ORF 27 (SEQ ID NO: 55) is the polynucleotide drawn from residues 32257 to 32931 of SEQ ID NO: 1, and SEQ ID NO: 54 represents the polypeptide deduced from SEQ ID NO: 55. ORF 28 (SEQ ID NO: 57) is the polynucleotide drawn from residues 32943 to 33644 of SEQ ID NO: 1, and SEQ ID NO: 56 represents the polypeptide deduced from SEQ ID NO: 57. ORF 29 (SEQ ID NO: 59) is the polynucleotide drawn from residues 34377 to 33637 of SEQ ID NO: 1, and SEQ ID NO: 58 represents the polypeptide deduced from SEQ ID NO: 59. ORF 30 (SEQ ID NO: 61) is the polynucleotide drawn from residues 34572 to 34907 of SEQ ID NO: 1, and SEQ ID NO: 60 represents the polypeptide deduced from SEQ ID NO: 61. ORF 31 (SEQ ID NO: 63) is the polynucleotide drawn from residues 34904 to 36583 of SEQ ID NO: 1, and SEQ ID NO: 62 represents the polypeptide deduced from SEQ ID NO: 63. ORF 32 (SEQ ID NO: 66) is the polynucleotide drawn from residues 23 to 1621 of SEQ ID NO: 64, and SEQ ID NO: 65 represents the polypeptide deduced from SEQ ID NO: 66. ORF 33 (SEQ ID NO: 68) is the polynucleotide drawn from residues 1702 to 2973 of SEQ ID NO: 64, and SEQ ID NO: 67 represents the polypeptide deduced from SEQ ID NO: 68. ORF 34 (SEQ ID NO: 70) is the polynucleotide drawn from residues 3248 to 4270 of SEQ ID NO: 64, and SEQ ID NO: 69 represents the polypeptide deduced from SEQ ID NO: 70. ORF 35 (SEQ ID NO: 72) is the polynucleotide drawn from residues 4452 to 5933 of SEQ ID NO: 64, and SEQ ID NO: 71 represents the polypeptide deduced from SEQ ID NO: 72. ORF 36 (SEQ ID NO: 75) is the polynucleotide drawn from residues 30 to 398 of SEQ ID NO: 73, and SEQ ID NO: 74 represents the polypeptide deduced from SEQ ID NO: 75. ORF 37 (SEQ ID NO: 77) is the polynucleotide drawn from residues 395 to 1372 of SEQ ID NO: 73, and SEQ ID NO: 76 represents the polypeptide deduced from SEQ ID NO: 77. ORF 38 (SEQ ID NO: 79) is the polynucleotide drawn from residues 3388 to 1397 of SEQ ID NO: 73, and SEQ ID NO: 78 represents the polypeptide deduced from SEQ ID NO: 79. ORF 39 (SEQ ID NO: 81) is the polynucleotide drawn from residues 3565 to 5286 of SEQ ID NO: 73, and SEQ ID NO: 80 represents the polypeptide deduced from SEQ ID NO: 81. ORF 40 (SEQ ID NO: 83) is the polynucleotide drawn from residues 5283 to 7073 of SEQ ID NO: 73, and SEQ ID NO: 82 represents the polypeptide deduced from SEQ ID NO: 83. ORF 41 (SEQ ID NO: 85) is the polynucleotide drawn from residues 7108 to 8631 of SEQ ID NO: 73, and SEQ ID NO: 84 represents the polypeptide deduced from SEQ ID NO: 85. ORF 42 (SEQ ID NO: 87) is the polynucleotide drawn from residues 9371 to 8673 of SEQ ID NO: 73, and SEQ ID NO: 86 represents the polypeptide deduced from SEQ ID NO: 87. ORF 43 (SEQ ID NO: 89) is the polynucleotide drawn from residues 9762 to 9364 of SEQ ID NO: 73, and SEQ ID NO: 88 represents the polypeptide deduced from SEQ ID NO: 89.

[0312]Some open reading frames provided in the Sequence Listing, namely ORF 2 (SEQ ID NO: 5), ORF 5 (SEQ ID NO: 11), ORF 12 (SEQ ID NO: 25), ORF 13 (SEQ ID NO: 27), ORF 15 (SEQ ID NO: 31), ORF 17 (SEQ ID NO: 35), ORF 19 (SEQ ID NO: 39), ORF 20 (SEQ ID NO: 41), ORF 22 (SEQ ID NO: 45), ORF 24 (SEQ ID NO: 49), ORF 26 (SEQ ID NO: 53) and ORF 27 (SEQ ID NO: 55) initiate with non-standard initiation codons (eg. GTG-Valine, or CTG-Leucine) rather than standard initiation codon ATG methionine. All ORFs are listed with the appropriate M, V or L amino acids at the amino-terminal position to indicate the specificity of the first codon of the ORF. It is expected, however, that in all cases the biosynthesized protein will contain a methionine residue, and more specifically a formylmethionine residue, at the amino terminal position, in keeping with the widely accepted principle that protein synthesis in bacteria initiate with methionine (formylmethionine) even when the encoding gene specifies a non-standard initiation codon (e.g. Stryer BioChemistry 3^rd edition, 1998, W.H. Freeman and Co., New York, pp. 752-754).

[0313]ORF 32 (SEQ ID NO: 65) is incomplete and contains a truncation of 10 to 20 amino acids from its carboxy terminus. This is due to incomplete sequence information between Contigs 2 and 3 (SEQ ID NOS: 64 and 73, respectively).

[0314]Deposits of E. coli DH10B vectors, each harbouring a cosmid clone (designated in FIG. 12 as 046KM and 046KQ respectively) of a partial biosynthetic locus for the compound of Formula II from Micromonospora sp. strain 046-ECO11 and together spanning the full biosynthetic locus for production of the compound of Formula II have been deposited with the International Depositary Authority of Canada, Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2 on Feb. 25, 2003. The cosmid clone designated 046KM was assigned deposit accession numbers IDAC 250203-06, and the cosmid clone designated 046KQ was assigned deposit accession numbers IDAC 250203-07. Cosmid 046KM covers residue 1 to residue 32,250 of Contig 1 (SEQ ID NO: 1). Cosmid 046KQ covers residue 21,700 of Contig 1 (SEQ ID NO: 1) to residue 9,762 of Contig 3 (SEQ ID NO: 73). The sequence of the polynucleotides comprised in the deposited strains, as well as the amino acid sequence of any polypeptide encoded thereby are controlling in the event of any conflict with any description of sequences herein.

[0315]The deposit of the deposited strains has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure. The deposited strains will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposited strains are provided merely as convenience to those skilled in the art and are not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. §112. A license may be required to make, use or sell the deposited strains, and compounds derived therefrom, and no such license is hereby granted.

[0316]In order to identify the function of the proteins coded by the genes forming the biosynthetic locus for the production of the compound of Formula II the gene products of ORFs 1 to 43, namely SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 were compared, using the BLASTP version 2.2.10 algorithm with the default parameters, to sequences in the National Center for Biotechnology Information (NCBI) nonredundant protein database and the DECIPHER® database of microbial genes, pathways and natural products (Ecopia BioSciences Inc. St.-Laurent, QC, Canada).

[0317]The accession numbers of the top GenBank® hits of this BLAST analysis are presented in Table 14 along with the corresponding E values. The E value relates the expected number of chance alignments with an alignment score at least equal to the observed alignment score. An E value of 0.00 indicates a perfect homolog. The E values are calculated as described in Altschul et al. J. Mol. Biol., 215, 403-410 (1990). The E value assists in the determination of whether two sequences display sufficient similarity to justify an inference of homology.

TABLE-US-00009 TABLE 14 ORF Family # aa GenBank homology Probability % Identity % Similarity Proposed function of GenBank match 1 ABCC 571 NP_736627.1, 590aa 1.00E-107 222/496 (44.76%) 278/496 (56.05%) ABC transporter Corynebacterium efficiens NP_600638.1, 510aa 5.00E-80 184/500 (36.8%) 260/500 (52%) ABC transporter Corynebacterium efficiens NP_600638.1, 510aa 3.00E-12 58/195 (29.74%) 84/195 (43.08%) ABC transporter Corynebacterium efficiens 2 RECH 689 CAC93719.1, 923aa 3.00E-17 57/158 (36.08%) 87/158 (55.06%) regulator[Lechevalieria aerocolonigenes] BAC55205.1, 943aa 3.00E-12 51/170 (30%) 81/170 (47.65%) transcriptional activator [Streptomyces sp. NP_631154.1, 932aa 3.00E-07 29/63 (46.03%) 40/63 (63.49%) regulator. [Streptomyces coelicolor A3(2) 3 REGD 895 CAC93719.1, 923aa 3.00E-20 92/330 (27.88%) 142/330 (43.03%) regulator [Lechevalieria aerocolonigenes] BAC55205.1, 943aa 1.00E-15 80/277 (28.88%) 101/277 (36.46%) activator [Streptomyces sp. TP-A0274] NP_733725.1, 908aa 3.00E-12 95/339 (28.02%) 140/339 (41.3%) regulator [Streptomyces coelicolor A3(2)] 4 IDSA 362 NP_601376.2, 371aa 2.00E-80 158/321 (49.22%) 208/321 (64.8%) GGPP synthase [Corynebacterium glutamicum NP_738677.1, 366aa 3.00E-79 158/330 (47.88%) 204/330 (61.82%) polyprenyl synthase, Corynebacterium efficiens NP_216689.1, 352aa 2.00E-78 153/331 (46.22%) 203/331 (61.33%) idsA2 [Mycobacterium tuberculosis H37Rv] 5 MVKA 354 BAB07790.1, 345aa 2.00E-71 150/326 (46.01%) 193/326 (59.2%) mevalonate kinase [Streptomyces sp. CL190] BAB07817.1, 334aa 5.00E-66 145/324 (44.75%) 185/324 (57.1%) mevalonate kinase [Kitasatospora griseola] NP_720650.1, 332aa 3.00E-36 95/327 (29.05%) 157/327 (48.01%) mevalonate kinase [Streptococcus mutans 6 DMDA 346 BAB07791.1, 350aa 2.00E-88 177/305 (58.03%) 199/305 (65.25%) diphosphomevalonate decarboxylase [Streptomyces sp. BAB07818.1, 300aa 2.00E-69 145/275 (52.73%) 168/275 (61.09%) mevalonate diPH decaroboxylase [Kitasatospora griseola] NP_785307.1, 325aa 3.00E-44 105/307 (34.2%) 141/307 (45.93%) diphosphomevalonate decarboxylase [Lactobacillus plantarum 7 MVKP 369 BAB07792.1, 374aa 4.00E-93 183/365 (50.14%) 220/365 (60.27%) phosphomevalonate kinase [Streptomyces sp. CL190] BAB07819.1, 360aa 6.00E-77 171/358 (47.77%) 202/358 (56.42%) phosphomevalonate kinase [Kitasatospora griseola] AAG02442.1, 368aa 2.00E-31 102/354 (28.81%) 149/354 (42.09%) 3 phosphomevalonate kinase [Enterococcus faecalis] 8 IPPI 360 Q9KWF6, 364aa 1.00E-128 238/361 (65.93%) 269/361 (74.52%) Isopentenyl-diphosphate delta-isomerase Q9KWG2, 363aa 1.00E-128 230/349 (65.9%) 270/349 (77.36%) Isopentenyl-diphosphate delta-isomerase NP_814639.1, 347aa 5.00E-73 154/348 (44.25%) 212/348 (60.92%) isopentenyl diphosphate isomerase [Enterococcus faecalis 9 HMGA 351 BAA70975.1, 353aa 1.00E-165 284/348 (81.61%) 317/348 (91.09%) 3-hydroxy-3-methylglutaryl coenzyme A reductase [Streptomyces sp.] BAA74565.1, 353aa 1.00E-160 282/347 (81.27%) 310/347 (89.34%) 3-hydroxy-3-methylglutaryl coenzyme A reductase [Kitasatospora griseola] BAA74566.1, 353aa 1.00E-155 277/347 (79.83%) 299/347 (86.17%) 3-hydroxy-3-methylglutaryl coenzyme A reductase [Streptomyces sp.] 10 KASH 391 BAB07795.1, 389aa 1.00E-148 260/386 (67.36%) 300/386 (77.72%) 3-hydroxy-3-methylglutaryl CoA synthase [Streptomyces sp. CL190] BAB07822.1, 346aa 1.00E-136 239/343 (69.68%) 268/343 (78.13%) HMG-CoA synthase [Kitasatospora griseola] CAD24420.1, 388aa 6.00E-79 166/385 (43.12%) 210/385 (54.55%) HMG-CoA synthase [Paracoccus zeaxanthinifaciens] 11 IPTN 290 NP_631248.1, 295aa 5.00E-22 79/282 (28.01%) 124/282 (43.97%) hypothetical protein [Streptomyces coelicolor A3(2)] AAN65239.1, 324aa 5.00E-06 70/278 (25.18%) 112/278 (40.29%) cloQ [Streptomyces roseochromogenes subsp. oscitans] 12 SPKG 370 AAM78435.1, 344aa 5.00E-48 112/208 (53.85%) 131/208 (62.98%) two-component sensor [Streptomyces coelicolor A3(2)] NP_630507.1, 382aa 5.00E-48 112/208 (53.85%) 131/208 (62.98%) sensor kinase [Streptomyces coelicolor A3(2)] ZP_00058991.1, 407aa 9.00E-34 88/198 (44.44%) 114/198 (57.58%) Signal transduction histidine kinase [Thermobifida fusca] 13 RREB 220 NP_630508.1, 224aa 3.00E-79 148/220 (67.27%) 179/220 (81.36%) regulatory protein [Streptomyces coelicolor A3(2)] ZP_00058992.1, 221aa 4.00E-67 129/218 (59.17%) 163/218 (74.77%) Response regulator [Thermobifida fusca] NP_625364.1, 221aa 6.00E-66 134/222 (60.36%) 164/222 (73.87%) response regulator [Streptomyces coelicolor A3(2)] 14 UNES 131 No hit -- -- -- -- 15 UNEZ 154 NP_649459.2, 628aa 7.60E-02 21/55 (38.18%) 33/55 (60%) CG1090-PB [Drosophila melanogaster] NP_730819.1, 473aa 7.60E-02 21/55 (38.18%) 33/55 (60%) CG1090-PA [Drosophila melanogaster] AAM11079.1, 428aa 7.60E-02 21/55 (38.18%) 33/55 (60%) GH23040p [Drosophila melanogaster] 16 OXDS 661 NP_242948.1, 500aa 1.00E-52 129/433 (29.79%) 197/433 (45.5%) unknown conserved protein [Bacillus halodurans] ZP_00091617.1, 480aa 3.00E-32 123/426 (28.87%) 175/426 (41.08%) Putative multicopper oxidases [Azotobacter vinelandii] NP_252457.1, 463aa 1.00E-31 115/408 (28.19%) 170/408 (41.67%) metallo-oxidoreductase [Pseudomonas aeruginosa PA01] 17 UNFD 129 NP_437360.1, 127aa 7.00E-33 73/121 (60.33%) 87/121 (71.9%) bleomycin resistance protein family [Sinorhizobium meliloti] AAO91879.1, 123aa 1.00E-31 68/117 (58.12%) 86/117 (73.5%) unknown [uncultured bacterium] NP_103287.1, 131aa 1.00E-23 59/122 (48.36%) 76/122 (62.3%) unknown protein [Mesorhizobium loti] 18 UNFA 178 19 CSMB 661 ZP_00137697.1, 769aa 1.00E-166 319/622 (51.29%) 408/622 (65.59%) Anthranilate/para-aminobenzoate synthase [Pseudomonas aeruginosa NP_250594.1, 627aa 1.00E-166 319/622 (51.29%) 408/622 (65.59%) phenazine biosynthesis protein PhzE [Pseudomonas aeruginosa PA01] ZP_00137701.1, 687aa 1.00E-166 319/622 (51.29%) 408/622 (65.59%) Anthranilate/para-aminobenzoate synthas [Pseudomonas aeruginosa 20 AAKD 427 P41403, 421aa 1.00E-64 161/420 (38.33%) 214/420 (50.95%) Aspartokinase (Aspartate kinase) ZP_00057166.1, 445aa 2.00E-64 154/415 (37.11%) 218/415 (52.53%) Aspartokinases [Thermobifida fusca] AAD49567.1, 421aa 6.00E-64 152/412 (36.89%) 216/412 (52.43%) aspartokinase subunit A [Amycolatopsis mediterranei] 21 ALDB 274 NP_275722.1, 266aa 2.00E-53 104/231 (45.02%) 147/231 (63.64%) conserved protein [Methanothermobacter thermautotrophicus] NP_614692.1, 270aa 2.00E-52 104/240 (43.33%) 146/240 (60.83%) Fructose-1,6-bisphosphate aldolase [Methanopyrus kandleri AV19] NP_615406.1, 267aa 2.00E-50 99/231 (42.86%) 141/231 (61.04%) fructose-bisphosphate aldolase [Methanosarcina acetivorans str. C2A] 22 UNFC 367 NP_275723.1, 378aa 4.00E-46 116/308 (37.66%) 171/308 (55.52%) conserved protein [Methanothermobacter thermautotrophicus] NP_614691.1, 402aa 2.00E-45 115/295 (38.98%) 163/295 (55.25%) alternative 3-dehydroquinate synthase [Methanopyrus kandleri NP_248244.1, 361aa 2.00E-43 103/255 (40.39%) 150/255 (58.82%) conserved hypothetical protein [Methanococcus jannaschii 23 HYDK 253 NP_577771.1, 247aa 4.00E-14 55/178 (30.9%) 87/178 (48.88%) metal-dependent hydrolase [Pyrococcus furiosus DSM 3638] NP_142108.1, 247aa 1.00E-12 50/151 (33.11%) 78/151 (51.66%) hypothetical protein PH0093 [Pyrococcus horikoshii] NP_125791.1, 248aa 1.00E-11 42/151 (27.81%) 76/151 (50.33%) hypothetical protein [Pyrococcus abyssi] 24 ADSA 438 NP_070499.1, 433aa 2.00E-41 122/347 (35.16%) 171/347 (49.28%) coenzyme F390 synthetase [Archaeoglobus fulgidus NP_618724.1, 434aa 5.00E-41 119/345 (34.49%) 171/345 (49.57%) coenzyme F390 synthetase [Methanosarcina acetivorans NP_632700.1, 437aa 7.00E-41 121/345 (35.07%) 171/345 (49.57%) Coenzyme F390 synthetase [Methanosarcina mazei Goe1] 25 HOXV 396 ZP_00027430.1, 442aa 8.00E-76 152/358 (42.46%) 211/358 (58.94%) 2-polyprenyl-6-methoxyphenol hydroxylase [Burkholderia fungorum] NP_627457.1, 420aa 1.00E-71 161/420 (38.33%) 216/420 (51.43%) salicylate hydroxylase [Streptomyces coelicolor A3(2)] ZP_00033877.1, 403aa 2.00E-68 146/395 (36.96%) 200/395 (50.63%) 2-polyprenyl-6-methoxyphenol hydroxylase [Burkholderia fungorum] 26 SDRA 261 NP_391080.1, 261aa 6.00E-58 119/261 (45.59%) 149/261 (57.09%) 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase [Bacillus subtilis] ZP_00059512.1, 260aa 1.00E-55 116/259 (44.79%) 144/259 (55.6%) Dehydrogenase [Thermobifida fusca] AAG31126.1, 257aa 9.00E-55 117/257 (45.53%) 144/257 (56.03%) MxcC [Stigmatella aurantiaca] 27 DHBS 224 Q51790, 207aa 7.00E-60 110/198 (55.56%) 142/198 (71.72%) isochorismatase Q51518, 207aa 1.00E-58 110/198 (55.56%) 140/198 (70.71%) isochorismatase NP_391077.1, 312aa 2.00E-58 106/203 (52.22%) 139/203 (68.47%) isochorismatase [Bacillus subtilis] 28 SDRA 233 NP_103491.1, 242aa 9.00E-21 74/230 (32.17%) 112/230 (48.7%) acyl-carrier protein reductase [Mesorhizobium loti] AAL14912.1, 245aa 1.00E-15 65/229 (28.38%) 100/229 (43.67%) short-chain dehydrogenase [Rhizobium leguminosarum bv. trifolii] NP_902480.1, 235aa 7.00E-15 67/229 (29.26%) 100/229 (43.67%) oxidoreductase [Chromobacterium violaceum 29 UNIQ 246 S18541, 281aa 4.50E-02 43/146 (29.45%) 63/146 (43.15%) hypothetical protein 3 - Streptomyces coelicolor NP_629228.1, 281aa 5.90E-02 43/146 (29.45%) 63/146 (43.15%) hypothetical protein [Streptomyces coelicolor A3(2)] 30 UNFE 111 ZP_00058149.1, 130aa 1.00E-10 35/97 (36.08%) 47/97 (48.45%) membrane protein [Thermobifida fusca] NP_737701.1, 120aa 1.00E-09 37/111 (33.33%) 51/111 (45.95%) hypothetical protein [Corynebacterium efficiens NP_827629.1, 118aa 7.00E-09 35/105 (33.33%) 51/105 (48.57%) hypothetical protein [Streptomyces avermitilis MA-4680] 31 EFFT 559 ZP_00058148.1, 537aa 2.00E-67 165/517 (31.91%) 253/517 (48.94%) Predicted symporter [Thermobifida fusca] NP_626090.1, 544aa 4.00E-66 162/521 (31.09%) 257/521 (49.33%) transport protein [Streptomyces coelicolor A3(2)] NP_827630.1, 549aa 7.00E-63 160/523 (30.59%) 256/523 (48.95%) sodium-dependent symporter [Streptomyces avermitilis 32 HOYH 532 AAM96655.1, 544aa 2.00E-92 206/526 (39.16%) 279/526 (53.04%) 2,4-dihydroxybenzoate monooxygenase [Sphingobium chlorophenolicum] ZP_00029353.1, 543aa 1.00E-73 188/539 (34.88%) 263/539 (48.79%) 2-polyprenyl-6-methoxyphenol hydroxylase [Burkholderia fungorum] NP_769326.1, 569aa 5e-62 173/519 (33.33%) 251/519 (48.36%) blr2686 [Bradyrhizobium japonicum] dbj 33 DAHP 423 T03226, 391aa 1.00E-111 207/383 (54.05%) 259/383 (67.62%) hypothetical protein - Streptomyces hygroscopicus ZP_00137693.1, 405aa 3.00E-87 172/385 (44.68%) 233/385 (60.52%) DAHP synthase [Pseudomonas aeruginosa UCBPP-PA14] NP_250592.1, 405aa 1.00E-86 169/380 (44.47%) 232/380 (61.05%) phenazine biosynthesis protein PhzC [Pseudomonas aeruginosa 34 REGG 340 BAC53615.1, 346aa 1.00E-67 142/307 (46.25%) 192/307 (62.54%) regulator protein [Streptomyces kasugaensis] S44506, 424aa 3.00E-66 141/305 (46.23%) 182/305 (59.67%) regulator protein - Streptomyces glaucescens AAK81822.1, 348aa 1.00E-65 141/323 (43.65%) 192/323 (59.44%) transcriptional regulator [Streptomyces lavendulae] 35 UNFJ 493 ZP_00073237.1, 678aa 7.00E-35 124/454 (27.31%) 197/454

(43.39%) RTX toxins [Trichodesmium erythraeum IMS101] NP_484716.1, 433aa 3.00E-05 109/470 (23.19%) 172/470 (36.6%) similar to vanadium chloroperoxidase [Nostoc sp. ZP_00067005.1, 667aa 7.40E-02 37/139 (26.62%) 52/139 (37.41%) hypothetical protein [Microbulbifer degradans 2-40] 36 RECI 112 NP_627088.1, 125aa 3.00E-17 48/100 (48%) 59/100 (59%) hypothetical protein [Streptomyces coelicolor A3(2)] NP_846017.1, 109aa 7.00E-15 40/101 (39.6%) 60/101 (59.41%) hypothetical protein [Bacillus anthracis str. Ames] NP_241272.1, 174aa 9.00E-15 39/106 (36.79%) 62/106 (58.49%) unknown conserved protein [Bacillus halodurans] 37 UNIQ 325 NP_422203.1, 187aa 1.00E-03 24/61 (39.34%) 36/61 (59.02%) hypothetical protein [Caulobacter crescentus CB15] 38 OXAH 663 ZP_00058724.1, 659aa 0.00E+00 370/647 (57.19%) 435/647 (67.23%) Acyl-CoA dehydrogenases [Thermobifida fusca] AAB97825.1, 433aa 5.00E-93 203/446 (45.52%) 251/446 (56.28%) acyl-CoA oxidase [Myxococcus xanthus] AAF14635.1, 694aa 5.00E-85 211/565 (37.35%) 292/565 (51.68%) 1 acyl-CoA oxidase [Petroselinum crispum] 39 ABCA 537 T14162, 574aa 9.00E-62 189/509 (37%) 240/509 (47%) hABC transport protein - Mycobacterium smegmatis NP_624808.1 4.00E-60 184/540 (35%) 251/540 (46%) ABC transporter [Streptomyces coelicolor A3(2)] NP_822745.1 8.00E-32 124/392 (31%) 168/392 (42%) ABC transportert [Streptomyces avermitilis MA-4680] 40 ABCA 596 T14180, 1122aa 1.00E-107 236/594 (39.73%) 300/594 (50.51%) exiT protein - Mycobacterium smegmatis AAC82548.1, 589aa 1.00E-107 234/583 (40.14%) 295/583 (50.6%) unknown [Mycobacterium smegmatis] NP_624810.1, 601aa 3.00E-97 222/593 (37.44%) 283/593 (47.72%) ABC-transporter [Streptomyces coelicolor A3(2)] 41 UNIQ 507 NP_831570.1, 676aa 8.00E-07 62/262 (23.66%) 116/262 (44.27%) methyltransferases [Bacillus cereus NP_655735.1, 676aa 2.00E-06 61/262 (23.28%) 116/262 (44.27%) ubiE/COQ5 methyltransferase family [Bacillus anthracis NP_844290.1, 681aa 2.00E-06 61/262 (23.28%) 116/262 (44.27%) hypothetical protein [Bacillus anthracis str. Ames] 42 232 NP_830809.1, 208aa 8.00E-08 46/210 (21.9%) 74/210 (35.24%) Transporter, LysE family [Bacillus cereus] NP_844737.1, 210aa 2.00E-07 46/210 (21.9%) 74/210 (35.24%) homoserine/threonine efflux protein [Bacillus anthracis NP_655752.1, 208aa 1.00E-06 47/210 (22.38%) 75/210 (35.71%) LysE, LysE type translocator [Bacillus anthracis 43 132 NP_827272.1, 127aa 4.00E-09 38/107 (35.51%) 52/107 (48.6%) hypothetical protein [Streptomyces avermitilis MA-4680] NP_246491.1, 112aa 5.90E-02 21/94 (22.34%) 44/94 (46.81%) unknown [Pasteurella multocida]

[0318]The ORFs encoding proteins involved in the biosynthesis of compounds of Formula II are assigned a putative function and grouped together in families based on sequence similarity to known proteins. To correlate structure and function, the protein families are given a four-letter designation used throughout the description and figures as indicated in Table 15. The meaning of the four letter designations is as follows: AAKD designates an amino acid kinase; ABCA and ABCC designate ABC transporters; ADSA designates an amide synthetase; ALDB designates an aldolase function; CSMB designates a chorismate transaminase; DAHP designates a 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate synthase activity; DHBS designates a 2,3-dihydro-2,3-dihydroxybenzoate synthase activity; DMDA designates a diphosphomevalonate decarboxylase; EFFT designates an efflux protein; HMGA designates a 3-hydroxy-3-methylglutaryl-CoA reductase; HOXV designates a monooxygenase activity; HOYH designates a hydroxylase/decarboxylase activity; HYDK designates a hydrolase activity; IDSA designates an isopentenyl diphosphate synthase; IPPI designates an isopentenyl diphosphate isomerase; IPTN designates an isoprenyltransferase; KASH designates 3-hydroxy-3-methylglutaryl-CoA synthase; MVKA designates a mevalonate kinase; MVPK designates a phosphomevalonate kinase; OXAH designates an acylCoA oxidase; OXDS designates an oxidoreductase; RECH, RECI, REGD, REGG and RREB designate regulators; SDRA designates a dehydrogenase/ketoreductase, SPKG designates a sensory protein kinase; UNES, UNEZ, UNFA, UNFC, UNFD, UNFE, UNFJ and UNIQ designate proteins of unknown function.

TABLE-US-00010 TABLE 15 FAMILY FUNCTION AAKD amino acid kinase; strong homology to primary aspartate kinases, converting L- aspartate to 4-phospho-L-aspartate ABCA ABC transporter ABCC ABC transporter ADSA adenylating amide synthetase ALDB aldolase; similarity to fructose-1,6-biphosphate aldolase that generates D- glyceraldehyde-3Ph, precursor of D-erythrose-4Ph involved in the shikimate pathway CSMB chorismate transaminase, similarity to anthranilate synthase DAHP DAHP synthase, class II; involved in formation of aminoDAHP from PEP and erythrose-4-phosphate DHBS 2,3-dihydro-2,3-dihydroxybenzoate synthase (isochorismatase) DMDA diphosphomevalonate decarboxylase (mevalonate pyrophosphate decarboxylase) EFFT efflux protein HMGA HMG-CoA reductase; converts 3-hydroxy-3-methylglutaryl-CoA to mevalonate plus CoA in isoprenoid biosynthesis HOXV FAD monooxygenase; shows homology to a variety of monooxygenases including salicylate hydroxylases, zeaxanthin epoxidases HOYH hydroxylase/decarboxylase; FAD-dependent monooxygenase HYDK hydrolase IDSA isoprenyl diphosphate synthase, catalyzes the addition of 2 molecules of isopentenyl pyrophosphate to dimethylallyl pyrophosphate to generate GGPP IPPI isopentenyl diphosphate isomerase, catalyzes the isomerization of IPP to produce dimethylallyl diphosphate IPTN isoprenyltransferase; catalyzes covalent N-terminal attachment of isoprenyl units to amide groups of nitrogen-containing heterocycle rings KASH HMG-CoA synthase; condenses acetyl-CoA with acetoacetyl-CoA to form 3- hydroxy-3-methylglutaryl-CoA MVKA mevalonate kinase; converts mevalonate to 5-phosphomevalonate in the mevalonate pathway of isoprenoid biosynthesis MVKP phosphomevalonate kinase; converts 5-phosphomevalonate to 5- diphosphomevalonate in the mevalonate pathway of isoprenoid biosynyhesis OXAH acyl CoA oxidase OXDS oxidoreductase RECH regulator RECI regulator; similarity to PadR transcriptional regulators involved in repression of phenolic acid metabolism REGD transcriptional regulator; relatively large regulators with an N-terminal ATP-binding domain containing Walker A and B motifs and a C-terminal LuxR type DNA-binding domain REGG regulator RREB response regulator; similar to response regulators that are known to bind DNA and act as transcriptional activators SDRA dehydrogenase/ketoreductase, NAD-dependent SPKG sensory protein kinase, two component system UNES unknown function UNEZ unknown function UNFA unknown function UNFC unknown function UNFD unknown function UNFE putative membrane protein UNFJ unknown function UNIQ unknown function

[0319]Biosynthesis of the compound of Formula II involves the action of various enzymes that synthesize the three building blocks of the compound, namely the farnesyl-diphosphate component (FIG. 13), the 3-hydroxy-anthranilate-adenylate component (FIG. 14a) and the 2-amino-6-hydroxy-benzoquinone component (FIG. 14b) that are subsequently condensed to form the final compound (FIG. 15).

[0320]The farnesyl-diphosphate biosynthesis involves the concerted action of seven enzymes (FIG. 13). ORF 10 (KASH) (SEQ ID NO: 20) encodes a hydroxymethylglutaryl-CoA synthase that catalyzes an aldol addition of acetyl-CoA onto acetoacyl-CoA to yield 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). This product is subsequently reduced through the action of ORF 9 (HMGA) (SEQ ID NO: 18) to form mevalonic acid (MVA). ORF 5 (MVKA) (SEQ ID NO: 10) phosphorylates mevalonate to 5'-phosphomevalonate using ATP as the phosphate donor. The next step in the farnesyl-diphosphate biosynthesis is the phosphorylation reaction of the 5'-phosphomevalonate to 5'-pyrophosphomevalonate (DPMVA) that is catalyzed by ORF 7 (MVKP) (SEQ ID NO: 14). Subsequent decarboxylation of 5'-pyrophosphomevalonate catalyzed by ORF 6 (DMDA) (SEQ ID NO: 12) yields isopentenyl diphosphate (IPP) which is then converted to dimethylallyldiphosphate (DMADP) through the action of ORF 8 (IPPI) (SEQ ID NO: 16) that has isomerase enzymatic activity. The final step in the biosynthesis of farnesyl-diphosphate is the condensation of one molecule of dimethylallyldiphosphate with two molecules of isopentenyl diphosphate catalyzed by the isoprenyl diphosphate synthase ORF 4 (IDSA) (SEQ ID NO: 8). The described pathway involved in synthesis of farnesyl-diphosphate is entirely consistent with related mevalonate pathways described in other actinomycete species (Takagi et al., J. Bacteriol. 182, 4153-4157, (2000)).

[0321]Biosynthesis of the 3-hydroxy-anthranilate component involves the use of precursors derived from the shikimate pathway (FIG. 14a). Chorismic acid is transaminated through the action of ORF 19 (CSMB) (SEQ ID NO: 38) to form aminodeoxyisochorismic acid. This enzyme resembles anthranilate synthases and is likely to catalyze specifically the transfer of the amino group using glutamine as the amino donor. The next step involves isochorismatase activity and is mediated by ORF 27 (DHBS) (SEQ ID NO: 54). This reaction consists in the removal of the pyruvate side chain from aminodeoxyisochorismic acid to form 6-amino-5-hydroxy-cyclohexa-1,3-dienecarboxylic acid. This compound is subsequently oxidized through the action of ORF 26 (SDRA) (SEQ ID NO: 52) yielding 3-hydroxy-anthranilic acid. ORF 24 (ADSA) (SEQ ID NO: 48) catalyzes the activation of 3-hydroxy-anthranilic acid through adenylation generating the 3-hydroxy-anthranilate-adenylate component (FIG. 14a).

[0322]Biosynthesis of the 2-amino-6-hydroxy-benzoquinone component of the compound of Formula II, requires components derived from the aminoshikimate pathway. FIG. 14b depicts the series of enzymatic reactions involved in the biosynthesis of this constituent. ORF 21 (ALDB) (SEQ ID NO: 42) resembles aldolases involved in the generation of precursors of D-erythrose-4-phosphate which is part of the aminoshikimate pathway used for the generation of 2-amino-6-hydroxy-[1,4]-benzoquinone. ORF 33 (DAHP) (SEQ ID NO: 67) catalyzes the initial step in the aminoshikimate pathway that corresponds to the formation of 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate (amino DAHP) from phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E-4Ph). Subsequent reactions leading to 3-amino-5-hydroxy-benzoic acid are catalyzed by enzymes provided by primary metabolism biosynthetic pathways present in Micromonospora sp. strain 046-ECO11. ORF 25 (HOXV) (SEQ ID NO: 50) hydroxylates 3-amino-5-hydroxy-benzoic acid at position 2, generating 3-amino-2,5-dihydroxy-benzoic acid. This intermediate is further modified by ORF 32 (HOYH) (SEQ ID NO: 65) that catalyzes a decarboxylative oxidation reaction yielding 6-amino-benzene-1,2,4-triol. A final oxidation reaction is performed by ORF 16 (OXDS) (SEQ ID NO: 32) yielding 2-amino-6-hydroxy-[1,4]-benzoquinone (FIG. 14b).

[0323]Assembly of the three components resulting in the compound of Formula II is catalyzed by ORFs 24 and 11 (FIG. 15). ORF 24 (ADSA) (SEQ ID NO: 48) catalyzes the condensation of the adenylated 3-hydroxy-anthranilate with the 2-amino-6-hydroxy-[1,4]-benzoquinone component. A spontaneous condensation between the free amino group of the 3-hydroxy-anthranilate and one of the carbonyl groups present on the 2-amino-6-hydroxy-[1,4]-benzoquinone component occurs yielding a dibenzodiazepinone intermediate. This compound is further modified through transfer of the farnesyl group of the farnesyl-diphosphate intermediate onto the nitrogen of the amide of the dibenzodiazepinone catalyzed by ORF 11 (IPTN) (SEQ ID NO: 22) and resulting in the formation of the compound of Formula II (FIG. 15).

[0324]Additional ORFs, namely ORF 2 (RECH) (SEQ ID NO: 4), ORF 3 (REGD) (SEQ ID NO: 6), ORF 12 (SPKG) (SEQ ID NO: 24), ORF 13 (RREB) (SEQ ID NO: 26), ORF 34 (REGG) (SEQ ID NO: 69) and ORF 36 (RECI) (SEQ ID NO: 74) are involved in the regulation of the biosynthetic locus encoding the compound of Formula II. Other ORFs, namely ORF 1 (ABCC) (SEQ ID NO: 2), ORF 31 (EFFT) (SEQ ID NO: 62), ORFs 39 and 40 (ABCA) (SEQ ID NOS: 80 and 82, respectively) and ORF 42 (SEQ ID NO: 86) are involved in transport. Other ORFs involved in the biosynthesis of the compound of Formula II include ORF 20 (AAKD) (SEQ ID NO: 40), ORF 23 (HYDK) (SEQ ID NO: 46), ORF 38 (OXAH) (SEQ ID NO: 78) as well as ORFs 14, 15, 17, 18, 22, 29, 30, 35, 37, 41 and 43 (SEQ ID NOS: 28, 30, 34, 34, 44, 58, 60, 71, 76, 84 and 88, respectively) of unknown function.

TABLE-US-00011 TABLE 16 PREFERRED MEDIA COMPOSITION FOR PRODUCTION OF ECO-04601 COMPONENT QB MA KH RM JA FA pH*⁵ 7.2 7.5 7 6.85 7.3 7.0 Glucose 12 10 10 10 Sucrose 100 Lactose Cane molasses 15 Corn starch 30 Soluble starch 10 25 Potato dextrin 20 40 Corn steep solid Corn steep 5 15 Dried yeast 2 Yeast extract 5 Malt extract 35 Pharmamedia ® 10 15 Glycerol NZ-Amine 5 10 Soybean 15 Soybean flour Meat extract Bacto-peptone MgSO₄•7H₂O 1 MgCl₂•6H₂O CaCO₃ 4 1 2 2 NaCl 5 (NH₄)₂ SO₄ 2 K₂SO₄ 0.25 MnCl₂•4H₂O MgCl₂•6H₂O 10 FeCl₂•4H₂O ZnCl₂ Na₂HPO₄ 3 Thiamine Casamino acid 0.1 Proflo oil 4 MOPS 21 Trace element 2 solution*³ ml/L Unless otherwise indicated all the ingredients are in gm/L. *³Trace elements solution contains: ZnCl₂ 40 mg; FeCl₃ 6H₂O (200 mg); CuCl₂ 2H₂O (10 mg); MnCl₂•4H₂O; Na₂B₄O₇•10H₂O (10 mg); (NH₄)₆ MO₇O₂₄•4H₂O (10 mg) per litre. *⁵The pH is to adjusted as marked prior to the addition of CaCO₃.

[0325]All patents, patent applications, and published references cited herein are hereby incorporated by reference in their entirety. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Sequence CWU 1

89136602DNAMicromonospora sp. strain 046-ECO11 1ccggtgcacc gggttctcca ggatcgccgt cgcgcccacc ggccccgaca ggtagacgac 60gttcagggac ttgccgcgcc cttcgtagtt ggcccgcacc acctgcgcgt cgccgatccg 120gcccctggtc tccagcgtgc ggttctccca cacctgccat ccgacgaagg tcaggaacag 180cgcggtgaac agggacgtga cgagcagcca gaggccagct gtcagcacgg tcgccccctc 240gccccgtagc aggccgagga cgacctcctc gtagcgcgag gggcggccga cggggccggt 300gcccgctccg tcgacagcca tcccgccgct ccttcgccga ctgccccgga catccacggt 360agccagcgag tccagtccgg tgaggaaggg gtggcgagaa gtcgatatga ctgagaggca 420tatttatgac tcccagtcat atcgctcgga agtgaccgaa cgacctgacg ccgccggggc 480tgtgagcggc agcgtgggcc aggccgcgag gtcctggagc atctgccggt cgtgggtggc 540gacgaccacc gccgcccggg tcgtcagcag ggcggcggtg aggtcgtcga ccagcggcgc 600cgacaggtgg ttcgtcggtt cgtcgaggat cagcaggtcg ggacgttcgg ccaggcgcag 660cgccaggttc agccgccgtt gctgtccgtg cgacatccgg ccgacggggg tacgccgggc 720ctcggcgtcg agcaggttcg tcgcgctcag cggcagggcc gtgccggagc cgacgcgccc 780gctggagcgg agccggccca cgtgctgctc gtacaggtcg tgcgcgagca gcgccggcgg 840ccagtcgggc acctcctgac cgaggtacgc gacgcgcgcg ccggacaggt gccggacctc 900cccggtcgac ggcgtgaggt cgccggccag cacggagagc agcgtcgact tgcccgcgcc 960gttgggtccg gtcaccagca ggcggtcccc gccgtcgagc gtgagcgtga cccgggtacg 1020caggcgcccg gccaccgtga cgtcgtggca tcgcaggatg ggcagtccgg cacgggtgtc 1080cagcggcggc cagcgcagcg gctgcggtgg ctccggcacg gtgacgcggt gcgcgtcgag 1140cgcctcctgc cggcggcgca gcgcctggac cagtccgggc gcgcgggact ggcgctggtg 1200cttgccgtgc cccttctccg gccgccagcc ggtgctgagc cggtcccgcg cctcccgtac 1260cccgtcggcc agccgctggt gctcggcctg ctgcgcctcg tggtcgcgta cccagtgcgc 1320gaagtcgcgg cggcgcccgt cctgccaggc gacgtagtcc ccggcgtagc ggcgcgggcg 1380cccgtccgcg ctggggtcga ggtccaggaa ctccgtggcg acgtcccgca gcagggcgcg 1440gtcgtgggtc accagcacga cgccgcccgg gtggtcgcgt agccgggcgg tgaggaaggc 1500caggctgtcg gcgtcgaggt ggttcgtcgg ctcgtccagc atcagcaggt cgaccctcgc 1560tcccagcagg cacgccagcc gtacccggta gcgctggccg acggacaacg tggccagctg 1620ccggtcccgg tccgggcacg cgtcgaggcc ggccagcgcc acgtcgacgc gccgctgcgc 1680gtcccaggcg tccagccggg tcgccgcgtc gagcgcggcc gcgtacgcgt cgtccgcgcc 1740cgcccggccc tcggtgagcg cgatcgtcgc ctcgtcgagc gcccgcagcg cgcgttcgga 1800ctcccggatc gcctcccgga cgagcgtgcc cactgtctcg ccgtggcgcg actccaggtt 1860ctgccgggcg acgccgatcg tgcccagccg ttccaccacg ccctggtcgg gcgcgatgag 1920gccggccagc acgtgcagca gggtggtctt gccgcggccg ttctcgccga cgactgcgag 1980gcgggaagcg gcggagacgg tcacgctgac gtcggacagc acgacccggc cgccgcgtac 2040gacgcggacg ccgtcggccc gcacgtgcgc ccggtgcccg gcgggcagcg aaccgcccga 2100ggtggatggg gaggaaggaa tgttgtcgag gttgtgcaca gtccgctctt cggctcgtcg 2160tggagccggg cagcgcgagg acaccgcccg gcgggaacgc cgggacggcg gagcagagct 2220ggtacgtcag aagaagccgg tcaccctgcc gccgtcagcg gagggaccag ggcttcatga 2280cagcggcgta gaacctcatg cggtcaacac tacccggggc cgggccggag atcgccgcag 2340ttatcggcgg cggcgggcgt cggcctcggc gtcgagcagg tcgttcaccg ccagcgccga 2400gttgatcaga gcgaggtggc tgaacgcctg cggatccacg gttgcggtac tccatttgca 2460gtacacctgt cggtatccgg tcagcgccgt atcctgcgct ttctctgtcg gcagagcggc 2520gcggtcgccc gccgcccgcc gacgtggctg cggggccggt cgggctcgga ccgctcggtg 2580cggcgtcgcg gcccggccgt agcatgtttc acctgttcag agcggcttcc gggcgctcgg 2640gccgtcggcc gcggtggtta ccggcgaggg ctatttcggt catgcgagag ggttctgcca 2700atcgtggcat tgtttagtta agtccgatat cagcgggatg ctgcctgata tatgacggct 2760gcgcccgggc ctgccggata gctatgatga gcgacgacgg tgatcgatgg caaatgttgt 2820tgctgtgggg tagcgtcacc gccgagtcca ggcttttctt gagctgtgtg cgcatattcc 2880ggggggatta tgacaacggg acggccgggg gagaaccggg cgacagacgc ggcacgaaat 2940ccggggtggg ccgccggggg gccggcgtcc cagccatggg gcggggggaa cgacgagcag 3000gtcctgcgcg agatcctcgg ggtcgacgtg caccgcgagc tgattgactt cgcgggtggt 3060gccggcggaa atccgcacct ggtcgccgaa ctcgcgcgcg ggctcgccga agagggattg 3120attcgggaga caaacggtcg ggcggaattg gtgtcccggc gaattccccg gcgcgtgctg 3180agttttgtca tgcgtcgatt gaatgatgtc agcgccggct gccagcagtt cttgaaggtt 3240gccgcggcat tgggcagatc cttcatgctg gaggacgttt cgagaatgct gggccgatcg 3300tcggcggccc tgctcccgcc ggtggacgag gcgatcgcat cgggcttcgt cgtcgccgcc 3360gagcatcaac tcgcctttca gagcgacttc ctgctgcgcg gcatcatcga gtccattccc 3420gggcccgccc gcgacgcctt acgacgtgag gcgatgagcc tttccgggcg acggcgcccg 3480gcggccgacc agaatcgccg gttggacgcg gcgcctaccg cgccggtgag cgcgaccggg 3540gaggacgcca ccggatcctg ttcccgggcg caccgcctga taatgaacgg gaacgcgaag 3600gccggcattc gcgtcgccga ggcggttctc gccggcccgg ccgcgtcgct cgctgcccgg 3660cgtgacgcgg aggcgtgtct ggtgctggcc gatctgctgc tcggcgggga gggcggcggc 3720ccgatgaccg aggcgatcct gcgcgaacgc gacgccgagt ccggtgacgc cgcactggcg 3780atggcgctga ccgcccggtc caccgggctg tggtcggcgg gaaagctggc ggagggcctg 3840aagctgggac gggcggcggt gcgggcgggc gcggaggccg aaccggtgtg gcgtctgcac 3900gcccagctcg cgctcgccgg gaaactcgcg aacctccgcg agttcgacga ggccgaggcg 3960ttgatcaacg aggcggaagc gggcctgcgc ggactgcccg cgccgatctg gacggccgcg 4020acggcggtga tgcggtcccg gttgctgctc caggcggggc ggatcgggga ggcgcgtcgg 4080gaggcggcgc tggccaccac cgccgtggag ggggacgcgg tgccgatgct gcggcctctc 4140gcctacgcgg tgctcagcac cgcctccttc tacatggggg acctgcccgc cgcgatcgag 4200tacctcaggc gggggcagcg ggacgcggac cgccacgtgg tcctcgactc ggtgcagtac 4260tcgtgggcgg aagtgctgat cacggtcaag caggaaggcc cgcgggccgc cgcccagctg 4320ctcgcgggca agcaccaccg cctgcccacg cagcgccgcc tctacgtcga ggtgccgagc 4380gccgccgcct tcctggtcct gctcgcccgc gacgtggacg accgtgacct cgaacgccgc 4440gtcctcgaca cggtcaacgg gctcgccgcg gacaacccca ggatccaggt cgtcagcctc 4500accgccatgc acgcccacgc gctggcgaac agcgctccgg ccgccctggc gctcatcatc 4560gtgcagtcac gggacccgat ctcggtggcg ctggccaccg aggaactcgc caagctctac 4620gccgcgcagg cccaggcggg gggacggccg gcgacgccgg cccgcgccga ggaggccgcc 4680accccgccgg cgagctgctg gtcgaccctg tccgacatgg agcagcggat cgcctacctg 4740gtgagcgtgg gtctgacgaa ccggcagatc gccaagcagg tccacctgtc cgcgcacacc 4800gtcaactacc acctgcggaa gatctaccgg aaactgggtt tcaacacccg ggccgagctg 4860gcgcacgccg cggccacgta ctccggccgg gcggcgatct actccatgag cggcgaccag 4920gactggggcg ccggatccat gaccggcaag gccagctgaa ccgcattccc ggcgtccgcc 4980ggctgaaccg cgccccggcg tacgccggcc ggttcagccg gcggacgccg gctggcgtgt 5040ggtggccagc gccggccgga ccgcctcgtg cgcgatgaag cagcgggtca gttccacccg 5100gctgttgatg tcgagcttgg agaagacgcg ccgcaggtga ctgtcgacgg tgtgcgggga 5160caggaacagc gaactcgccg cctcgcggtt ggtcatcccg tccacgatgg cccgcacgac 5220ccgcagctcc gcgctggtca ggctctccca ccccgaccgg ggccggtcgg ggaccagcgg 5280gcggacgttg tgagccggca ggccacgcag ctcggcctcc acgcgctcca ggtcgcgtcg 5340cgcgccgcac tcccggtagc cgtccgtcgc ggcctcgagc agacgggtgg cctcggcccg 5400gtcccgggtg ctgcgggccg cgtcctccac cgcgccggcc gccgcgagcg tacggccggc 5460gagccggtgc agatccgcgg cccgcagcag cgccgccgga tcgtcgcgca ggagacccgc 5520ggcgtgttcc gccgccgccg ccagcgactg gacgaacggg ttgccgcggg cgacgcgccg 5580ggcgacctcc acggcgcgct cggcctccgc gtcgagcccc gcccggcggg cctggcgtac 5640gagcgtcgcc gcagcggccg gcgcctcggt gaacagcagc ggatcgggtg cgacctgtcc 5700ggcgacgttg atcagcgtct gcaccatcat cgccggacgg ccgctggcag cgtggaaccg 5760ggccagcgcc cagtccatcc gcgccgagtc gtcggcggag gccagccgct cggccgcccg 5820caactggtcg ctggccgtgg cgaggtcacc gtggtgcacg ccgaggtggg ccaggaccag 5880gcgcgccggc acgcagtcgc ccggccggga gtggtcggcg gctcgcagcg ccgcctccgc 5940ctcggcgcgt gcctcgtcca gccgtccggc cgctgccagc agctcggccc ggtggccgcg 6000ccagagcgac tccgagccgg tgtgactggg ctcctgcgcc agcggtcgta cggtgtccag 6060caccgcctgc gcctcgtcga gctgatcggc cgcgcccagc gcccggacca gccaggtcca 6120cagcggccgc cggccgggcg cgcagcccgg ggactggtgc cggggctcca gctcggcgga 6180ggaggcaccg cccaggtgct tcgtggtgtc cgcgagcgcc cggtccagct tggcgcggtc 6240cagctcgcac acgtcgtggc gggcctgcgt ccggcgcagg aagccggccg ccaggcggtg 6300gctgccggcg gcccgcatcc cgtgtcccag ttcgagcacg agctgcgcct cgacgtccgc 6360cgcgaggtcg cggcggagca tcacctccgc gaggcggccg gcctcggcgg cccgccccgc 6420cccggccagc aggcgcagcg cacgggccag tgctcgtggc gcctcggcgg atccgttctc 6480caggtgggac acggcggctg ccgccacgtc gtcgcacccg caccggcccg agcgcggccc 6540cgccgtcgcg gcgggcgtgg ccgcgtccgg cgcggagcgc gtgacgcgta cgccggcggg 6600ggagtggggc gtcccgggcc gcggatcggg ccgcccgcgc cggaccgggt cgcccgccgc 6660cggtgccggc gcggatccgg gctcggcacg ctccggttcc gggtacgcgg cgtggcgaag 6720cgcctctccg agcaccgggt gggcgaaggt cagctccgcg ccgtcgcgtc gtatcagccc 6780gacccgcacc gcctcgtcga tcgcggcgga cacgtcggcg gccgagccgt ccagcaggcc 6840cgtcacccgg tcgacgggaa acgtgtggcc gagccggccg ccggccgcga gcaggcgccg 6900cagcgggggc ggcagctcct ccagcagccc gcgaacggcg gcgaggacac cgtcgggcag 6960ctcgtcggac accaccgacg ccgccccgtc cacgatgatc atctggccgg ccttgatgaa 7020cgcgctgaag acgatctcca tcaccttcgg gttgccgccg cagcgggccg cccagcgcag 7080gacggaggcg tccggccggg cgccgaggat gccggcgcac aggtcggcca ccgcctcctc 7140gcccggctcg cgcagccgta cccgtaccgc gacgtgctcg gccagccagt cgacggcgtg 7200ctgagcgatc gacccggcgg cgaccggccg gcgggccagc agccagagca ccggcgagga 7260cgccaggcgc ggcacgagcc cgcgcagggc cagggcgctg acgtcgtcga tgcgctgggc 7320gtcgtccagg gcgaccacga gcgggcgccg gcgcgccgcg acctcgacca gatcgccgac 7380ccggtcgatc agccagaacg ggttggcgcc cggcagggcg agctgctcga ccgccgcttc 7440gccgggcatc gcgtggcgca ggaagttgac gagcaggtgt acgggcaccg gctgatccgt 7500gacgcttgcc cgcccggcca ccactgtcag cccgcgggcc gccgcctcca ggccggtgac 7560cttcagcagg tgggtcttgc cgatgccgaa cggcccgtcg acgacgacgc agcccccgga 7620tccccgcatg gtggcgtcga gcagttcccc caatgaggac aattcctgcc cgcgccccgc 7680catgcgattc atgatgacca tcccgttttc ctctgctgaa tcgtccgacg tgcgccgcga 7740gccgatgtcc caccgcgttc gaccgtccgt tctggacagt tgaacgccgg atcggggcgg 7800gctactcagt tatacgggat ctgcggccgt tcgtcggcga cgtcgctggc agcgcgcact 7860actcgcgtga gtagtgggca gggtgtcagg ccgcgattac tgtcaggcca tgccgggctc 7920ggcgtgccgg cgcggacgaa atggcgacgc cgatggggag atcggcgtcg tttccgcgcc 7980ggcgcaaaac gtccggaacg gaatcgacta atcgccgctc gacgcgactg gtccagcgaa 8040tccaggggag tccgagatgc gtgagtgtaa tggtgaccgc cgtcttgatc gggagacgcg 8100ggcatgaccg tcggatatct cgggacggtc accgactcgg cgcccgtcga cgccgcgctg 8160cgcgacttct tcgccgagcg ccgcgccgag gcacgcgagc tcggcgacga cttcgcggcc 8220ctggtcgccg agctggagag ctacgtcctg cggggcggca agcgcatccg gcccgccttc 8280gcctggctgg gctggatcgg cgccggcggc gacccggagg acccggtggc gaccgcggtg 8340ctgaacgcct gcgccgggtt cgagctgctg cacgcgtccg gcctcatcca cgacgacatc 8400atcgacgcgt cgcagacccg ccgcggccat cccgccgcgc acgtcgcgta cgccgaacgg 8460catcgggcgc ggcgcttctc cggtgacccg ggaacgttcg gcaccggcac cgccatcctg 8520atcggagacc tcgtcctgat ctgggccgac gtcctggtcc gcgcctccgg cctgccggcc 8580gacgcgcacg tgcgggtctc gccggtgtgg tcggcggtgc gctccgaggt catgtacggc 8640cagctgctcg atctgatcag ccaggtgagc cggagcgagg acgtcgacgc ggcgctgcgc 8700atcaaccagt acaagaccgc gtcgtacacg gtggagcggc cactgcagtt cggcgcggcg 8760atcgccggcg cggacgacga cctcttcgcg gcctaccgcg ccttcggcgc cgacgtgggt 8820attgccttcc agctgcgcga cgacctgctc ggcgtgttcg gcgacccggt ggtgacgggc 8880aagccgtccg gcgacgacct gcgggagggc aagcggacgg tcctgctcgc cacggcgctc 8940aagcgcgccg acgaacggga cccggacgcg gcggcctacc tgcgggcgaa ggtcggcacg 9000gacctcgcgg acgaggagat cgcccgcatc cgcgccatct tccgcgacgt cggcgcggtc 9060gaggagatcg agcggcagat ctcgcagcgc accgaccggg cgctggccgc gctggaggcg 9120agcagcgcca ccgcccccgc gaagcatcag ctcgccgaca tggcgatcaa ggccacccag 9180cgggcccagt gatgtccacg gaaccggtga ccgtcgtcgc ccgcggcgtt ctcgacggcc 9240ggggtgacgg gccgggccgc ctcggcaccg gccgcgccca cggcaaggcc atcctgctgg 9300gcgaacacgc cgtcgtgtac ggcgctccgg cgctcgccgt cccggtgccg caactgaccg 9360ccgtggccaa ggcgcggcgg gccggcggcg acggcggcga cgaggtctcc ttcgccatcg 9420ccgggctgga gagcccggag gtgacgtcgc ttccgaccga cggcctgcaa catctggtga 9480cggagttccg gcagcgggcc gccgtcaccg agccgatgcg cgtcgacgtg ctcgtggact 9540gcgccatccc gcagggccgg gggctcgggt cgagcgccgc ctgcgcccgc gccgcggtgc 9600tggccctcgc ggacgcgttc gaccgccgcc tcgacgccgc cacggtgttc gatctggtgc 9660agacctcgga gaacgtggcg cacggccggg ccagcggcat cgacgccctg gccaccggtg 9720cgaccgcgcc gctgatcttc cgcaacggcg tgggccggga actgccggtc gccatggcgg 9780gcgccgcgcg tgccgcgcga gggtcgggcc cggccggctt cgacgcggtg ctcgtcatcg 9840ccgacagcgg cgtcagcggc agcacccggg acgcggtgga gctgctgcgg ggtgccttcg 9900agcgctcccc gcgcacgcgc gacgagttcg tcagccgggt gaccagcctg accgaggcgg 9960cggcgcacga cctgctccag ggccgggtcg ccgacttcgg cgcgcggctg accgagaacc 10020accggctgtt gcgcgaggtc ggcatcagca ccgaacggat cgaccggatg gtcgacgccg 10080cgctcgcggc gggcagcccg ggcgccaaga tcagcggcgg tggcctgggc ggctgcatga 10140tcgcactggc ccgggaccgc caggaatccg cggcggtggt gcggagcgtc cagcaggccg 10200gcgccgtccg cacctggacc gtcccgatgg ggaggttcac cggccatgac gactgaccac 10260cgggcggagc cgtccgagcc ggcgctcgac cggcccgcga ccgccgtggc ccatccgaac 10320atcgcgctga tcaagtactg gggcaagcgc gacgagcagc tgatgatccc gtacgccgac 10380agcctgtcga tgacgctcga cgtcttcccg accaccacca ccgtccggat cgacagcggc 10440gcggcggccg acgaggtcgt cctcgacggc tcgcccgccg acggcgaacg gcgacagcgc 10500gtcgtcacct tcctggacct ggtacgcaag ctggccgggc gcacggaacg ggcctgcgtc 10560gacacccgca actccgtgcc caccggcgcc ggcctggcgt cctcggcgag cggattcgcc 10620gccctcgccc tcgccggcgc cgccgcgtac ggcctcgacc tggacaccac cgcgctgtcc 10680cgcctggccc ggcggggatc cgtgtcggcc tcccggtcgg tcttcggcgg cttcgcgatg 10740tgccacgcag gccccggcgc cgggaccgcc gcggacctcg gctcctacgc cgagccggtg 10800cccgtcgcgc ccctcgacgt cgcgctggtg atcgcgatcg tcgacgccgg gccgaaggcg 10860gtgtcgagcc gcgaggggat gcggcgaacc gtccggacct ccccgctcta tcagtcgtgg 10920gtcgcctccg gccgcgccga cctggccgag atgcgggccg cgctgctcca gggagacctg 10980gacgcggtcg gcgagatcgc cgaacgcaac gccctcggca tgcacgccac catgctggcc 11040gcccggccgg cggtgcgcta cctggcgccg gtcactgtcg ccgtgctcga cagcgtgctg 11100cgcctgcgcg ccgacggcgt ctccgcctac gccacgatgg acgcgggacc gaacgtcaag 11160gtgctctgcc gccgcgcgga cgccgaccgg gtcgccgaca ccctgcgcga cgccgcgccg 11220agctgcgccg tggtcgtcgc cggaccgggg ccggcggccc ggccggaccc gggcagccgg 11280ccgtgaccgg cccgggcgcc gtgcgccgcc acgcgccggg caagctgttc gtcgccggtg 11340agtacgcggt gctggagccg ggccacccgg cgctgctggt ggcggtcgac aggggagtgg 11400acgtcaccgt ctccggcgcc gacgcccacc tcgttgtcga ctccgacctc tgcccggagc 11460aggcgtgcct gcggtggcag gacggccggc tcgtcggcgc gggcgacggg cagccggcgc 11520ccgacgccct cggcgccgtg gtctcggcga tcgaggtggt cggcgaactc ctgaccggac 11580gagggctgcg cccgctgccc atgcgggtgg cgatcaccag ccggctgcac cgcgacggca 11640cgaagttcgg cctcgggtcg agcggggcgg tgacagtcgc cacggtgacc gcagtggccg 11700cgtaccacgg ggtggagctg tcgctcgaat cgcggttccg gctggcgatg ctggcgacgg 11760tgcgtgacgg cgccgacgcc tccggcggtg atctggccgc gagcgtctgg ggcggctgga 11820tcgcctacca ggcgcccgac cgcgcggccg tgcgcgagat ggcgcggcgg cgcggcgtcg 11880aggagacgat gcgcgcgccc tggccgggcc tgcgggtccg gcggctgcca ccaccgcgtg 11940gcctcgcgct ggaggtgggc tggaccggcg agccggcgag cagcagctcg ttgaccgggc 12000ggctggccgc ctcccggtgg cggggcagcc cggcgcggtg gagcttcacc agccgtagcc 12060aggagtgtgt gcgtaccgcc atcgacgcgc tggagcgggg cgacgaccag gaactgctgc 12120accaggtccg gcgggcccgg cacgtgcttg ccgagctgga cgacgaggtc cggctcggga 12180tcttcacccc ccggctgacg gcgctgtgcg acgccgccga gaccgtcggc ggcgcggcca 12240aaccgtccgg cgccggtggc ggggactgcg gcatcgcgtt gctggacgcc accgccgcga 12300cgcggaccgc gcggctgcgc gagcagtggg ccgccgccgg ggtgctcccc atgccgatcc 12360aggtccatca gacgaacggg agcgcgcgat gatcgccaac cgcaaggacg accacgtccg 12420gctcgccgcc gagcagcagg gccggctcgg cggtcaccac gagttcgacg acgtgtcctt 12480cgtgcaccac gccctggccg gcatcgaccg gtccgacgtc tcgctggcca cgtcgttcgg 12540cggcatcgac tggccggtgc cgctgtgcat caacgcgatg accggcggca gcaccaagac 12600cggcctgatc aaccgggacc tggcgatcgc ggcccgggag accggcgtac cgatcgccac 12660cgggtcgatg agcgcctact tcgccgacga gtcggtggcc gagagtttca gcgtgatgcg 12720ccgggagaac cccgacgggt tcatcatggc caacgtcaac gccaccgcct ccgtcgaacg 12780ggcccggcgg gctgtcgacc tgatgcgggc cgacgcgctg cagatccacc tgaacaccat 12840ccaggagacg gtgatgccgg agggggaccg gtcgttcgcc gcctgggggc cgcggatcga 12900acagatcgtc gccggcgtcg gtgtgccggt gatcgtcaag gaggtcggct tcgggctcag 12960ccgcgaaacg ctgctgcggc tgcgggacat gggcgtccgg gtggccgacg tcgccggccg 13020cggcggcacg aacttcgcgc gcatcgagaa cgaccggcgg gacgccgccg actactcctt 13080cctcgacggg tggggacagt cgacacccgc ctgcctgctg gacgcccagg gcgtggacct 13140gcccgtgctg gcctccggcg gcatccgcaa cccgctcgac gtggtccgcg ggctggcgct 13200cggcgccggc gcggccgggg tgtccggact gttcctgcgc acgctcctgg acggcggcgt 13260gccggcgctg ctgtcgctgc tgtccacctg gctcgaccag atcgaagccc tgatgaccgc 13320cctgggcgcg cggaccccgg ccgacctgac ccgctgcgac ctgctgatcc agggtcggct 13380gagcgcgttc tgcgcggccc ggggcatcga cacccaccgc ctcgccaccc gttccggcgc 13440cacccacgag atgatcggag gcattcgatg aacgacgcga tcgccggtgt gcccatgaaa 13500tgggtaggtc ccgtgcggat ctcgggaaac gtggcgcaga tcgagacgga ggttccgctc 13560gccacgtacg agtcgccgct ctggccgtcc gtcggccggg gcgcgaagat ctcccggatg 13620gtcgaggcgg gcatcgtcgc cacgctcgtc gacgagcgca tgacccgctc ggtgttcgtg 13680cgcgccaagg acgcgcagac cgcctacctg gcctcgcttg aggtcgacgc gcggttcgac 13740gaactgcgtg acatcgtgcg cacctgcggc aggttcgtcg agctgatcgg gttccaccac 13800gagatcaccg cgaacctgct gttcctgcgg ttcagtttca ccaccggcga cgcgtccggg 13860cacaacatgg cgacgctggc cgccgacgcg ctgctgaagc acatcctgga caccattccg 13920ggcatctcgt acggctcgat ctcgggcaac tactgcaccg acaagaaggc caccgcgata 13980aacggcattc tcggccgggg caagaacgtg gtcaccgagc tggtcgtgcc gcgggagatc 14040gtccacgaca gcctgcacac gacggcggcg gcgatcgccc agctgaacgt gcacaagaac 14100atgatcggca cgttgctcgc cggcggtatc cgctcggcca acgcccacta cgcgaacatg 14160ctgctcgggt tctacctggc cacgggtcag gacgccgcga acatcgtcga gggctcccag 14220ggcgtgacgg tcgccgagga ccgcgacggc gacctctact tctcctgcac gctgcccaac 14280ctgatcgtgg gcaccgtcgg caacggcaag gggctcggct tcgtcgagga gaacctggag 14340cggctcggct gccgcgcctc gcgtgatccg ggcgagaacg cccggcggct cgcggtcatc 14400gcggccgcga cggtgctctg cggcgagctg tccctgctcg ccgcgcagac caacccgggc 14460gagctgatgc gggcgcacgt ccggctcgaa cgcccgaccg agaccacgaa gatcggagcc 14520tgacgatggc cgagagaccc gccgtcggca tccacgacct gtccgccgcg acggcgcatc 14580acgtgctgac acacgagacc ctggccgcga gcaacggcgc cgacgtggcc aagtaccacc 14640gtggcatcgg gctgcgggcg atgagcgtgc ccgccccgga cgaggacatc gtgacgatgg 14700ctgctgccgc cgccgcgccg gtggtcgccc gccacggcac cgaccggatc cggaccgtcg 14760tgttcgccac ggagtcgtcg gtcgaccagg cgaaggcggc cgggatacac gtccactccc 14820tgctcggcct cccctcggcc acccgggtgg tcgagctgaa gcaggcctgc tacggcggta 14880cggcgggact gcagttcgcc atcggcctgg tgcaccgtga cccgtcgcag caggtcctgg 14940tgatcgccag cgacgtgtcg aagtacgcgc tgggtgagcc cggcgaggcg acccagggcg 15000ccgcggcggt cgccatgctc

gtcggcgcgg acccggcgct ggtacgcgtc gaggacccgt 15060cgggcatgtt caccgccgac gtcatggact tctggcggcc gaactaccgc accaccgccc 15120tggtcgacgg gcacgagtcc atctccgcct acctgcaggc gctggagggc tcgtggaagg 15180actacaccga gcgcggcggt cgcaccctgg acgagttcgg cgcgttctgc taccaccagc 15240cgttcccgag gatggccgac aaggcgcacc ggcacctgct caactactgc gggcgcgacg 15300tcgacgacgc gctggtggcc ggggccatcg ggcacaccac cgcgtacaac gccgagatcg 15360gcaacagcta cacggcgtcg atgtatctcg ggctcgcggc actgctcgac accgccgacg 15420acctgaccgg ccggaccgtc ggcttcctca gctacgggtc cggcagcgtc gccgagttct 15480tcgccggcac tgtcgtgccc gggtaccgcg cgcacacgcg acccgaccag caccgcgcgg 15540cgatcgaccg gcggcaggag atcgactacg cgacgtaccg ggagttgcac gagcacgcct 15600tcccggtcga cggcggcgac tatccggcgc cggaggtgac caccgggccg taccggctgg 15660ccgggctctc cggtcacaag cgcgtctacg agccgcgata ggaccggcca cgccggccgc 15720cctgaccgaa cgaaccatgc ttggaggatc gatgtccgga actcccgagg tggccgagct 15780ctactcgacc atcgaggaat cggcccggca actggacgtg ccgtgttcgc gcgaccgggt 15840ctggcccatc ctgtccgcgt acggcgacgc gttcgcccat cccgaggcgg tggtcgcctt 15900ccgggtggcg accgcgctgc gtcacgcggg cgagctggac tgccggttcc ggacgcatcc 15960ggacgaccgg gacccgtacg cctcggcgct cgcccggggc ctcaccccgc gcacggacca 16020ccccgtcggc gcgctgctct ccgaggtcca ccggcgctgc ccggtggaga gccacggcat 16080cgacttcggg gtggtcggcg gcttcaagaa gatctacgcg gccttcgccc cggacgagct 16140gcaggtggcc acgtcgctcg ccggcattcc ggcgatgccc cgcagcctcg ccgcgaacgc 16200cgacttcttc acccggcacg gcctcgacga ccgggtcggc gtgctgggat tcgactaccc 16260ggcccggacc gtgaacgtct acttcaacga cgtgccgcgt gagtgcttcg agccggagac 16320catccggtcg acgctgcgcc ggaccgggat ggccgagccg agcgagcaga tgctccggct 16380cggcaccggg gcgttcgggc tctacgtcac gctgggctgg gactccccgg agatcgagcg 16440gatctgctac gccgcggcga ccacggacct gaccacgctt ccggtacccg tggaaccgga 16500gatcgagaag ttcgtgaaaa gcgttccgta cggcggcggg gaccggaagt tcgtctacgg 16560cgtggcgctg acccccaagg gggagtacta caaactcgag tcgcactaca aatggaagcc 16620gggcgcggtg aacttcattt gaacagcggc cggttccgcc gcccgggcgg cggaaccggg 16680atcaatgcct gttcgctcgg gttcaacact ggcgcgctcc gctaaagtgc gaacatgacg 16740actggactgt ccagtgtgtg ggcccgggtg aagaactggg tcgtcgcgtt ggctgtggcg 16800gcggtgctga tgatcagcgc gctggccggt gaccatcctg cccccgaggg cctcggtctg 16860ctcggcttcg cgctggtggc ggcgagcggc ctggcgctgg ccgccagtcg tcgggccccg 16920atcgccgtgc tggtcgccac cgggctgtgc gtggtgggct acaacgcgat cggcttcggg 16980gtgcccgcca tcgcgtacct gttcgcggtc tacgcggcgg tccgggccgg gcaccggctc 17040gtcacgctcg gggcgagcgc cgccctgctc gtcgtcctgc cgctggcgat catggtctcg 17100cccgcggacg gcgccctcaa ggaggcgctc gcgcagtcgc ggggcgtgct ggaactggcc 17160tggctgatcg ccgcggcggc ggccggtgag gcgctgcggc aggccgaacg gcgagcggac 17220gaggcggaac ggacccgcga ggagaccgcc cggctgcgcg ccacccagga gcggctgcac 17280atcgcacggg agctgcacga ctcgctcacc caccagatct cgatcatcaa ggtgcaggcg 17340gaggtggcgg tccacctggc ccgcaagcgg ggcgagcagg tgccggagtc gctgctggcg 17400atccaggagg ccggccgggc ggcgactcgc gagctgcgcg cgaccctgga gacgctgcgt 17460gacctgacca agtccccgtc gcacgggctc gaccacctcc cggagctgct ggccggggcc 17520gagaagatcg gcctggccac cacgctgacc atcgagggcg accagcggga cgtgccggag 17580gcggtgggcc gcaccgcgta ccggatcgtg caggagtcgc tcaccaacac cgcccggcac 17640gcctccgccg cggccgccgc ggtccggatc gactaccgcc cggacgcgct gagcatccgg 17700atcgacgacg acgggacggc ccggccgggc gccgccccgg tgcccggcgt cgggctgctg 17760gggatgcacg agcgcgtcct cgcgctgggc ggccggctgc gggcggaacc ccgcaccggc 17820ggaggcttca ccgtccaggc cgaactcccg gtggtgcgcg tcccatgatc aggatcatgc 17880tgctcgacga ccagccgctg ctgcgcagcg ggttccgcgc gctcctcgac gccgaggacg 17940acatcgaggt ggtggccgag ggcgggaacg gccgggaggg cctggcgctg gcccggcagc 18000acctgcccga tctcgccctg atcgacatcc agatgccggt catggacggc gtcgagacga 18060cccggcagat cgtcgcggat ccggcgctgg ccggggtacg cgtcgtcatc ctcaccaact 18120acggcctcga cgagtacgtc ttccacgcgc tgcgcgccgg cgccaccggc ttcctggtca 18180aggacatcga gccggacgac ctgctgcacg ccgtgcgggt cgccgcgcgc ggtgacgcgc 18240tgctcgcgcc gtcgatcacc cggatgctga tcaacaggta cgtgtcggag ccgctctgcg 18300cggacgtcac gcccggcatg gaggagctga ccaaccggga acgcgaggcg gtcgccctgg 18360ccgcccgggg cctgtccaac gacgagatcg ccgatcgcat ggtgatcagc ccgctgaccg 18420cgaagaccca cgtcaaccgc gccatgacca agctgcaggc ccgcgaccgc gcccagctgg 18480tggtgttcgc ctacgagtcc ggcctggtgt cacccggcaa tcgctgaccg ggcagcccgc 18540ccggtctgtc gcctcggcag tgctgcggct gcggtatgcg gctgctcccg gcgcagacgc 18600cggagcccgt ggataccgtc accgcagtag atcgatcgat tgtctccttc ggcatgacga 18660cccgtagcgg ggtcgttacc tacgctggcg cagatgcctg ttcccgcagc cgaaggggct 18720tccatgttca tccgtcgttt gctcaccgcc gccgcagccg gcgtcctcgg tgggctcgca 18780ctcgtcgcac cggcggccgc gcaggtgacg gccgccgacg gtgacggtgg ttccggccgc 18840gccggatccg tgctggcgct cgcgctcgcg ttgctcggcc tcgtcctggg cgggtgggcg 18900ttgcgctccg cggggcgcgg cggcggtcgt ggcaacgcga tcgccgcgct ggtgctcgcg 18960gtggccggcc tgatcgccgg cgtggtcgcc ctggccggct ccgacggtgg tgtcggcagc 19020ggcaacggcc gtggtggcgc catcgtggcc gtcgtgctgg cgctgatcgg gatcgccgtc 19080ggcggcctgg cattcacccg ctcccggcgc gccgcctgac cggcgctgcc gaccgaacac 19140cccggtgacc caaccgaacc cgaaggggag tcccatgcgc aaagtgttcg ccggactggc 19200agcgttcctg ctgctcgtgc tcgtggtgca gttcttcctg gccgccagcg gcgcgttcag 19260caacgaggcc aacgaggagg cgttccgccc tcaccggatc ctgggcctgg ggagcatcct 19320cgtcgccgtg gtgctgacgg tggccgccgc ggtgatgcgg atgcccggcc ggatcatcgg 19380cctgtccggc ctggtcgccg ggctgggcat cctgcaggcc ctgatcgcgg tcatcgccaa 19440ggcgttcggc gactcggccg gtgactcggc cgtcggccgg tacgtgttcg gcctgcacgc 19500ggtcaacgga ctggtgatgg tggccgtcgc ccgcgtcatc ctgcgcagcg tccgggcggc 19560gccggacacg accaccacgc ccggcgtgga cacgacggtc accggtccgg cggccgactc 19620ggcgcgaacg gcgtcatgag cacgctccaa tggatcctcg tggaccacgt cgtggcgctg 19680ctcggtgtcg cgacgtggtt cgcaacgggt gtcacggcag ctctcggccg ccaccggatc 19740gcgttggcgc tcctcggcgc cgcggtgctg gtgacagtcg cccgcctggg caccgtggcg 19800ctgctggccg accgcggctg gtggttcgtc caggagaagg ttctgctggg gctgccgatg 19860ctcggcgccg cggggctcgt cgcggtgctc ctggccggcc cgcgcctgct cgcggcccgg 19920cagtcaccgg cggcggacct gccggccggc gcgctggtcg cggtgctgac cgccggcttc 19980gccgcgctgg ccggcctggt ggtgacgttc accgccgggt acccgctgac gtggagcacc 20040gcgctgatcg ccgtcgccct cgtctgcgcc gccgcgctgc tcaccgcgcg ggtggtcgga 20100cgacccgccg ccccggccgc ggaggccggc tccccggagc acacgccggc ggcggccggg 20160cccacggcgc tgtcccgccg ccggttcctc ggcgtggccg ggggagtggt cgcggcgggc 20220gccggcgcca ccggcgtcgg cctgctcttc cgcgacccgg aggcgatggt caccggaggc 20280ggccccggac acgccggtgg cgcccgcccc aaggtctccg tggcggacct gcgcggcccc 20340ggcgctccgg cggcgggcgg cacggcgcga cgccacgtgc tcaccgcccg gacgggcacc 20400gtcacgattc cgtccggacg tccgatcgac gcctggagct acgagggccg cctgcccggg 20460ccggccatca ccgcgaccga gggcgacctg atcgaggtga cgctccgcaa cgccgacatc 20520gaggacggcg tcaccgtgca ctggcacggg tacgacgtgc cgtgcggcga ggacggcgcg 20580ccgggcgcca cgcagcacgc ggtgcagccc ggcggcgagt tcgtctaccg gttccaggcg 20640gaccaggtgg ggacgtactg gtaccacacc caccaggcgt cgcaccccgc cgtgcgcaaa 20700gggctgtacg ggacgctcgt cgtgacgccg cgcgaggacc ggccggaagc ggagcgcggg 20760ctggacctga cgctgccggt gcacacgttc gacgacgtca cgatcctcgg cgaccaggag 20820ggacgcgccg tccacgacgt ccgccccggc cagccggtgc gactgcgtct gatcaacacc 20880gactccaacc cgcactggtt cgccgtcgtc ggctcgccct tccgcgtggt ggccgtcgac 20940ggccgcgacc tcaaccagcc gggcgaggta cgcgaggtcg ggctccgcct gcccgccgga 21000ggccggtacg acctgaccct ggccatgccg gacgccaagg tcacgctgct gctcgacaac 21060gactccgacc agggcgtcct gctgcgcccg ccgggcgtcg gcggtggtga ccgcccgctg 21120ccggacaccg ccgactggcc cgagttcgac ctgctgggct acggcgagcc ggcgcccgtg 21180ccgttcgacg ccgacgacgc cgaccgccac ttcaccatcg tcctcgaccg ggccctggcc 21240atggtcgacg gcaagcccgc gtacgcccag accgtcgacg gtcgcgcaca tccctccgtc 21300cccgaccagc tcgtccggga gggggacgtc gtgcgcttca cggtggtcaa ccggagcctc 21360gaaacccacc cgtggcacct gcacggccat ccggtgctga tcctgtcccg cgacggccgg 21420ccgtactccg gcagcccgct gtggatggac accttcgacg tgcggccggg agaggtgtgg 21480gaggtggcgt tccgggcgga caatccgggt gtctggatga accactgcca caacctgccg 21540caccaggagc agggcatgat gctgcggctc gtctacgacg gtgtcaccac gcccttcgcc 21600agcacgagcc acgcacactg aggggactcg catgaccgca gacctgcacg gcctggccag 21660cgtccgctac atcgtcgacg acgtgtcggc ggcgatcgag ttctacacca cccacctggg 21720tttcacggtg tcgaccgcgt tcccgccggc cttcgccgac gtggtgcgcg ggccgctgcg 21780gctcctgctg tccgggccga ccagctcggg cgcccgggtc accccggcgg acgcggccgg 21840gtgcgggcgc aaccgcatcc acctgatcgt cgacgatctc gacgccgaac gggagcggct 21900ggagcgcgcc ggggtgacgt tgcgcagcga cgtcgtggcc gggccgggcg gccgtcagtt 21960cctgatcgcc gacccggcgg gcaacctggt cgaggtgttc gagccggcag cccgcggctg 22020aaccgccgac ggacgccctc ccacctcgcg acgcccgaag cccgacacct ggccgcgtcg 22080cggccacgat caccgtggcc gcgacgcggt gacggggtgc cttaccgggg cggggtgggc 22140gcggcgagcc gcgcggccag gatggagatg atcacggcgc cggcgatcac gtgggtgccg 22200gcgaggacga gctgcgtcga caccggggtg tcccgggcga aggcgggcgc ggcgagggac 22260agcacggtga acgcgacggt gccggccacg aaggcacgca cgggccgccg ggcccgccgc 22320gccacgacca ccgccaggac gattccgccg atcgaccaga gcacgacgct gcgggcgatg 22380gcccccaccg ggatcgcctg cgcctgctcc tcccagacgc cggccgcctc catcggtacg 22440ccgaagcccc gggcggcgag cgtgaacgcc tccgcggcca cggccccggc gagggtggcc 22500agcacgccga ccagccacac cggagcggtg gccggcgacc aggtgggccg tgccgcgacg 22560ggagttcggg gagtggcctc atccacggcg tcgcctccgg tcgggtgcct cgatgtgttc 22620tcgggagaat gcggggacgc cacgacggca gtcaacatgg acagttgaac gccctggcgt 22680cacgggcggt tcccgcgccg gcccgccgcc tcggccgcgg cggcggccgt gccgtcggcg 22740agcagggaga ccagcaggtc gcccaggatc cgtgggccgt gctgggtgag gacggactcc 22800aggtggaact ggacggaacg gaatcccggg ccgcgcagcg cgtgcacgtc cccgctgtcc 22860gggctgcggc tgatctcgat cgggccccgc cggccaccgg ccaccacgtc gtgcgcggag 22920cgggcggtgt aggtgttgta gaaccccacg agttccggcc ggccgaacag gtcgatccgc 22980ttctgcacac cctggttggg caccgcgcgc cgggcgaggg ggaaccccag ttcggcggcg 23040agcacctggt ggcccaggca gatggacagg aacggcaccg ttccggcgag caggtcgcgg 23100gtgagcccgc gcagggtccg catacgcggg tcggtcaggt cgcccgggtc gccggggccg 23160ggaccgacga cgacgaggtc gtgtccgtcc ggccgcagcc ggctgtcgaa ccgggcgatg 23220ctcgaccgca gcccgagggc ccgcaactgg tggtcgagca tggccatgaa cgtgtcctcg 23280ttgtcgacga cgagcacgcg gcgtccggtc agcgccgggt tcggggtgcg ccgctccgcg 23340ccgtcgagcc agaacctcga cagtgtggtg ttgcgctcgc gcaacgcccg ccgtacccgg 23400gggtcggtgg ccagggacga acgagcccgc gcggccgtgg tccgcccgcc gtccgggccg 23460tccgggtcga cgccgaggcc gagcgccgcg cgcatggcgc ccgccttggc ccgcgtctcg 23520gccacctccg actccggctt ggagtcccgc acgagggtgg cgccgacgcc caggcgcagc 23580gtgcccgcgt cgtcgatctc ggcggtgcgg atcatgatgg ccgagtcgag cgtacggctg 23640ccggccgagt cacggcccat caacgcgagc acgccgccgt agtagccgcg gccggtcgtc 23700tcgtggcggg tgatgacccg gaacgcgttc tcgatcgggc tgccggtgac cgtcggcgcg 23760agcagggtct cccgcagcac gtcgcgcacg tccaggtcgc tgcggccggt caggatgtac 23820tcggagtgcg tcacccgcgc catttccttg aggaacgggc cgtgcacctg gccgccggag 23880gcgcacatcc gcgccatcat tttcagttcc tcgtcgacga ccatgtagag ttcgttagcc 23940tctttcgggt cgttcaggaa ttccagcaga ccggaaacgg ccgggccgtt cggggggtgc 24000cggtaggtcc cgctgatggg attcatcgag acggttccgt cgatcatgct gacgtgtcgt 24060tccggtgacg cgccgatgaa cgtgccggcg ccggagtgga acagaaacgt ccagtaggaa 24120cccagttcgc cggtcagcaa ccggcggaag agcgccagtt ccgtggcgat cgagtagtcg 24180gccagccgcg cggtgaaggt gcgccggatg acgaagttgg atccggcgcc cagcccgatc 24240tcgtcaccca ccacccgctt gacgatcgcg gcgtagtcct cgtcgctgag gtcgaagtcg 24300gcgtcggtca ccggcacacc gcgttcgggc aggcccgcca gcgcctgtcc gcggtcgagc 24360ccgaactgct cgtggacgcg catcgcgagc agcggcgcgc cgtcgtcgtg gcagtcgaac 24420ccccgttcgg tgacctgccg gtacggcacc gccacgagca ggtcgtgccg cgcgccggtc 24480gccggctcgg tgggcagggg cagctcgccg agagtgtcca cgtcgcacac ctcgccggtc 24540agaacctcca cgtacgcgca cccggccgcg ccgggccggt gcagcagggc gaaggcgcgc 24600ccgtcgccgc cgagaccgga cagcagatcg gggaatccgg tcacgttcga ttccgtcccg 24660tccatgtcgc tccctttgcc tgagagatcg cctgtcgata ctgcgtccgg caaaaggcgt 24720cgcacatgac gtgaagtcgc cgacggcatc acgtgtttcc ggtaacgcgc cgacgttatg 24780gcgtgaacga ctgaatcggc gggctactac tcgggcgagt agtgcccacg cagatcgacc 24840gcgattactg tcgaccgcaa tgccgatacg acgagggcgg tgaagacgac tgtggacgtg 24900ctggtccaga aatacggggg cacctcgctg cagaccctcg accgcgttcg gcacgccgcg 24960ctgcggatcg ccgaggcgcg gcggcacggc tccgccgtga cagtggtcgt gtcggcgcgc 25020ggcagccgga ccgacgacct gctgcggctg gcggccgacg tcggcgccgc gggtccgtcc 25080cgggaactcg accagttgct cgcagtcggc gagtccgagt cggcggcgct gatggcgctg 25140gcgttgaccg ggctgggagt gccggccgtc tcgctgaccg ggcaccaggc ggagatccac 25200accaccgacc ggcacggcga cgcgctgatc tcgcggatcg gggcggcgcg ggtggaagcg 25260gcgctgggcc gtggcgaggt cgccgtggtc accggattcc agggcatcga ccgggccggt 25320gacgtcgcca cgctggggcg cggcggctcc gacacgacag cggtggcgct cgcggcccgg 25380ctccgcgcgt cggcgtgcga gatctacacc gacgtggacg gcgtcttcag cgccgacccc 25440cgcatccttc cggcggcgcg ttgcctgccg tgggtggagc ccggcgtcat ggcggagatg 25500gcgttcgccg gcgcgcgggt cctgcacacc cgatgcatcg agctggccgc catggaaggg 25560gtcgaagtgc gcgtgcgcaa cgcgtcgtcg caggcgcccg gaacgatagt cgtggaccgg 25620cccgacgacc ggccgctgga gacccggcgg gccgtggtgg cggtcaccca cgacaccgat 25680gtcgtccgcg tgctggtgca ctgccgcgac ggccgccggg acatggcacc cgacgtgttc 25740gaggtgctgg ccgcccatgg ggcggtggcg gacctggtgg cccggtccgg gccctacgag 25800agcgagttcc ggatggggtt caccatccgc cgcagccagg ccgaagcggt gcggaccgcg 25860ctgcacgacc tcaccgcgtc cttcgacggc ggggtccact tcgacgagaa cgtcggcaag 25920gtgtccgtgg tcggcatggg cctgctcagc cgccccgagc acacggcccg gctgatggcg 25980gcgctggccg cggcggggat ctcgacgagc tggatctcca cctcccagat gcggctgtcg 26040gtgatcgtgt cgcgggaccg caccgtcgac gccgtcgaag ccctgcaccg cgcgttccgc 26100ctggaccggt ccgagccggc ggacgccacg tccctgacct cccgccgttc cgccaccgcc 26160tgagagaggt aggaaaccgt ggccgtactc aacgcttcgt tcgctcgtgg cctgcgtctg 26220cgccgactgt tccgacgcgg cgacggacgc ctgctcgtcg tcccgctcga ccactccgtc 26280accgacgggc cgctgcgccg cggcgacctg aactcgctgc tcggtgagct cgccggcacc 26340ggcgtggacg ccgtggtgct gcacaagggc agcctgcggc acgtcgacca cggctggttc 26400ggcgacatgt cgctgatcgt gcatctgagc gtgagcaccc ggcacgcccc ggacccggac 26460gcgaagtacc tggtcgcgca cgtggaggag gcgctgcggc tgggcgccga cgcggtcagc 26520gtgcacgtca acctcggctc accgcaggag gcgcggcaga tcgccgacct ggcggcggtg 26580gcgggggagt gcgaccgctg gaacgtcccg ctgctggcca tggtgtacgc ccgcgggccg 26640cagatcaccg actcccgggc accggagctg gtggcgcacg ccgcgacgct cgccgcggac 26700ctcggcgccg acatcgtcaa gaccgactac gtgggcacgc ccgagcagat ggccgaggtg 26760gtgcgcggct gcccgatccc gctgatcgtg gccggcggcc cgcgctcggc cgacactccg 26820acggtgctcg cctacgtctc ggacgcgctg cgcggcggcg tggccgggat ggccatgggc 26880cgcaacgtgt tccaggccga gcagcccggc ctgatggccg ccgccgtggc acggctggtg 26940cacgagccac ggcacgtgcc ggaccggtac gacgtcgacg accggctcgc ccttacgtcc 27000tgagactccc tgaccgtcca ccgaggagaa acccgtgaag ctgtgctggc tggacatccg 27060taacgtcaac ggcgccaagg aggcaatcgt cgaggaggcg gtccaccagc gggtggacgc 27120cgtcgtggcg gccgatccgg ccgacctgga gacgcttccc ccgacggtga agaaggtgct 27180gttcccgcag ggcgggccgc tgccggagaa gctggaaccg gccgacctgg tgatcgtcga 27240gccggcccgg cacggcgagc ccgccgagct ggcggcccgg tacccggagg tggagttcgg 27300ccggttcgtc gagatcgtcg acgcggacag cctggaggac gcctgccggt ccgcgcgcca 27360cgaccggtgg agcctgctgt acttccgcga ccccaccaag atcccgctgg agatcgtgct 27420ggcggccgcg gcgggcgcgg agggcagcat catcacccag gtcgccgacg tcgaggaggc 27480ggagatcgtc ttcggcgtcc tggagcacgg ctcggacgga gtgatgctgg cgccccgcgc 27540cgtgggggag gccaccgagc tgcggaccgc cgcggtgagc acggcggcgg acctgtcgct 27600cgtggagctg gaggtcaccg gcatccggcg ggtgggcatg ggcgagcgcg cctgcgtcga 27660cacgtgcacg aacttccgtc tggacgaggg catcctggtc ggctcgcact ccaccggcat 27720gatcctgtgc tgcagcgaga cgcatccgct gccgtacatg ccgacccggc cgttccgggt 27780caacgccggc gcgctgcact cgtacacgct ctccgccggc gggcggacca actacctcag 27840cgagctggtc tccggcggcc gggtgctcgc cgtggactcg caggggaagt cccgcgtcgt 27900cacagtggga cgggtcaaga tcgagacgcg tccgctgctg gcgatcgacg cggtctcccc 27960ctccgggaca cgcgtcaacc tcatcgtcca ggacgactgg cacgtgcgcg tgctcgggcc 28020gggcggcacc gtgctcaacg tgaccgagct gaccgccggc acgaaggtgc tcggttacct 28080gccggtggag aagcggcacg tcggctaccc gatcgacgag ttctgcatcg agaagtgaca 28140ggcggcggga aggggagcgg gcgatgaccg cgcagccggt gctggacttc cacgtacgcc 28200tggcgccccg gcccggggcg cgggagcggc tgctcgccgc gctgcgcgag tgcgggctgg 28260cgcgggcggt ggtgtgcgcg ggcggcacca tcgacctgga ccggctgtcc cgccagctcg 28320tcaccggcgg ccacgtcgag accgacgccg acaacgacgc ggtggcggcg gcctgcgccg 28380gcaccgacgg ccggctggtg ccgttcttct tcgccaaccc gcaccggccg gccgaggcgt 28440accgggcccg cgccgccgag ttccgcggcc tggagatctc acccgccgtc cacggcgtcg 28500ccctgaccga cccgcgggtc gccgacctcg tggccgtggc ggcggagttc gaccatccgg 28560tgtacgtggt ctgcctggac cgacccggcg cgggcgtggc cgacctggtc ggcctgagcc 28620gccggttccc gcaggtgagc ttcgtgctcg ggcacagcgg cgtcggcaac atcgacctct 28680acgccctgac cctgatccag gacgagccga acatctcgct ggagacctcc ggcggctaca 28740cctgcgtggc cgaggcggcg ctacgccgcc tcggcgacga ccgggtggtg ttcggctccg 28800agtacccgct gcagcacccg gccgtggaac tggccaagtt ccaggcgttg cgactgccgc 28860cggagcggtg gcggcggatc gcctgggaca acgcgcatcg actgctagga gaggagaagc 28920ggtgagcgag ccaagttcga gcctgccccg gctcggccag tggcacggcc tcgaggacct 28980gcggcgcctc caggagaagc aactggcgga gacgttcacc tgggcggccc ggtcgccgtt 29040ctaccgggcg cggctggcct ccggcgcgcc gccggtgacg cccgccgacc tggccgacct 29100gccgctgacc accaagcagg acctgcggga caactacccc ttcggcatgc tcgccgtgcc 29160ccgcgaacgg ctggcgacct accacgagtc gagcgggacc gccgggaagc ccaccccctc 29220ctactacacc gcggaggact ggaccgacct ggcggagcgc ttcgcccgca agtggatcgg 29280catgtccgcc gacgacgtct tcctggtccg cacgccgtac gcgctgctgc tgaccgggca 29340tctcgcccac gccgcagccc ggctgcgtgg ggccacggtg gtacctggcg acaaccggtc 29400gctggcgatg ccgtacgccc gggtggtccg ggtgatgcac gacctggacg tcacgctcac 29460ctggtcggtg ccgacggagt gcctgatctg ggccgccgcg gcgatcgcgg ccgggcaccg 29520gcccgacatc gacttcccgg cgctgcgcgc gctgttcgtc ggcggcgagc cgatgaccga 29580cgcccgccgg cggcggatca gccgcctgtg gggggtgccg gtcatcgagg agtacggctc 29640gacggagacc ggcagcctgg ccggggagtg ccccgaggga cgcctgcacc tgtgggccga 29700ccgggcgctg ttcgaggtgt acgacccgga caccggcgcc gtccgcgcgg acggcgacgg 29760ccagctcgtg gtcacgccgc tgttccggga ggcgatgccg ctgctgcggt acaacctgga 29820ggacaacgtg tcggtctcct acgacgactg cggatgcggc tggaagctgc ccaccgtgcg 29880ggtgctcggc cggtcggcgt tcggctaccg ggtcggcggc accaccatca cccagcacca 29940gctggaggaa ctggtcttct ccctgccgga ggcgcaccgg gtgatgttct ggcgggccaa 30000ggcggagccg gcgctgttgc gggtcgagat cgaggtggcc gccgcgcacc gggtcgccgc 30060cgaggcggag ctgaccgccg

cgatccgggc cgccttcggc gtggacagcg aggtcaccgg 30120cctggcgccg ggaaccctga tcccgctcga cgcgctgacc agcatgccgg acgtggtgaa 30180gccacgcagc ctgttcggtc cggacgagga ctggagcaaa gcgctcctct actactgagg 30240gaaccgacat gccgcagatg agggtcgccg tggccggcgc cggcatcgcc gggctcgcct 30300tcgccgccgc cctgcgccgg accgggatcg actgccacgt gtacgaacag gccgaccagc 30360tcatggaggt gggcgcgggc gtgcaggtcg cgccgaacgc cacccggctg ctgcaccggc 30420tgggcctgcg tgaccgcctg cgtacggtgg ctgtcgcgcc gcaggcgatc gagatgcgcc 30480gctgggacga cggcacgctg ctgcaacgca cccagctggg cagcgtgtgc ggacgccgct 30540tcggcgcgcc gtactacgtg gtgcaccgcg cggacctgca cagcagcctg ctgtcgctgg 30600tgccgccgga ccgggtgcac ctgggcgccc gcctcaccgc cgtgacgcag accgccgacg 30660aggcgtacct gcacctgtcc aacggcacca cggtcgcggc ggatctcgtc gtgggcgccg 30720acggcatcca ctcggtcgcg cgggagcaga tcgtggcgga ccggccgcgc ttctccggac 30780agtccatcta ccgcgggctg gtgccggccg agcgggtgcc gttcctgctc accgaacccc 30840gggtgcagtt gtggttcggg ccggaccagc actgcgtctg ctacccggtg tccgccggcc 30900ggcaggtgag cttcggcgcg acggtgcccg ccaccgactg gcggcaggag tcgtggtcgg 30960gccggggcga cgtgacgcaa ctcgcggccg cgtacgcggg ctggcacccg gacgtcaccc 31020ggctgatcgc cgcggccgac cgggtcggca ggtgggcgct gcacgaccgg gacagcatcg 31080accggctcag cgcgggacgg gtgaccctga tcggcgacgc cgcgcacccg atgctgccgt 31140tccaggcgca gggcgcgaac caggccgtcg aggacgcggt ggtgctcgcg gtctgcctgg 31200ccggcgtgga accggcgggc ctgggcgccg cgctgcgccg ctacgaacgg atccgcctgc 31260cccggaccac ccggatccag cggcagtccc gggccaacgc cgagatgttc cacctggccg 31320acggcgccga ccagcgccgc cgggacgtcg ccgcacaatc ctcgtccggc ctggaccgcc 31380acgaatggct cttcgggtac gacgccgaga aagccaccac gaccagcggg agcgcctgat 31440ggaactgacc ggaatcgagt cgaaggtcgc cctggtcacg ggcgcggggc agggcatcgg 31500cgccgccgtg gccggtgtcc tggcgagggc gggcgcgcag gtggcggcgg tggaccgcaa 31560cgccgaggcg ctgaccaccg tcgtgacgaa gctcgccgcc gagggcgact cggcgcgcgc 31620ctactgcgtc gacgtgtgcg acagcgaggc ggtggacgcg ctggtgcgcc gggtcgagga 31680cgagatgggg ccggtcgcca tcctggtcaa cgccgccggc gtgctgcaca ccggacgggt 31740cgtcgagctg tcggaccggc agtggcgccg gaccttctcg gtgaacgccg acggcgtgtt 31800ccacgtgtcc cgggcggtgg cgcggcggat ggtgggccgc cgtcgtggcg cgatcgtcac 31860cgtggcgtcg aacgccgccg gggtgccgcg taccgagatg gccgcgtacg ccgcctccaa 31920ggccgcgtcc gcgcagttca cccgctgcct ggggcttgag ctgtccggct acggcatccg 31980gtgcaacgtg gtctcgcccg gctccaccga cacccccatg ctgcgggcca tgctcggcga 32040gggcgccgac ccgagcgcgg tgatcgaggg cacgccgggc gcgtaccgcg tcggcatccc 32100gctgcgcaag ctggcccagc cgcgcgacgt ggccgaggcg gtcgcctatc tggtgtccga 32160ccaggcgggc cacgtgacca tgcacgacct gtacgtcgac ggcggcgcgg ccctgcacgt 32220gtgacgccct cgcacggaaa ccggaggcga gaaccgatgg ccatgacccc gatcgcgccg 32280taccgcatgc ccggcgacgg cgacctgccc ggcaccgcgc tgccctggcg tccgcacccg 32340gaccgggccg ccgtgctggt gcacgacctg caacgctact tcctgcgccc gttcgaggcc 32400ggggagtccc cgatggccga actgctcccc aacgtcgcga agctgctcgc cacggcgcgg 32460gcggccggcg tgccggtgct gtacaccgcg cagcccggcg gcatgagccg gcaggaccgc 32520gggttgctgc acgacctgtg gggccccggc atgagcagcg ccgaggacga ccggggcatc 32580gtcgacgacg tcgccccgca gccgggcgac acggtgctga ccaagtggcg ctacagcgcg 32640ttcttccgca gcgacctgga ggagcgactg cgcggtgcgg gacgggacca gctcgtggtc 32700tgcggcgtgt acgcgcacat ggggtgcctg atcaccgcct gcgacgcgtt cagccgcgac 32760atcgaggcgt tcctggtggc ggacgcgctg gccgacctat cgcgcgagga ccacctgatg 32820gcgctgcgct acgccgcgga ccgctgcgcg gtgccgttgt ggacggcgga tgtgctggac 32880gggctggcgg acgccgccgg gcgtccggat cagagcagca cccaacgatg aggagaacat 32940cgatgtcgga tcggacccgg gtcgtggtcg tcggcggaac ctcggggatc gggcggcact 33000tcgcccgatt ctgcgccgaa cgcggagacg acgtggtgat caccggccgt tcggcggccc 33060ggaccaagac cgtggcggac gagatcggcg ggcggacccg tgggctcgct ctcgacctgg 33120ccgagccgga gacgatcgcg gacgcgctcg ccgacgtgcc gcacgtcgac cggctcgtgg 33180tcgcggcgct ggaccgcgac tacaacaccg tccgcgcgta ccggccgggc gacgcggcgc 33240ggctgctgac cgtcaagctg gtcggctaca cggcggtcct gcacgccctc gccccgcgga 33300tgaccgacga gagcgcagtc gtgctgctcg gcggcctggc cagccaccgg ccgtatcccg 33360gctccacctc cgtcacgacc gccaacggcg ggatcagcgc gctggtgcgg accctggctg 33420tggaactctc gccggtccgg gtcaacgccc tgcacccgag catcgtctcc gacacgccgt 33480tctggagcga caagcccgcc gcgcgggagg ccgccgcgac ccgcgcgctc agccgacggc 33540cggtcaccat gcaggactgc gccgaggcga tcgacttcct gctgacgaac cgctcgataa 33600acggggtcaa cctgaacatc gacggcgggg acgtgctcat ctgacgccgg aggcgatccg 33660ccacggcccc caccacccgg tcgcgccctg cccgtgctcc cgctgctcgc gggggtaccg 33720ggccaggtcg cgggcggaga agagcgccat gccggcgtgg aatccggtca ccggcaccgg 33780gacccgcgcc cagtaggcga gccggccgtc gacgtggaac tccacctccg acgtcggcgc 33840ccggtaggtg atggcgtatc cgtgcgcccg gcccggctcc gtcggcacgt ccaggaccac 33900ccggtggatg tagtgctcgt gcggctgggt cacgccgggc agcaccaggc gctcgaccgt 33960cgcgtacacg gtgtcgttcg tggcggcggc gttgaacacg acgccggtct ccaggtcgaa 34020caggttcacc gtgccgaacg cgtccagcag gtcgtgcggg atctgccggt acgtccgcac 34080gcccatctcc acctcgacgg tcagcgagcc ctccgccggc acggcgaagc gccgcaccga 34140ccggtacatc tgcttggcgt tgttctgccg gggatcggtg tcgtggaagc gggtgaacgg 34200gtcgacggtc agctccagcc gcccgtcgcc ggtgcggacc tgggcgttgc ggtcctggta 34260cctgtgggtc tgcccgtccg cgccggcgat cgacatgatc gcccagcggg cggggtccag 34320ctcgcggctg gtgaagtcgt cgtacgtcca cgcgctggtt ctcagtgccg acgtcatgca 34380gtcaccatcg gacgccggcc gggcgcgggc atcacccgtt cacgcggttc ggccggaccc 34440ggcacgccaa tgcgccggcc acgccccgga aatcccgtga ttaagccatg ccggagcgtg 34500aacggtcgcc gagactgacg ccgcacccat ctccgcatcg tctgcgacgt tctcaccagg 34560gggagagagc aatggacacg gcagctccgg caacggacgg cggtcgctac ctcgccgtcc 34620atcacagcgc agagttcagg gaactacggc gacgatcgag cacgttcacg ctctgggcca 34680gcgtcgcctt cttcggctgg tggttcctcg gcagcctgct cgccacctac gcgccggact 34740tcttccggga gaaggtggcc ggcccggtca acgtgggtct gctcttcgtc ttcctgtcgt 34800tcgccttcgt ggtgacgctc gccgccttct acctgcgtta cgcccgcacg catctcgatc 34860cgctcagcga gaagatccgt gccgacctgg aaggagcgtc ccgatgagcg tcatcctcgc 34920cgacccgcca cccccggtcg acaacacgtg ggcgacgccc gcgatcgccg tgccggtcac 34980catcgtcctc gcgctcgcgg tgctctacct ggtccggtcg gcgcgcgcca gcaccaccac 35040cgcggacggc ttcctgctgg ccgaccggcg gatcgggccg gtgcagaacg cgctggcggt 35100ggcctccgcg ccgctgatgt actcgacgat gtacatcatc accggccaca tcgcgctcag 35160cggctacgac gccatcctgc tgatgaccgc cttcaccatg ggcaccatgc tcgcgctgtt 35220cctcttcgcc gggccggtgc gcaacgtggg cggctacacg ctcggtgacc tgctcgcggt 35280ccgtacccgg gagcggccgg cgcggatcgc gtcggcggtg ctcacgctgc tgacgtacgt 35340catgctgacg gtgatcatga tggccgccat cgcgttcatc ttcaaccgct ggttcggcgt 35400cgacgccctc gtcggcctgg tcctcccggt gttcgtcgtc ggtctgatca cggtggggta 35460cgtgtacctc ggcgggatgc tcggggtcac ccgcatcctg gtgttcaagc tggtgctgtc 35520ggtggtcgtc gtgggcgtgc tgaccgcctg ggtgctggcc cgcttcgacc tgaacctctt 35580cagcctgctg gagcgggccg aggcgaacgc ggcgccggtg cccagcggca gcgacctgct 35640gggcccgggc cggctgttcg gcgagggcgc gaccacgctc gtgcacctgt cgaagctgtt 35700cgccatcgcc gtcggagtgg cggccattcc gttcctgttc atgcgcaact tcgcggtgac 35760cagcgggcgg gacgcgcgcc ggtcgaccgg gtgggcgtcg atgatcatcg tcgggttcta 35820cctgtgcctg tccgtcgtcg ggctcggtgc cgtcgcgatc ctcggccggg acaacatcgg 35880cgtcatcaag gcccaccgcg acatcagctt ccccaagctc gccgacgagc tcggcggtcc 35940ggtgatggtc ggctccctgg ccggcgtcgc ggtcctgacg atcgtcggcg tcttcgcgcc 36000gctgctgcac agcgccgtga cgacggtgac caaggacctg aacgtgatcc gcggccggcg 36060gctggatccg gccgccgagc tgcgggacat caagcgcaac accctgatca tcggcgtcgg 36120ctccgtgctg ctggcggtcg tgatgctgcc ggtacggacc cacatcttca tcccgacctc 36180gatcgacatt gccggcgcgg tggtcctgcc gatcgtcgtc tacgcgttgt tctggcggcg 36240tttcaacacc cgcggactgc agtggacggt ctacggcggc ctcgcgctca ccgcgttcct 36300ggtgctgttc tccaacggtg tctcgggcga gccggacgcc atcttcccgg accgcaactt 36360caagttcgtg gacgtcgagc ccgcgctgat cacggtgccg gtcggcttcc tgctcggcta 36420cctcggctcg atcaccagcc gggagcgcga cgacgccgcg ttcgccgaga tgcaggtccg 36480gtccctcacc ggagctgtcg tcacgggacc gccgcggccg gccgccgtgg acgacgagga 36540ccgcgacggc cgccaggacc gggcgcccag cccggtgagc tgaacatccg caacggtgtg 36600gg 366022571PRTMicromonospora sp. strain 046-ECO11 2Val His Asn Leu Asp Asn Ile Pro Ser Ser Pro Ser Thr Ser Gly Gly1 5 10 15Ser Leu Pro Ala Gly His Arg Ala His Val Arg Ala Asp Gly Val Arg 20 25 30Val Val Arg Gly Gly Arg Val Val Leu Ser Asp Val Ser Val Thr Val 35 40 45Ser Ala Ala Ser Arg Leu Ala Val Val Gly Glu Asn Gly Arg Gly Lys 50 55 60Thr Thr Leu Leu His Val Leu Ala Gly Leu Ile Ala Pro Asp Gln Gly65 70 75 80Val Val Glu Arg Leu Gly Thr Ile Gly Val Ala Arg Gln Asn Leu Glu 85 90 95Ser Arg His Gly Glu Thr Val Gly Thr Leu Val Arg Glu Ala Ile Arg 100 105 110Glu Ser Glu Arg Ala Leu Arg Ala Leu Asp Glu Ala Thr Ile Ala Leu 115 120 125Thr Glu Gly Arg Ala Gly Ala Asp Asp Ala Tyr Ala Ala Ala Leu Asp 130 135 140Ala Ala Thr Arg Leu Asp Ala Trp Asp Ala Gln Arg Arg Val Asp Val145 150 155 160Ala Leu Ala Gly Leu Asp Ala Cys Pro Asp Arg Asp Arg Gln Leu Ala 165 170 175Thr Leu Ser Val Gly Gln Arg Tyr Arg Val Arg Leu Ala Cys Leu Leu 180 185 190Gly Ala Arg Val Asp Leu Leu Met Leu Asp Glu Pro Thr Asn His Leu 195 200 205Asp Ala Asp Ser Leu Ala Phe Leu Thr Ala Arg Leu Arg Asp His Pro 210 215 220Gly Gly Val Val Leu Val Thr His Asp Arg Ala Leu Leu Arg Asp Val225 230 235 240Ala Thr Glu Phe Leu Asp Leu Asp Pro Ser Ala Asp Gly Arg Pro Arg 245 250 255Arg Tyr Ala Gly Asp Tyr Val Ala Trp Gln Asp Gly Arg Arg Arg Asp 260 265 270Phe Ala His Trp Val Arg Asp His Glu Ala Gln Gln Ala Glu His Gln 275 280 285Arg Leu Ala Asp Gly Val Arg Glu Ala Arg Asp Arg Leu Ser Thr Gly 290 295 300Trp Arg Pro Glu Lys Gly His Gly Lys His Gln Arg Gln Ser Arg Ala305 310 315 320Pro Gly Leu Val Gln Ala Leu Arg Arg Arg Gln Glu Ala Leu Asp Ala 325 330 335His Arg Val Thr Val Pro Glu Pro Pro Gln Pro Leu Arg Trp Pro Pro 340 345 350Leu Asp Thr Arg Ala Gly Leu Pro Ile Leu Arg Cys His Asp Val Thr 355 360 365Val Ala Gly Arg Leu Arg Thr Arg Val Thr Leu Thr Leu Asp Gly Gly 370 375 380Asp Arg Leu Leu Val Thr Gly Pro Asn Gly Ala Gly Lys Ser Thr Leu385 390 395 400Leu Ser Val Leu Ala Gly Asp Leu Thr Pro Ser Thr Gly Glu Val Arg 405 410 415His Leu Ser Gly Ala Arg Val Ala Tyr Leu Gly Gln Glu Val Pro Asp 420 425 430Trp Pro Pro Ala Leu Leu Ala His Asp Leu Tyr Glu Gln His Val Gly 435 440 445Arg Leu Arg Ser Ser Gly Arg Val Gly Ser Gly Thr Ala Leu Pro Leu 450 455 460Ser Ala Thr Asn Leu Leu Asp Ala Glu Ala Arg Arg Thr Pro Val Gly465 470 475 480Arg Met Ser His Gly Gln Gln Arg Arg Leu Asn Leu Ala Leu Arg Leu 485 490 495Ala Glu Arg Pro Asp Leu Leu Ile Leu Asp Glu Pro Thr Asn His Leu 500 505 510Ser Ala Pro Leu Val Asp Asp Leu Thr Ala Ala Leu Leu Thr Thr Arg 515 520 525Ala Ala Val Val Val Ala Thr His Asp Arg Gln Met Leu Gln Asp Leu 530 535 540Ala Ala Trp Pro Thr Leu Pro Leu Thr Ala Pro Ala Ala Ser Gly Arg545 550 555 560Ser Val Thr Ser Glu Arg Tyr Asp Trp Glu Ser 565 57031716DNAMicromonospora sp. strain 046-ECO11 3 gtgcacaacc tcgacaacat tccttcctcc ccatccacct cgggcggttc gctgcccgcc 60gggcaccggg cgcacgtgcg ggccgacggc gtccgcgtcg tacgcggcgg ccgggtcgtg 120ctgtccgacg tcagcgtgac cgtctccgcc gcttcccgcc tcgcagtcgt cggcgagaac 180ggccgcggca agaccaccct gctgcacgtg ctggccggcc tcatcgcgcc cgaccagggc 240gtggtggaac ggctgggcac gatcggcgtc gcccggcaga acctggagtc gcgccacggc 300gagacagtgg gcacgctcgt ccgggaggcg atccgggagt ccgaacgcgc gctgcgggcg 360ctcgacgagg cgacgatcgc gctcaccgag ggccgggcgg gcgcggacga cgcgtacgcg 420gccgcgctcg acgcggcgac ccggctggac gcctgggacg cgcagcggcg cgtcgacgtg 480gcgctggccg gcctcgacgc gtgcccggac cgggaccggc agctggccac gttgtccgtc 540ggccagcgct accgggtacg gctggcgtgc ctgctgggag cgagggtcga cctgctgatg 600ctggacgagc cgacgaacca cctcgacgcc gacagcctgg ccttcctcac cgcccggcta 660cgcgaccacc cgggcggcgt cgtgctggtg acccacgacc gcgccctgct gcgggacgtc 720gccacggagt tcctggacct cgaccccagc gcggacgggc gcccgcgccg ctacgccggg 780gactacgtcg cctggcagga cgggcgccgc cgcgacttcg cgcactgggt acgcgaccac 840gaggcgcagc aggccgagca ccagcggctg gccgacgggg tacgggaggc gcgggaccgg 900ctcagcaccg gctggcggcc ggagaagggg cacggcaagc accagcgcca gtcccgcgcg 960cccggactgg tccaggcgct gcgccgccgg caggaggcgc tcgacgcgca ccgcgtcacc 1020gtgccggagc caccgcagcc gctgcgctgg ccgccgctgg acacccgtgc cggactgccc 1080atcctgcgat gccacgacgt cacggtggcc gggcgcctgc gtacccgggt cacgctcacg 1140ctcgacggcg gggaccgcct gctggtgacc ggacccaacg gcgcgggcaa gtcgacgctg 1200ctctccgtgc tggccggcga cctcacgccg tcgaccgggg aggtccggca cctgtccggc 1260gcgcgcgtcg cgtacctcgg tcaggaggtg cccgactggc cgccggcgct gctcgcgcac 1320gacctgtacg agcagcacgt gggccggctc cgctccagcg ggcgcgtcgg ctccggcacg 1380gccctgccgc tgagcgcgac gaacctgctc gacgccgagg cccggcgtac ccccgtcggc 1440cggatgtcgc acggacagca acggcggctg aacctggcgc tgcgcctggc cgaacgtccc 1500gacctgctga tcctcgacga accgacgaac cacctgtcgg cgccgctggt cgacgacctc 1560accgccgccc tgctgacgac ccgggcggcg gtggtcgtcg ccacccacga ccggcagatg 1620ctccaggacc tcgcggcctg gcccacgctg ccgctcacag ccccggcggc gtcaggtcgt 1680tcggtcactt ccgagcgata tgactgggag tcataa 17164689PRTMicromonospora sp. strain 046-ECO11 4Met Thr Thr Gly Arg Pro Gly Glu Asn Arg Ala Thr Asp Ala Ala Arg1 5 10 15Asn Pro Gly Trp Ala Ala Gly Gly Pro Ala Ser Gln Pro Trp Gly Gly 20 25 30Gly Asn Asp Glu Gln Val Leu Arg Glu Ile Leu Gly Val Asp Val His 35 40 45Arg Glu Leu Ile Asp Phe Ala Gly Gly Ala Gly Gly Asn Pro His Leu 50 55 60Val Ala Glu Leu Ala Arg Gly Leu Ala Glu Glu Gly Leu Ile Arg Glu65 70 75 80Thr Asn Gly Arg Ala Glu Leu Val Ser Arg Arg Ile Pro Arg Arg Val 85 90 95Leu Ser Phe Val Met Arg Arg Leu Asn Asp Val Ser Ala Gly Cys Gln 100 105 110Gln Phe Leu Lys Val Ala Ala Ala Leu Gly Arg Ser Phe Met Leu Glu 115 120 125Asp Val Ser Arg Met Leu Gly Arg Ser Ser Ala Ala Leu Leu Pro Pro 130 135 140Val Asp Glu Ala Ile Ala Ser Gly Phe Val Val Ala Ala Glu His Gln145 150 155 160Leu Ala Phe Gln Ser Asp Phe Leu Leu Arg Gly Ile Ile Glu Ser Ile 165 170 175Pro Gly Pro Ala Arg Asp Ala Leu Arg Arg Glu Ala Met Ser Leu Ser 180 185 190Gly Arg Arg Arg Pro Ala Ala Asp Gln Asn Arg Arg Leu Asp Ala Ala 195 200 205Pro Thr Ala Pro Val Ser Ala Thr Gly Glu Asp Ala Thr Gly Ser Cys 210 215 220Ser Arg Ala His Arg Leu Ile Met Asn Gly Asn Ala Lys Ala Gly Ile225 230 235 240Arg Val Ala Glu Ala Val Leu Ala Gly Pro Ala Ala Ser Leu Ala Ala 245 250 255Arg Arg Asp Ala Glu Ala Cys Leu Val Leu Ala Asp Leu Leu Leu Gly 260 265 270Gly Glu Gly Gly Gly Pro Met Thr Glu Ala Ile Leu Arg Glu Arg Asp 275 280 285Ala Glu Ser Gly Asp Ala Ala Leu Ala Met Ala Leu Thr Ala Arg Ser 290 295 300Thr Gly Leu Trp Ser Ala Gly Lys Leu Ala Glu Gly Leu Lys Leu Gly305 310 315 320Arg Ala Ala Val Arg Ala Gly Ala Glu Ala Glu Pro Val Trp Arg Leu 325 330 335His Ala Gln Leu Ala Leu Ala Gly Lys Leu Ala Asn Leu Arg Glu Phe 340 345 350Asp Glu Ala Glu Ala Leu Ile Asn Glu Ala Glu Ala Gly Leu Arg Gly 355 360 365Leu Pro Ala Pro Ile Trp Thr Ala Ala Thr Ala Val Met Arg Ser Arg 370 375 380Leu Leu Leu Gln Ala Gly Arg Ile Gly Glu Ala Arg Arg Glu Ala Ala385 390 395 400Leu Ala Thr Thr Ala Val Glu Gly Asp Ala Val Pro Met Leu Arg Pro 405 410 415Leu Ala Tyr Ala Val Leu Ser Thr Ala Ser Phe Tyr Met Gly Asp Leu 420 425 430Pro Ala Ala Ile Glu Tyr Leu Arg Arg Gly Gln Arg Asp Ala Asp Arg 435 440 445His Val Val Leu Asp Ser Val Gln Tyr Ser Trp Ala Glu Val Leu Ile 450 455 460Thr Val Lys Gln Glu Gly Pro Arg Ala Ala Ala Gln Leu Leu Ala Gly465 470 475 480Lys His His Arg Leu Pro Thr Gln Arg Arg Leu Tyr Val Glu Val Pro 485 490 495Ser Ala Ala Ala Phe Leu Val Leu Leu Ala Arg Asp Val Asp Asp Arg 500 505 510Asp Leu Glu Arg Arg Val

Leu Asp Thr Val Asn Gly Leu Ala Ala Asp 515 520 525Asn Pro Arg Ile Gln Val Val Ser Leu Thr Ala Met His Ala His Ala 530 535 540Leu Ala Asn Ser Ala Pro Ala Ala Leu Ala Leu Ile Ile Val Gln Ser545 550 555 560Arg Asp Pro Ile Ser Val Ala Leu Ala Thr Glu Glu Leu Ala Lys Leu 565 570 575Tyr Ala Ala Gln Ala Gln Ala Gly Gly Arg Pro Ala Thr Pro Ala Arg 580 585 590Ala Glu Glu Ala Ala Thr Pro Pro Ala Ser Cys Trp Ser Thr Leu Ser 595 600 605Asp Met Glu Gln Arg Ile Ala Tyr Leu Val Ser Val Gly Leu Thr Asn 610 615 620Arg Gln Ile Ala Lys Gln Val His Leu Ser Ala His Thr Val Asn Tyr625 630 635 640His Leu Arg Lys Ile Tyr Arg Lys Leu Gly Phe Asn Thr Arg Ala Glu 645 650 655Leu Ala His Ala Ala Ala Thr Tyr Ser Gly Arg Ala Ala Ile Tyr Ser 660 665 670Met Ser Gly Asp Gln Asp Trp Gly Ala Gly Ser Met Thr Gly Lys Ala 675 680 685Ser 52070DNAMicromonospora sp. strain 046-ECO11 5atgacaacgg gacggccggg ggagaaccgg gcgacagacg cggcacgaaa tccggggtgg 60gccgccgggg ggccggcgtc ccagccatgg ggcgggggga acgacgagca ggtcctgcgc 120gagatcctcg gggtcgacgt gcaccgcgag ctgattgact tcgcgggtgg tgccggcgga 180aatccgcacc tggtcgccga actcgcgcgc gggctcgccg aagagggatt gattcgggag 240acaaacggtc gggcggaatt ggtgtcccgg cgaattcccc ggcgcgtgct gagttttgtc 300atgcgtcgat tgaatgatgt cagcgccggc tgccagcagt tcttgaaggt tgccgcggca 360ttgggcagat ccttcatgct ggaggacgtt tcgagaatgc tgggccgatc gtcggcggcc 420ctgctcccgc cggtggacga ggcgatcgca tcgggcttcg tcgtcgccgc cgagcatcaa 480ctcgcctttc agagcgactt cctgctgcgc ggcatcatcg agtccattcc cgggcccgcc 540cgcgacgcct tacgacgtga ggcgatgagc ctttccgggc gacggcgccc ggcggccgac 600cagaatcgcc ggttggacgc ggcgcctacc gcgccggtga gcgcgaccgg ggaggacgcc 660accggatcct gttcccgggc gcaccgcctg ataatgaacg ggaacgcgaa ggccggcatt 720cgcgtcgccg aggcggttct cgccggcccg gccgcgtcgc tcgctgcccg gcgtgacgcg 780gaggcgtgtc tggtgctggc cgatctgctg ctcggcgggg agggcggcgg cccgatgacc 840gaggcgatcc tgcgcgaacg cgacgccgag tccggtgacg ccgcactggc gatggcgctg 900accgcccggt ccaccgggct gtggtcggcg ggaaagctgg cggagggcct gaagctggga 960cgggcggcgg tgcgggcggg cgcggaggcc gaaccggtgt ggcgtctgca cgcccagctc 1020gcgctcgccg ggaaactcgc gaacctccgc gagttcgacg aggccgaggc gttgatcaac 1080gaggcggaag cgggcctgcg cggactgccc gcgccgatct ggacggccgc gacggcggtg 1140atgcggtccc ggttgctgct ccaggcgggg cggatcgggg aggcgcgtcg ggaggcggcg 1200ctggccacca ccgccgtgga gggggacgcg gtgccgatgc tgcggcctct cgcctacgcg 1260gtgctcagca ccgcctcctt ctacatgggg gacctgcccg ccgcgatcga gtacctcagg 1320cgggggcagc gggacgcgga ccgccacgtg gtcctcgact cggtgcagta ctcgtgggcg 1380gaagtgctga tcacggtcaa gcaggaaggc ccgcgggccg ccgcccagct gctcgcgggc 1440aagcaccacc gcctgcccac gcagcgccgc ctctacgtcg aggtgccgag cgccgccgcc 1500ttcctggtcc tgctcgcccg cgacgtggac gaccgtgacc tcgaacgccg cgtcctcgac 1560acggtcaacg ggctcgccgc ggacaacccc aggatccagg tcgtcagcct caccgccatg 1620cacgcccacg cgctggcgaa cagcgctccg gccgccctgg cgctcatcat cgtgcagtca 1680cgggacccga tctcggtggc gctggccacc gaggaactcg ccaagctcta cgccgcgcag 1740gcccaggcgg ggggacggcc ggcgacgccg gcccgcgccg aggaggccgc caccccgccg 1800gcgagctgct ggtcgaccct gtccgacatg gagcagcgga tcgcctacct ggtgagcgtg 1860ggtctgacga accggcagat cgccaagcag gtccacctgt ccgcgcacac cgtcaactac 1920cacctgcgga agatctaccg gaaactgggt ttcaacaccc gggccgagct ggcgcacgcc 1980gcggccacgt actccggccg ggcggcgatc tactccatga gcggcgacca ggactggggc 2040gccggatcca tgaccggcaa ggccagctga 20706895PRTMicromonospora sp. strain 046-ECO11 6Met Val Ile Met Asn Arg Met Ala Gly Arg Gly Gln Glu Leu Ser Ser1 5 10 15Leu Gly Glu Leu Leu Asp Ala Thr Met Arg Gly Ser Gly Gly Cys Val 20 25 30Val Val Asp Gly Pro Phe Gly Ile Gly Lys Thr His Leu Leu Lys Val 35 40 45Thr Gly Leu Glu Ala Ala Ala Arg Gly Leu Thr Val Val Ala Gly Arg 50 55 60Ala Ser Val Thr Asp Gln Pro Val Pro Val His Leu Leu Val Asn Phe65 70 75 80Leu Arg His Ala Met Pro Gly Glu Ala Ala Val Glu Gln Leu Ala Leu 85 90 95Pro Gly Ala Asn Pro Phe Trp Leu Ile Asp Arg Val Gly Asp Leu Val 100 105 110Glu Val Ala Ala Arg Arg Arg Pro Leu Val Val Ala Leu Asp Asp Ala 115 120 125Gln Arg Ile Asp Asp Val Ser Ala Leu Ala Leu Arg Gly Leu Val Pro 130 135 140Arg Leu Ala Ser Ser Pro Val Leu Trp Leu Leu Ala Arg Arg Pro Val145 150 155 160Ala Ala Gly Ser Ile Ala Gln His Ala Val Asp Trp Leu Ala Glu His 165 170 175Val Ala Val Arg Val Arg Leu Arg Glu Pro Gly Glu Glu Ala Val Ala 180 185 190Asp Leu Cys Ala Gly Ile Leu Gly Ala Arg Pro Asp Ala Ser Val Leu 195 200 205Arg Trp Ala Ala Arg Cys Gly Gly Asn Pro Lys Val Met Glu Ile Val 210 215 220Phe Ser Ala Phe Ile Lys Ala Gly Gln Met Ile Ile Val Asp Gly Ala225 230 235 240Ala Ser Val Val Ser Asp Glu Leu Pro Asp Gly Val Leu Ala Ala Val 245 250 255Arg Gly Leu Leu Glu Glu Leu Pro Pro Pro Leu Arg Arg Leu Leu Ala 260 265 270Ala Gly Gly Arg Leu Gly His Thr Phe Pro Val Asp Arg Val Thr Gly 275 280 285Leu Leu Asp Gly Ser Ala Ala Asp Val Ser Ala Ala Ile Asp Glu Ala 290 295 300Val Arg Val Gly Leu Ile Arg Arg Asp Gly Ala Glu Leu Thr Phe Ala305 310 315 320His Pro Val Leu Gly Glu Ala Leu Arg His Ala Ala Tyr Pro Glu Pro 325 330 335Glu Arg Ala Glu Pro Gly Ser Ala Pro Ala Pro Ala Ala Gly Asp Pro 340 345 350Val Arg Arg Gly Arg Pro Asp Pro Arg Pro Gly Thr Pro His Ser Pro 355 360 365Ala Gly Val Arg Val Thr Arg Ser Ala Pro Asp Ala Ala Thr Pro Ala 370 375 380Ala Thr Ala Gly Pro Arg Ser Gly Arg Cys Gly Cys Asp Asp Val Ala385 390 395 400Ala Ala Ala Val Ser His Leu Glu Asn Gly Ser Ala Glu Ala Pro Arg 405 410 415Ala Leu Ala Arg Ala Leu Arg Leu Leu Ala Gly Ala Gly Arg Ala Ala 420 425 430Glu Ala Gly Arg Leu Ala Glu Val Met Leu Arg Arg Asp Leu Ala Ala 435 440 445Asp Val Glu Ala Gln Leu Val Leu Glu Leu Gly His Gly Met Arg Ala 450 455 460Ala Gly Ser His Arg Leu Ala Ala Gly Phe Leu Arg Arg Thr Gln Ala465 470 475 480Arg His Asp Val Cys Glu Leu Asp Arg Ala Lys Leu Asp Arg Ala Leu 485 490 495Ala Asp Thr Thr Lys His Leu Gly Gly Ala Ser Ser Ala Glu Leu Glu 500 505 510Pro Arg His Gln Ser Pro Gly Cys Ala Pro Gly Arg Arg Pro Leu Trp 515 520 525Thr Trp Leu Val Arg Ala Leu Gly Ala Ala Asp Gln Leu Asp Glu Ala 530 535 540Gln Ala Val Leu Asp Thr Val Arg Pro Leu Ala Gln Glu Pro Ser His545 550 555 560Thr Gly Ser Glu Ser Leu Trp Arg Gly His Arg Ala Glu Leu Leu Ala 565 570 575Ala Ala Gly Arg Leu Asp Glu Ala Arg Ala Glu Ala Glu Ala Ala Leu 580 585 590Arg Ala Ala Asp His Ser Arg Pro Gly Asp Cys Val Pro Ala Arg Leu 595 600 605Val Leu Ala His Leu Gly Val His His Gly Asp Leu Ala Thr Ala Ser 610 615 620Asp Gln Leu Arg Ala Ala Glu Arg Leu Ala Ser Ala Asp Asp Ser Ala625 630 635 640Arg Met Asp Trp Ala Leu Ala Arg Phe His Ala Ala Ser Gly Arg Pro 645 650 655Ala Met Met Val Gln Thr Leu Ile Asn Val Ala Gly Gln Val Ala Pro 660 665 670Asp Pro Leu Leu Phe Thr Glu Ala Pro Ala Ala Ala Ala Thr Leu Val 675 680 685Arg Gln Ala Arg Arg Ala Gly Leu Asp Ala Glu Ala Glu Arg Ala Val 690 695 700Glu Val Ala Arg Arg Val Ala Arg Gly Asn Pro Phe Val Gln Ser Leu705 710 715 720Ala Ala Ala Ala Glu His Ala Ala Gly Leu Leu Arg Asp Asp Pro Ala 725 730 735Ala Leu Leu Arg Ala Ala Asp Leu His Arg Leu Ala Gly Arg Thr Leu 740 745 750Ala Ala Ala Gly Ala Val Glu Asp Ala Ala Arg Ser Thr Arg Asp Arg 755 760 765Ala Glu Ala Thr Arg Leu Leu Glu Ala Ala Thr Asp Gly Tyr Arg Glu 770 775 780Cys Gly Ala Arg Arg Asp Leu Glu Arg Val Glu Ala Glu Leu Arg Gly785 790 795 800Leu Pro Ala His Asn Val Arg Pro Leu Val Pro Asp Arg Pro Arg Ser 805 810 815Gly Trp Glu Ser Leu Thr Ser Ala Glu Leu Arg Val Val Arg Ala Ile 820 825 830Val Asp Gly Met Thr Asn Arg Glu Ala Ala Ser Ser Leu Phe Leu Ser 835 840 845Pro His Thr Val Asp Ser His Leu Arg Arg Val Phe Ser Lys Leu Asp 850 855 860Ile Asn Ser Arg Val Glu Leu Thr Arg Cys Phe Ile Ala His Glu Ala865 870 875 880Val Arg Pro Ala Leu Ala Thr Thr Arg Gln Pro Ala Ser Ala Gly 885 890 89572688DNAMicromonospora sp. strain 046-ECO11 7atggtcatca tgaatcgcat ggcggggcgc gggcaggaat tgtcctcatt gggggaactg 60ctcgacgcca ccatgcgggg atccgggggc tgcgtcgtcg tcgacgggcc gttcggcatc 120ggcaagaccc acctgctgaa ggtcaccggc ctggaggcgg cggcccgcgg gctgacagtg 180gtggccgggc gggcaagcgt cacggatcag ccggtgcccg tacacctgct cgtcaacttc 240ctgcgccacg cgatgcccgg cgaagcggcg gtcgagcagc tcgccctgcc gggcgccaac 300ccgttctggc tgatcgaccg ggtcggcgat ctggtcgagg tcgcggcgcg ccggcgcccg 360ctcgtggtcg ccctggacga cgcccagcgc atcgacgacg tcagcgccct ggccctgcgc 420gggctcgtgc cgcgcctggc gtcctcgccg gtgctctggc tgctggcccg ccggccggtc 480gccgccgggt cgatcgctca gcacgccgtc gactggctgg ccgagcacgt cgcggtacgg 540gtacggctgc gcgagccggg cgaggaggcg gtggccgacc tgtgcgccgg catcctcggc 600gcccggccgg acgcctccgt cctgcgctgg gcggcccgct gcggcggcaa cccgaaggtg 660atggagatcg tcttcagcgc gttcatcaag gccggccaga tgatcatcgt ggacggggcg 720gcgtcggtgg tgtccgacga gctgcccgac ggtgtcctcg ccgccgttcg cgggctgctg 780gaggagctgc cgcccccgct gcggcgcctg ctcgcggccg gcggccggct cggccacacg 840tttcccgtcg accgggtgac gggcctgctg gacggctcgg ccgccgacgt gtccgccgcg 900atcgacgagg cggtgcgggt cgggctgata cgacgcgacg gcgcggagct gaccttcgcc 960cacccggtgc tcggagaggc gcttcgccac gccgcgtacc cggaaccgga gcgtgccgag 1020cccggatccg cgccggcacc ggcggcgggc gacccggtcc ggcgcgggcg gcccgatccg 1080cggcccggga cgccccactc ccccgccggc gtacgcgtca cgcgctccgc gccggacgcg 1140gccacgcccg ccgcgacggc ggggccgcgc tcgggccggt gcgggtgcga cgacgtggcg 1200gcagccgccg tgtcccacct ggagaacgga tccgccgagg cgccacgagc actggcccgt 1260gcgctgcgcc tgctggccgg ggcggggcgg gccgccgagg ccggccgcct cgcggaggtg 1320atgctccgcc gcgacctcgc ggcggacgtc gaggcgcagc tcgtgctcga actgggacac 1380gggatgcggg ccgccggcag ccaccgcctg gcggccggct tcctgcgccg gacgcaggcc 1440cgccacgacg tgtgcgagct ggaccgcgcc aagctggacc gggcgctcgc ggacaccacg 1500aagcacctgg gcggtgcctc ctccgccgag ctggagcccc ggcaccagtc cccgggctgc 1560gcgcccggcc ggcggccgct gtggacctgg ctggtccggg cgctgggcgc ggccgatcag 1620ctcgacgagg cgcaggcggt gctggacacc gtacgaccgc tggcgcagga gcccagtcac 1680accggctcgg agtcgctctg gcgcggccac cgggccgagc tgctggcagc ggccggacgg 1740ctggacgagg cacgcgccga ggcggaggcg gcgctgcgag ccgccgacca ctcccggccg 1800ggcgactgcg tgccggcgcg cctggtcctg gcccacctcg gcgtgcacca cggtgacctc 1860gccacggcca gcgaccagtt gcgggcggcc gagcggctgg cctccgccga cgactcggcg 1920cggatggact gggcgctggc ccggttccac gctgccagcg gccgtccggc gatgatggtg 1980cagacgctga tcaacgtcgc cggacaggtc gcacccgatc cgctgctgtt caccgaggcg 2040ccggccgctg cggcgacgct cgtacgccag gcccgccggg cggggctcga cgcggaggcc 2100gagcgcgccg tggaggtcgc ccggcgcgtc gcccgcggca acccgttcgt ccagtcgctg 2160gcggcggcgg cggaacacgc cgcgggtctc ctgcgcgacg atccggcggc gctgctgcgg 2220gccgcggatc tgcaccggct cgccggccgt acgctcgcgg cggccggcgc ggtggaggac 2280gcggcccgca gcacccggga ccgggccgag gccacccgtc tgctcgaggc cgcgacggac 2340ggctaccggg agtgcggcgc gcgacgcgac ctggagcgcg tggaggccga gctgcgtggc 2400ctgccggctc acaacgtccg cccgctggtc cccgaccggc cccggtcggg gtgggagagc 2460ctgaccagcg cggagctgcg ggtcgtgcgg gccatcgtgg acgggatgac caaccgcgag 2520gcggcgagtt cgctgttcct gtccccgcac accgtcgaca gtcacctgcg gcgcgtcttc 2580tccaagctcg acatcaacag ccgggtggaa ctgacccgct gcttcatcgc gcacgaggcg 2640gtccggccgg cgctggccac cacacgccag ccggcgtccg ccggctga 26888362PRTMicromonospora sp. strain 046-ECO11 8Met Thr Val Gly Tyr Leu Gly Thr Val Thr Asp Ser Ala Pro Val Asp1 5 10 15Ala Ala Leu Arg Asp Phe Phe Ala Glu Arg Arg Ala Glu Ala Arg Glu 20 25 30Leu Gly Asp Asp Phe Ala Ala Leu Val Ala Glu Leu Glu Ser Tyr Val 35 40 45Leu Arg Gly Gly Lys Arg Ile Arg Pro Ala Phe Ala Trp Leu Gly Trp 50 55 60Ile Gly Ala Gly Gly Asp Pro Glu Asp Pro Val Ala Thr Ala Val Leu65 70 75 80Asn Ala Cys Ala Gly Phe Glu Leu Leu His Ala Ser Gly Leu Ile His 85 90 95Asp Asp Ile Ile Asp Ala Ser Gln Thr Arg Arg Gly His Pro Ala Ala 100 105 110His Val Ala Tyr Ala Glu Arg His Arg Ala Arg Arg Phe Ser Gly Asp 115 120 125Pro Gly Thr Phe Gly Thr Gly Thr Ala Ile Leu Ile Gly Asp Leu Val 130 135 140Leu Ile Trp Ala Asp Val Leu Val Arg Ala Ser Gly Leu Pro Ala Asp145 150 155 160Ala His Val Arg Val Ser Pro Val Trp Ser Ala Val Arg Ser Glu Val 165 170 175Met Tyr Gly Gln Leu Leu Asp Leu Ile Ser Gln Val Ser Arg Ser Glu 180 185 190Asp Val Asp Ala Ala Leu Arg Ile Asn Gln Tyr Lys Thr Ala Ser Tyr 195 200 205Thr Val Glu Arg Pro Leu Gln Phe Gly Ala Ala Ile Ala Gly Ala Asp 210 215 220Asp Asp Leu Phe Ala Ala Tyr Arg Ala Phe Gly Ala Asp Val Gly Ile225 230 235 240Ala Phe Gln Leu Arg Asp Asp Leu Leu Gly Val Phe Gly Asp Pro Val 245 250 255Val Thr Gly Lys Pro Ser Gly Asp Asp Leu Arg Glu Gly Lys Arg Thr 260 265 270Val Leu Leu Ala Thr Ala Leu Lys Arg Ala Asp Glu Arg Asp Pro Asp 275 280 285Ala Ala Ala Tyr Leu Arg Ala Lys Val Gly Thr Asp Leu Ala Asp Glu 290 295 300Glu Ile Ala Arg Ile Arg Ala Ile Phe Arg Asp Val Gly Ala Val Glu305 310 315 320Glu Ile Glu Arg Gln Ile Ser Gln Arg Thr Asp Arg Ala Leu Ala Ala 325 330 335Leu Glu Ala Ser Ser Ala Thr Ala Pro Ala Lys His Gln Leu Ala Asp 340 345 350Met Ala Ile Lys Ala Thr Gln Arg Ala Gln 355 36091089DNAMicromonospora sp. strain 046-ECO11 9atgaccgtcg gatatctcgg gacggtcacc gactcggcgc ccgtcgacgc cgcgctgcgc 60gacttcttcg ccgagcgccg cgccgaggca cgcgagctcg gcgacgactt cgcggccctg 120gtcgccgagc tggagagcta cgtcctgcgg ggcggcaagc gcatccggcc cgccttcgcc 180tggctgggct ggatcggcgc cggcggcgac ccggaggacc cggtggcgac cgcggtgctg 240aacgcctgcg ccgggttcga gctgctgcac gcgtccggcc tcatccacga cgacatcatc 300gacgcgtcgc agacccgccg cggccatccc gccgcgcacg tcgcgtacgc cgaacggcat 360cgggcgcggc gcttctccgg tgacccggga acgttcggca ccggcaccgc catcctgatc 420ggagacctcg tcctgatctg ggccgacgtc ctggtccgcg cctccggcct gccggccgac 480gcgcacgtgc gggtctcgcc ggtgtggtcg gcggtgcgct ccgaggtcat gtacggccag 540ctgctcgatc tgatcagcca ggtgagccgg agcgaggacg tcgacgcggc gctgcgcatc 600aaccagtaca agaccgcgtc gtacacggtg gagcggccac tgcagttcgg cgcggcgatc 660gccggcgcgg acgacgacct cttcgcggcc taccgcgcct tcggcgccga cgtgggtatt 720gccttccagc tgcgcgacga cctgctcggc gtgttcggcg acccggtggt gacgggcaag 780ccgtccggcg acgacctgcg ggagggcaag cggacggtcc tgctcgccac ggcgctcaag 840cgcgccgacg aacgggaccc ggacgcggcg gcctacctgc gggcgaaggt cggcacggac 900ctcgcggacg aggagatcgc ccgcatccgc gccatcttcc gcgacgtcgg cgcggtcgag 960gagatcgagc ggcagatctc gcagcgcacc gaccgggcgc tggccgcgct ggaggcgagc 1020agcgccaccg cccccgcgaa gcatcagctc gccgacatgg cgatcaaggc cacccagcgg 1080gcccagtga 108910354PRTMicromonospora sp. strain 046-ECO11 10Met Ser Thr Glu Pro Val Thr Val Val Ala Arg Gly Val Leu Asp Gly1 5 10 15Arg Gly Asp Gly Pro Gly Arg Leu Gly Thr Gly Arg Ala His Gly Lys 20 25

30Ala Ile Leu Leu Gly Glu His Ala Val Val Tyr Gly Ala Pro Ala Leu 35 40 45Ala Val Pro Val Pro Gln Leu Thr Ala Val Ala Lys Ala Arg Arg Ala 50 55 60Gly Gly Asp Gly Gly Asp Glu Val Ser Phe Ala Ile Ala Gly Leu Glu65 70 75 80Ser Pro Glu Val Thr Ser Leu Pro Thr Asp Gly Leu Gln His Leu Val 85 90 95Thr Glu Phe Arg Gln Arg Ala Ala Val Thr Glu Pro Met Arg Val Asp 100 105 110Val Leu Val Asp Cys Ala Ile Pro Gln Gly Arg Gly Leu Gly Ser Ser 115 120 125Ala Ala Cys Ala Arg Ala Ala Val Leu Ala Leu Ala Asp Ala Phe Asp 130 135 140Arg Arg Leu Asp Ala Ala Thr Val Phe Asp Leu Val Gln Thr Ser Glu145 150 155 160Asn Val Ala His Gly Arg Ala Ser Gly Ile Asp Ala Leu Ala Thr Gly 165 170 175Ala Thr Ala Pro Leu Ile Phe Arg Asn Gly Val Gly Arg Glu Leu Pro 180 185 190Val Ala Met Ala Gly Ala Ala Arg Ala Ala Arg Gly Ser Gly Pro Ala 195 200 205Gly Phe Asp Ala Val Leu Val Ile Ala Asp Ser Gly Val Ser Gly Ser 210 215 220Thr Arg Asp Ala Val Glu Leu Leu Arg Gly Ala Phe Glu Arg Ser Pro225 230 235 240Arg Thr Arg Asp Glu Phe Val Ser Arg Val Thr Ser Leu Thr Glu Ala 245 250 255Ala Ala His Asp Leu Leu Gln Gly Arg Val Ala Asp Phe Gly Ala Arg 260 265 270Leu Thr Glu Asn His Arg Leu Leu Arg Glu Val Gly Ile Ser Thr Glu 275 280 285Arg Ile Asp Arg Met Val Asp Ala Ala Leu Ala Ala Gly Ser Pro Gly 290 295 300Ala Lys Ile Ser Gly Gly Gly Leu Gly Gly Cys Met Ile Ala Leu Ala305 310 315 320Arg Asp Arg Gln Glu Ser Ala Ala Val Val Arg Ser Val Gln Gln Ala 325 330 335Gly Ala Val Arg Thr Trp Thr Val Pro Met Gly Arg Phe Thr Gly His 340 345 350Asp Asp 111065DNAMicromonospora sp. strain 046-ECO11 11atgtccacgg aaccggtgac cgtcgtcgcc cgcggcgttc tcgacggccg gggtgacggg 60ccgggccgcc tcggcaccgg ccgcgcccac ggcaaggcca tcctgctggg cgaacacgcc 120gtcgtgtacg gcgctccggc gctcgccgtc ccggtgccgc aactgaccgc cgtggccaag 180gcgcggcggg ccggcggcga cggcggcgac gaggtctcct tcgccatcgc cgggctggag 240agcccggagg tgacgtcgct tccgaccgac ggcctgcaac atctggtgac ggagttccgg 300cagcgggccg ccgtcaccga gccgatgcgc gtcgacgtgc tcgtggactg cgccatcccg 360cagggccggg ggctcgggtc gagcgccgcc tgcgcccgcg ccgcggtgct ggccctcgcg 420gacgcgttcg accgccgcct cgacgccgcc acggtgttcg atctggtgca gacctcggag 480aacgtggcgc acggccgggc cagcggcatc gacgccctgg ccaccggtgc gaccgcgccg 540ctgatcttcc gcaacggcgt gggccgggaa ctgccggtcg ccatggcggg cgccgcgcgt 600gccgcgcgag ggtcgggccc ggccggcttc gacgcggtgc tcgtcatcgc cgacagcggc 660gtcagcggca gcacccggga cgcggtggag ctgctgcggg gtgccttcga gcgctccccg 720cgcacgcgcg acgagttcgt cagccgggtg accagcctga ccgaggcggc ggcgcacgac 780ctgctccagg gccgggtcgc cgacttcggc gcgcggctga ccgagaacca ccggctgttg 840cgcgaggtcg gcatcagcac cgaacggatc gaccggatgg tcgacgccgc gctcgcggcg 900ggcagcccgg gcgccaagat cagcggcggt ggcctgggcg gctgcatgat cgcactggcc 960cgggaccgcc aggaatccgc ggcggtggtg cggagcgtcc agcaggccgg cgccgtccgc 1020acctggaccg tcccgatggg gaggttcacc ggccatgacg actga 106512346PRTMicromonospora sp. strain 046-ECO11 12Met Thr Thr Asp His Arg Ala Glu Pro Ser Glu Pro Ala Leu Asp Arg1 5 10 15Pro Ala Thr Ala Val Ala His Pro Asn Ile Ala Leu Ile Lys Tyr Trp 20 25 30Gly Lys Arg Asp Glu Gln Leu Met Ile Pro Tyr Ala Asp Ser Leu Ser 35 40 45Met Thr Leu Asp Val Phe Pro Thr Thr Thr Thr Val Arg Ile Asp Ser 50 55 60Gly Ala Ala Ala Asp Glu Val Val Leu Asp Gly Ser Pro Ala Asp Gly65 70 75 80Glu Arg Arg Gln Arg Val Val Thr Phe Leu Asp Leu Val Arg Lys Leu 85 90 95Ala Gly Arg Thr Glu Arg Ala Cys Val Asp Thr Arg Asn Ser Val Pro 100 105 110Thr Gly Ala Gly Leu Ala Ser Ser Ala Ser Gly Phe Ala Ala Leu Ala 115 120 125Leu Ala Gly Ala Ala Ala Tyr Gly Leu Asp Leu Asp Thr Thr Ala Leu 130 135 140Ser Arg Leu Ala Arg Arg Gly Ser Val Ser Ala Ser Arg Ser Val Phe145 150 155 160Gly Gly Phe Ala Met Cys His Ala Gly Pro Gly Ala Gly Thr Ala Ala 165 170 175Asp Leu Gly Ser Tyr Ala Glu Pro Val Pro Val Ala Pro Leu Asp Val 180 185 190Ala Leu Val Ile Ala Ile Val Asp Ala Gly Pro Lys Ala Val Ser Ser 195 200 205Arg Glu Gly Met Arg Arg Thr Val Arg Thr Ser Pro Leu Tyr Gln Ser 210 215 220Trp Val Ala Ser Gly Arg Ala Asp Leu Ala Glu Met Arg Ala Ala Leu225 230 235 240Leu Gln Gly Asp Leu Asp Ala Val Gly Glu Ile Ala Glu Arg Asn Ala 245 250 255Leu Gly Met His Ala Thr Met Leu Ala Ala Arg Pro Ala Val Arg Tyr 260 265 270Leu Ala Pro Val Thr Val Ala Val Leu Asp Ser Val Leu Arg Leu Arg 275 280 285Ala Asp Gly Val Ser Ala Tyr Ala Thr Met Asp Ala Gly Pro Asn Val 290 295 300Lys Val Leu Cys Arg Arg Ala Asp Ala Asp Arg Val Ala Asp Thr Leu305 310 315 320Arg Asp Ala Ala Pro Ser Cys Ala Val Val Val Ala Gly Pro Gly Pro 325 330 335Ala Ala Arg Pro Asp Pro Gly Ser Arg Pro 340 345131041DNAMicromonospora sp. strain 046-ECO11 13atgacgactg accaccgggc ggagccgtcc gagccggcgc tcgaccggcc cgcgaccgcc 60gtggcccatc cgaacatcgc gctgatcaag tactggggca agcgcgacga gcagctgatg 120atcccgtacg ccgacagcct gtcgatgacg ctcgacgtct tcccgaccac caccaccgtc 180cggatcgaca gcggcgcggc ggccgacgag gtcgtcctcg acggctcgcc cgccgacggc 240gaacggcgac agcgcgtcgt caccttcctg gacctggtac gcaagctggc cgggcgcacg 300gaacgggcct gcgtcgacac ccgcaactcc gtgcccaccg gcgccggcct ggcgtcctcg 360gcgagcggat tcgccgccct cgccctcgcc ggcgccgccg cgtacggcct cgacctggac 420accaccgcgc tgtcccgcct ggcccggcgg ggatccgtgt cggcctcccg gtcggtcttc 480ggcggcttcg cgatgtgcca cgcaggcccc ggcgccggga ccgccgcgga cctcggctcc 540tacgccgagc cggtgcccgt cgcgcccctc gacgtcgcgc tggtgatcgc gatcgtcgac 600gccgggccga aggcggtgtc gagccgcgag gggatgcggc gaaccgtccg gacctccccg 660ctctatcagt cgtgggtcgc ctccggccgc gccgacctgg ccgagatgcg ggccgcgctg 720ctccagggag acctggacgc ggtcggcgag atcgccgaac gcaacgccct cggcatgcac 780gccaccatgc tggccgcccg gccggcggtg cgctacctgg cgccggtcac tgtcgccgtg 840ctcgacagcg tgctgcgcct gcgcgccgac ggcgtctccg cctacgccac gatggacgcg 900ggaccgaacg tcaaggtgct ctgccgccgc gcggacgccg accgggtcgc cgacaccctg 960cgcgacgccg cgccgagctg cgccgtggtc gtcgccggac cggggccggc ggcccggccg 1020gacccgggca gccggccgtg a 104114369PRTMicromonospora sp. strain 046-ECO11 14Val Thr Gly Pro Gly Ala Val Arg Arg His Ala Pro Gly Lys Leu Phe1 5 10 15Val Ala Gly Glu Tyr Ala Val Leu Glu Pro Gly His Pro Ala Leu Leu 20 25 30Val Ala Val Asp Arg Gly Val Asp Val Thr Val Ser Gly Ala Asp Ala 35 40 45His Leu Val Val Asp Ser Asp Leu Cys Pro Glu Gln Ala Cys Leu Arg 50 55 60Trp Gln Asp Gly Arg Leu Val Gly Ala Gly Asp Gly Gln Pro Ala Pro65 70 75 80Asp Ala Leu Gly Ala Val Val Ser Ala Ile Glu Val Val Gly Glu Leu 85 90 95Leu Thr Gly Arg Gly Leu Arg Pro Leu Pro Met Arg Val Ala Ile Thr 100 105 110Ser Arg Leu His Arg Asp Gly Thr Lys Phe Gly Leu Gly Ser Ser Gly 115 120 125Ala Val Thr Val Ala Thr Val Thr Ala Val Ala Ala Tyr His Gly Val 130 135 140Glu Leu Ser Leu Glu Ser Arg Phe Arg Leu Ala Met Leu Ala Thr Val145 150 155 160Arg Asp Gly Ala Asp Ala Ser Gly Gly Asp Leu Ala Ala Ser Val Trp 165 170 175Gly Gly Trp Ile Ala Tyr Gln Ala Pro Asp Arg Ala Ala Val Arg Glu 180 185 190Met Ala Arg Arg Arg Gly Val Glu Glu Thr Met Arg Ala Pro Trp Pro 195 200 205Gly Leu Arg Val Arg Arg Leu Pro Pro Pro Arg Gly Leu Ala Leu Glu 210 215 220Val Gly Trp Thr Gly Glu Pro Ala Ser Ser Ser Ser Leu Thr Gly Arg225 230 235 240Leu Ala Ala Ser Arg Trp Arg Gly Ser Pro Ala Arg Trp Ser Phe Thr 245 250 255Ser Arg Ser Gln Glu Cys Val Arg Thr Ala Ile Asp Ala Leu Glu Arg 260 265 270Gly Asp Asp Gln Glu Leu Leu His Gln Val Arg Arg Ala Arg His Val 275 280 285Leu Ala Glu Leu Asp Asp Glu Val Arg Leu Gly Ile Phe Thr Pro Arg 290 295 300Leu Thr Ala Leu Cys Asp Ala Ala Glu Thr Val Gly Gly Ala Ala Lys305 310 315 320Pro Ser Gly Ala Gly Gly Gly Asp Cys Gly Ile Ala Leu Leu Asp Ala 325 330 335Thr Ala Ala Thr Arg Thr Ala Arg Leu Arg Glu Gln Trp Ala Ala Ala 340 345 350Gly Val Leu Pro Met Pro Ile Gln Val His Gln Thr Asn Gly Ser Ala 355 360 365Arg 151110DNAMicromonospora sp. strain 046-ECO11 15gtgaccggcc cgggcgccgt gcgccgccac gcgccgggca agctgttcgt cgccggtgag 60tacgcggtgc tggagccggg ccacccggcg ctgctggtgg cggtcgacag gggagtggac 120gtcaccgtct ccggcgccga cgcccacctc gttgtcgact ccgacctctg cccggagcag 180gcgtgcctgc ggtggcagga cggccggctc gtcggcgcgg gcgacgggca gccggcgccc 240gacgccctcg gcgccgtggt ctcggcgatc gaggtggtcg gcgaactcct gaccggacga 300gggctgcgcc cgctgcccat gcgggtggcg atcaccagcc ggctgcaccg cgacggcacg 360aagttcggcc tcgggtcgag cggggcggtg acagtcgcca cggtgaccgc agtggccgcg 420taccacgggg tggagctgtc gctcgaatcg cggttccggc tggcgatgct ggcgacggtg 480cgtgacggcg ccgacgcctc cggcggtgat ctggccgcga gcgtctgggg cggctggatc 540gcctaccagg cgcccgaccg cgcggccgtg cgcgagatgg cgcggcggcg cggcgtcgag 600gagacgatgc gcgcgccctg gccgggcctg cgggtccggc ggctgccacc accgcgtggc 660ctcgcgctgg aggtgggctg gaccggcgag ccggcgagca gcagctcgtt gaccgggcgg 720ctggccgcct cccggtggcg gggcagcccg gcgcggtgga gcttcaccag ccgtagccag 780gagtgtgtgc gtaccgccat cgacgcgctg gagcggggcg acgaccagga actgctgcac 840caggtccggc gggcccggca cgtgcttgcc gagctggacg acgaggtccg gctcgggatc 900ttcacccccc ggctgacggc gctgtgcgac gccgccgaga ccgtcggcgg cgcggccaaa 960ccgtccggcg ccggtggcgg ggactgcggc atcgcgttgc tggacgccac cgccgcgacg 1020cggaccgcgc ggctgcgcga gcagtgggcc gccgccgggg tgctccccat gccgatccag 1080gtccatcaga cgaacgggag cgcgcgatga 111016360PRTMicromonospora sp. strain 046-ECO11 16Met Ile Ala Asn Arg Lys Asp Asp His Val Arg Leu Ala Ala Glu Gln1 5 10 15Gln Gly Arg Leu Gly Gly His His Glu Phe Asp Asp Val Ser Phe Val 20 25 30His His Ala Leu Ala Gly Ile Asp Arg Ser Asp Val Ser Leu Ala Thr 35 40 45Ser Phe Gly Gly Ile Asp Trp Pro Val Pro Leu Cys Ile Asn Ala Met 50 55 60Thr Gly Gly Ser Thr Lys Thr Gly Leu Ile Asn Arg Asp Leu Ala Ile65 70 75 80Ala Ala Arg Glu Thr Gly Val Pro Ile Ala Thr Gly Ser Met Ser Ala 85 90 95Tyr Phe Ala Asp Glu Ser Val Ala Glu Ser Phe Ser Val Met Arg Arg 100 105 110Glu Asn Pro Asp Gly Phe Ile Met Ala Asn Val Asn Ala Thr Ala Ser 115 120 125Val Glu Arg Ala Arg Arg Ala Val Asp Leu Met Arg Ala Asp Ala Leu 130 135 140Gln Ile His Leu Asn Thr Ile Gln Glu Thr Val Met Pro Glu Gly Asp145 150 155 160Arg Ser Phe Ala Ala Trp Gly Pro Arg Ile Glu Gln Ile Val Ala Gly 165 170 175Val Gly Val Pro Val Ile Val Lys Glu Val Gly Phe Gly Leu Ser Arg 180 185 190Glu Thr Leu Leu Arg Leu Arg Asp Met Gly Val Arg Val Ala Asp Val 195 200 205Ala Gly Arg Gly Gly Thr Asn Phe Ala Arg Ile Glu Asn Asp Arg Arg 210 215 220Asp Ala Ala Asp Tyr Ser Phe Leu Asp Gly Trp Gly Gln Ser Thr Pro225 230 235 240Ala Cys Leu Leu Asp Ala Gln Gly Val Asp Leu Pro Val Leu Ala Ser 245 250 255Gly Gly Ile Arg Asn Pro Leu Asp Val Val Arg Gly Leu Ala Leu Gly 260 265 270Ala Gly Ala Ala Gly Val Ser Gly Leu Phe Leu Arg Thr Leu Leu Asp 275 280 285Gly Gly Val Pro Ala Leu Leu Ser Leu Leu Ser Thr Trp Leu Asp Gln 290 295 300Ile Glu Ala Leu Met Thr Ala Leu Gly Ala Arg Thr Pro Ala Asp Leu305 310 315 320Thr Arg Cys Asp Leu Leu Ile Gln Gly Arg Leu Ser Ala Phe Cys Ala 325 330 335Ala Arg Gly Ile Asp Thr His Arg Leu Ala Thr Arg Ser Gly Ala Thr 340 345 350His Glu Met Ile Gly Gly Ile Arg 355 360171083DNAMicromonospora sp. strain 046-ECO11 17atgatcgcca accgcaagga cgaccacgtc cggctcgccg ccgagcagca gggccggctc 60ggcggtcacc acgagttcga cgacgtgtcc ttcgtgcacc acgccctggc cggcatcgac 120cggtccgacg tctcgctggc cacgtcgttc ggcggcatcg actggccggt gccgctgtgc 180atcaacgcga tgaccggcgg cagcaccaag accggcctga tcaaccggga cctggcgatc 240gcggcccggg agaccggcgt accgatcgcc accgggtcga tgagcgccta cttcgccgac 300gagtcggtgg ccgagagttt cagcgtgatg cgccgggaga accccgacgg gttcatcatg 360gccaacgtca acgccaccgc ctccgtcgaa cgggcccggc gggctgtcga cctgatgcgg 420gccgacgcgc tgcagatcca cctgaacacc atccaggaga cggtgatgcc ggagggggac 480cggtcgttcg ccgcctgggg gccgcggatc gaacagatcg tcgccggcgt cggtgtgccg 540gtgatcgtca aggaggtcgg cttcgggctc agccgcgaaa cgctgctgcg gctgcgggac 600atgggcgtcc gggtggccga cgtcgccggc cgcggcggca cgaacttcgc gcgcatcgag 660aacgaccggc gggacgccgc cgactactcc ttcctcgacg ggtggggaca gtcgacaccc 720gcctgcctgc tggacgccca gggcgtggac ctgcccgtgc tggcctccgg cggcatccgc 780aacccgctcg acgtggtccg cgggctggcg ctcggcgccg gcgcggccgg ggtgtccgga 840ctgttcctgc gcacgctcct ggacggcggc gtgccggcgc tgctgtcgct gctgtccacc 900tggctcgacc agatcgaagc cctgatgacc gccctgggcg cgcggacccc ggccgacctg 960acccgctgcg acctgctgat ccagggtcgg ctgagcgcgt tctgcgcggc ccggggcatc 1020gacacccacc gcctcgccac ccgttccggc gccacccacg agatgatcgg aggcattcga 1080tga 108318351PRTMicromonospora sp. strain 046-ECO11 18Met Asn Asp Ala Ile Ala Gly Val Pro Met Lys Trp Val Gly Pro Val1 5 10 15Arg Ile Ser Gly Asn Val Ala Gln Ile Glu Thr Glu Val Pro Leu Ala 20 25 30Thr Tyr Glu Ser Pro Leu Trp Pro Ser Val Gly Arg Gly Ala Lys Ile 35 40 45Ser Arg Met Val Glu Ala Gly Ile Val Ala Thr Leu Val Asp Glu Arg 50 55 60Met Thr Arg Ser Val Phe Val Arg Ala Lys Asp Ala Gln Thr Ala Tyr65 70 75 80Leu Ala Ser Leu Glu Val Asp Ala Arg Phe Asp Glu Leu Arg Asp Ile 85 90 95Val Arg Thr Cys Gly Arg Phe Val Glu Leu Ile Gly Phe His His Glu 100 105 110Ile Thr Ala Asn Leu Leu Phe Leu Arg Phe Ser Phe Thr Thr Gly Asp 115 120 125Ala Ser Gly His Asn Met Ala Thr Leu Ala Ala Asp Ala Leu Leu Lys 130 135 140His Ile Leu Asp Thr Ile Pro Gly Ile Ser Tyr Gly Ser Ile Ser Gly145 150 155 160Asn Tyr Cys Thr Asp Lys Lys Ala Thr Ala Ile Asn Gly Ile Leu Gly 165 170 175Arg Gly Lys Asn Val Val Thr Glu Leu Val Val Pro Arg Glu Ile Val 180 185 190His Asp Ser Leu His Thr Thr Ala Ala Ala Ile Ala Gln Leu Asn Val 195 200 205His Lys Asn Met Ile Gly Thr Leu Leu Ala Gly Gly Ile Arg Ser Ala 210 215 220Asn Ala His Tyr Ala Asn Met Leu Leu Gly Phe Tyr Leu Ala Thr Gly225 230 235 240Gln Asp Ala Ala Asn Ile Val Glu Gly Ser Gln Gly Val Thr Val Ala 245 250 255Glu Asp Arg Asp Gly Asp Leu Tyr Phe Ser Cys Thr Leu Pro Asn Leu 260 265 270Ile Val Gly Thr Val Gly Asn Gly Lys Gly Leu Gly Phe Val Glu Glu 275 280 285Asn Leu Glu Arg Leu Gly Cys Arg Ala Ser Arg Asp Pro Gly Glu Asn 290

295 300Ala Arg Arg Leu Ala Val Ile Ala Ala Ala Thr Val Leu Cys Gly Glu305 310 315 320Leu Ser Leu Leu Ala Ala Gln Thr Asn Pro Gly Glu Leu Met Arg Ala 325 330 335His Val Arg Leu Glu Arg Pro Thr Glu Thr Thr Lys Ile Gly Ala 340 345 350191056DNAMicromonospora sp. strain 046-ECO11 19atgaacgacg cgatcgccgg tgtgcccatg aaatgggtag gtcccgtgcg gatctcggga 60aacgtggcgc agatcgagac ggaggttccg ctcgccacgt acgagtcgcc gctctggccg 120tccgtcggcc ggggcgcgaa gatctcccgg atggtcgagg cgggcatcgt cgccacgctc 180gtcgacgagc gcatgacccg ctcggtgttc gtgcgcgcca aggacgcgca gaccgcctac 240ctggcctcgc ttgaggtcga cgcgcggttc gacgaactgc gtgacatcgt gcgcacctgc 300ggcaggttcg tcgagctgat cgggttccac cacgagatca ccgcgaacct gctgttcctg 360cggttcagtt tcaccaccgg cgacgcgtcc gggcacaaca tggcgacgct ggccgccgac 420gcgctgctga agcacatcct ggacaccatt ccgggcatct cgtacggctc gatctcgggc 480aactactgca ccgacaagaa ggccaccgcg ataaacggca ttctcggccg gggcaagaac 540gtggtcaccg agctggtcgt gccgcgggag atcgtccacg acagcctgca cacgacggcg 600gcggcgatcg cccagctgaa cgtgcacaag aacatgatcg gcacgttgct cgccggcggt 660atccgctcgg ccaacgccca ctacgcgaac atgctgctcg ggttctacct ggccacgggt 720caggacgccg cgaacatcgt cgagggctcc cagggcgtga cggtcgccga ggaccgcgac 780ggcgacctct acttctcctg cacgctgccc aacctgatcg tgggcaccgt cggcaacggc 840aaggggctcg gcttcgtcga ggagaacctg gagcggctcg gctgccgcgc ctcgcgtgat 900ccgggcgaga acgcccggcg gctcgcggtc atcgcggccg cgacggtgct ctgcggcgag 960ctgtccctgc tcgccgcgca gaccaacccg ggcgagctga tgcgggcgca cgtccggctc 1020gaacgcccga ccgagaccac gaagatcgga gcctga 105620391PRTMicromonospora sp. strain 046-ECO11 20Met Ala Glu Arg Pro Ala Val Gly Ile His Asp Leu Ser Ala Ala Thr1 5 10 15Ala His His Val Leu Thr His Glu Thr Leu Ala Ala Ser Asn Gly Ala 20 25 30Asp Val Ala Lys Tyr His Arg Gly Ile Gly Leu Arg Ala Met Ser Val 35 40 45Pro Ala Pro Asp Glu Asp Ile Val Thr Met Ala Ala Ala Ala Ala Ala 50 55 60Pro Val Val Ala Arg His Gly Thr Asp Arg Ile Arg Thr Val Val Phe65 70 75 80Ala Thr Glu Ser Ser Val Asp Gln Ala Lys Ala Ala Gly Ile His Val 85 90 95His Ser Leu Leu Gly Leu Pro Ser Ala Thr Arg Val Val Glu Leu Lys 100 105 110Gln Ala Cys Tyr Gly Gly Thr Ala Gly Leu Gln Phe Ala Ile Gly Leu 115 120 125Val His Arg Asp Pro Ser Gln Gln Val Leu Val Ile Ala Ser Asp Val 130 135 140Ser Lys Tyr Ala Leu Gly Glu Pro Gly Glu Ala Thr Gln Gly Ala Ala145 150 155 160Ala Val Ala Met Leu Val Gly Ala Asp Pro Ala Leu Val Arg Val Glu 165 170 175Asp Pro Ser Gly Met Phe Thr Ala Asp Val Met Asp Phe Trp Arg Pro 180 185 190Asn Tyr Arg Thr Thr Ala Leu Val Asp Gly His Glu Ser Ile Ser Ala 195 200 205Tyr Leu Gln Ala Leu Glu Gly Ser Trp Lys Asp Tyr Thr Glu Arg Gly 210 215 220Gly Arg Thr Leu Asp Glu Phe Gly Ala Phe Cys Tyr His Gln Pro Phe225 230 235 240Pro Arg Met Ala Asp Lys Ala His Arg His Leu Leu Asn Tyr Cys Gly 245 250 255Arg Asp Val Asp Asp Ala Leu Val Ala Gly Ala Ile Gly His Thr Thr 260 265 270Ala Tyr Asn Ala Glu Ile Gly Asn Ser Tyr Thr Ala Ser Met Tyr Leu 275 280 285Gly Leu Ala Ala Leu Leu Asp Thr Ala Asp Asp Leu Thr Gly Arg Thr 290 295 300Val Gly Phe Leu Ser Tyr Gly Ser Gly Ser Val Ala Glu Phe Phe Ala305 310 315 320Gly Thr Val Val Pro Gly Tyr Arg Ala His Thr Arg Pro Asp Gln His 325 330 335Arg Ala Ala Ile Asp Arg Arg Gln Glu Ile Asp Tyr Ala Thr Tyr Arg 340 345 350Glu Leu His Glu His Ala Phe Pro Val Asp Gly Gly Asp Tyr Pro Ala 355 360 365Pro Glu Val Thr Thr Gly Pro Tyr Arg Leu Ala Gly Leu Ser Gly His 370 375 380Lys Arg Val Tyr Glu Pro Arg385 390211176DNAMicromonospora sp. strain 046-ECO11 21atggccgaga gacccgccgt cggcatccac gacctgtccg ccgcgacggc gcatcacgtg 60ctgacacacg agaccctggc cgcgagcaac ggcgccgacg tggccaagta ccaccgtggc 120atcgggctgc gggcgatgag cgtgcccgcc ccggacgagg acatcgtgac gatggctgct 180gccgccgccg cgccggtggt cgcccgccac ggcaccgacc ggatccggac cgtcgtgttc 240gccacggagt cgtcggtcga ccaggcgaag gcggccggga tacacgtcca ctccctgctc 300ggcctcccct cggccacccg ggtggtcgag ctgaagcagg cctgctacgg cggtacggcg 360ggactgcagt tcgccatcgg cctggtgcac cgtgacccgt cgcagcaggt cctggtgatc 420gccagcgacg tgtcgaagta cgcgctgggt gagcccggcg aggcgaccca gggcgccgcg 480gcggtcgcca tgctcgtcgg cgcggacccg gcgctggtac gcgtcgagga cccgtcgggc 540atgttcaccg ccgacgtcat ggacttctgg cggccgaact accgcaccac cgccctggtc 600gacgggcacg agtccatctc cgcctacctg caggcgctgg agggctcgtg gaaggactac 660accgagcgcg gcggtcgcac cctggacgag ttcggcgcgt tctgctacca ccagccgttc 720ccgaggatgg ccgacaaggc gcaccggcac ctgctcaact actgcgggcg cgacgtcgac 780gacgcgctgg tggccggggc catcgggcac accaccgcgt acaacgccga gatcggcaac 840agctacacgg cgtcgatgta tctcgggctc gcggcactgc tcgacaccgc cgacgacctg 900accggccgga ccgtcggctt cctcagctac gggtccggca gcgtcgccga gttcttcgcc 960ggcactgtcg tgcccgggta ccgcgcgcac acgcgacccg accagcaccg cgcggcgatc 1020gaccggcggc aggagatcga ctacgcgacg taccgggagt tgcacgagca cgccttcccg 1080gtcgacggcg gcgactatcc ggcgccggag gtgaccaccg ggccgtaccg gctggccggg 1140ctctccggtc acaagcgcgt ctacgagccg cgatag 117622290PRTMicromonospora sp. strain 046-ECO11 22Val Ala Glu Leu Tyr Ser Thr Ile Glu Glu Ser Ala Arg Gln Leu Asp1 5 10 15Val Pro Cys Ser Arg Asp Arg Val Trp Pro Ile Leu Ser Ala Tyr Gly 20 25 30Asp Ala Phe Ala His Pro Glu Ala Val Val Ala Phe Arg Val Ala Thr 35 40 45Ala Leu Arg His Ala Gly Glu Leu Asp Cys Arg Phe Arg Thr His Pro 50 55 60Asp Asp Arg Asp Pro Tyr Ala Ser Ala Leu Ala Arg Gly Leu Thr Pro65 70 75 80Arg Thr Asp His Pro Val Gly Ala Leu Leu Ser Glu Val His Arg Arg 85 90 95Cys Pro Val Glu Ser His Gly Ile Asp Phe Gly Val Val Gly Gly Phe 100 105 110Lys Lys Ile Tyr Ala Ala Phe Ala Pro Asp Glu Leu Gln Val Ala Thr 115 120 125Ser Leu Ala Gly Ile Pro Ala Met Pro Arg Ser Leu Ala Ala Asn Ala 130 135 140Asp Phe Phe Thr Arg His Gly Leu Asp Asp Arg Val Gly Val Leu Gly145 150 155 160Phe Asp Tyr Pro Ala Arg Thr Val Asn Val Tyr Phe Asn Asp Val Pro 165 170 175Arg Glu Cys Phe Glu Pro Glu Thr Ile Arg Ser Thr Leu Arg Arg Thr 180 185 190Gly Met Ala Glu Pro Ser Glu Gln Met Leu Arg Leu Gly Thr Gly Ala 195 200 205Phe Gly Leu Tyr Val Thr Leu Gly Trp Asp Ser Pro Glu Ile Glu Arg 210 215 220Ile Cys Tyr Ala Ala Ala Thr Thr Asp Leu Thr Thr Leu Pro Val Pro225 230 235 240Val Glu Pro Glu Ile Glu Lys Phe Val Lys Ser Val Pro Tyr Gly Gly 245 250 255Gly Asp Arg Lys Phe Val Tyr Gly Val Ala Leu Thr Pro Lys Gly Glu 260 265 270Tyr Tyr Lys Leu Glu Ser His Tyr Lys Trp Lys Pro Gly Ala Val Asn 275 280 285Phe Ile 29023873DNAMicromonospora sp. strain 046-ECO11 23gtggccgagc tctactcgac catcgaggaa tcggcccggc aactggacgt gccgtgttcg 60cgcgaccggg tctggcccat cctgtccgcg tacggcgacg cgttcgccca tcccgaggcg 120gtggtcgcct tccgggtggc gaccgcgctg cgtcacgcgg gcgagctgga ctgccggttc 180cggacgcatc cggacgaccg ggacccgtac gcctcggcgc tcgcccgggg cctcaccccg 240cgcacggacc accccgtcgg cgcgctgctc tccgaggtcc accggcgctg cccggtggag 300agccacggca tcgacttcgg ggtggtcggc ggcttcaaga agatctacgc ggccttcgcc 360ccggacgagc tgcaggtggc cacgtcgctc gccggcattc cggcgatgcc ccgcagcctc 420gccgcgaacg ccgacttctt cacccggcac ggcctcgacg accgggtcgg cgtgctggga 480ttcgactacc cggcccggac cgtgaacgtc tacttcaacg acgtgccgcg tgagtgcttc 540gagccggaga ccatccggtc gacgctgcgc cggaccggga tggccgagcc gagcgagcag 600atgctccggc tcggcaccgg ggcgttcggg ctctacgtca cgctgggctg ggactccccg 660gagatcgagc ggatctgcta cgccgcggcg accacggacc tgaccacgct tccggtaccc 720gtggaaccgg agatcgagaa gttcgtgaaa agcgttccgt acggcggcgg ggaccggaag 780ttcgtctacg gcgtggcgct gacccccaag ggggagtact acaaactcga gtcgcactac 840aaatggaagc cgggcgcggt gaacttcatt tga 87324370PRTMicromonospora sp. strain 046-ECO11 24Val Trp Ala Arg Val Lys Asn Trp Val Val Ala Leu Ala Val Ala Ala1 5 10 15Val Leu Met Ile Ser Ala Leu Ala Gly Asp His Pro Ala Pro Glu Gly 20 25 30Leu Gly Leu Leu Gly Phe Ala Leu Val Ala Ala Ser Gly Leu Ala Leu 35 40 45Ala Ala Ser Arg Arg Ala Pro Ile Ala Val Leu Val Ala Thr Gly Leu 50 55 60Cys Val Val Gly Tyr Asn Ala Ile Gly Phe Gly Val Pro Ala Ile Ala65 70 75 80Tyr Leu Phe Ala Val Tyr Ala Ala Val Arg Ala Gly His Arg Leu Val 85 90 95Thr Leu Gly Ala Ser Ala Ala Leu Leu Val Val Leu Pro Leu Ala Ile 100 105 110Met Val Ser Pro Ala Asp Gly Ala Leu Lys Glu Ala Leu Ala Gln Ser 115 120 125Arg Gly Val Leu Glu Leu Ala Trp Leu Ile Ala Ala Ala Ala Ala Gly 130 135 140Glu Ala Leu Arg Gln Ala Glu Arg Arg Ala Asp Glu Ala Glu Arg Thr145 150 155 160Arg Glu Glu Thr Ala Arg Leu Arg Ala Thr Gln Glu Arg Leu His Ile 165 170 175Ala Arg Glu Leu His Asp Ser Leu Thr His Gln Ile Ser Ile Ile Lys 180 185 190Val Gln Ala Glu Val Ala Val His Leu Ala Arg Lys Arg Gly Glu Gln 195 200 205Val Pro Glu Ser Leu Leu Ala Ile Gln Glu Ala Gly Arg Ala Ala Thr 210 215 220Arg Glu Leu Arg Ala Thr Leu Glu Thr Leu Arg Asp Leu Thr Lys Ser225 230 235 240Pro Ser His Gly Leu Asp His Leu Pro Glu Leu Leu Ala Gly Ala Glu 245 250 255Lys Ile Gly Leu Ala Thr Thr Leu Thr Ile Glu Gly Asp Gln Arg Asp 260 265 270Val Pro Glu Ala Val Gly Arg Thr Ala Tyr Arg Ile Val Gln Glu Ser 275 280 285Leu Thr Asn Thr Ala Arg His Ala Ser Ala Ala Ala Ala Ala Val Arg 290 295 300Ile Asp Tyr Arg Pro Asp Ala Leu Ser Ile Arg Ile Asp Asp Asp Gly305 310 315 320Thr Ala Arg Pro Gly Ala Ala Pro Val Pro Gly Val Gly Leu Leu Gly 325 330 335Met His Glu Arg Val Leu Ala Leu Gly Gly Arg Leu Arg Ala Glu Pro 340 345 350Arg Thr Gly Gly Gly Phe Thr Val Gln Ala Glu Leu Pro Val Val Arg 355 360 365Val Pro 370251113DNAMicromonospora sp. strain 046-ECO11 25gtgtgggccc gggtgaagaa ctgggtcgtc gcgttggctg tggcggcggt gctgatgatc 60agcgcgctgg ccggtgacca tcctgccccc gagggcctcg gtctgctcgg cttcgcgctg 120gtggcggcga gcggcctggc gctggccgcc agtcgtcggg ccccgatcgc cgtgctggtc 180gccaccgggc tgtgcgtggt gggctacaac gcgatcggct tcggggtgcc cgccatcgcg 240tacctgttcg cggtctacgc ggcggtccgg gccgggcacc ggctcgtcac gctcggggcg 300agcgccgccc tgctcgtcgt cctgccgctg gcgatcatgg tctcgcccgc ggacggcgcc 360ctcaaggagg cgctcgcgca gtcgcggggc gtgctggaac tggcctggct gatcgccgcg 420gcggcggccg gtgaggcgct gcggcaggcc gaacggcgag cggacgaggc ggaacggacc 480cgcgaggaga ccgcccggct gcgcgccacc caggagcggc tgcacatcgc acgggagctg 540cacgactcgc tcacccacca gatctcgatc atcaaggtgc aggcggaggt ggcggtccac 600ctggcccgca agcggggcga gcaggtgccg gagtcgctgc tggcgatcca ggaggccggc 660cgggcggcga ctcgcgagct gcgcgcgacc ctggagacgc tgcgtgacct gaccaagtcc 720ccgtcgcacg ggctcgacca cctcccggag ctgctggccg gggccgagaa gatcggcctg 780gccaccacgc tgaccatcga gggcgaccag cgggacgtgc cggaggcggt gggccgcacc 840gcgtaccgga tcgtgcagga gtcgctcacc aacaccgccc ggcacgcctc cgccgcggcc 900gccgcggtcc ggatcgacta ccgcccggac gcgctgagca tccggatcga cgacgacggg 960acggcccggc cgggcgccgc cccggtgccc ggcgtcgggc tgctggggat gcacgagcgc 1020gtcctcgcgc tgggcggccg gctgcgggcg gaaccccgca ccggcggagg cttcaccgtc 1080caggccgaac tcccggtggt gcgcgtccca tga 111326220PRTMicromonospora sp. strain 046-ECO11 26Met Ile Arg Ile Met Leu Leu Asp Asp Gln Pro Leu Leu Arg Ser Gly1 5 10 15Phe Arg Ala Leu Leu Asp Ala Glu Asp Asp Ile Glu Val Val Ala Glu 20 25 30Gly Gly Asn Gly Arg Glu Gly Leu Ala Leu Ala Arg Gln His Leu Pro 35 40 45Asp Leu Ala Leu Ile Asp Ile Gln Met Pro Val Met Asp Gly Val Glu 50 55 60Thr Thr Arg Gln Ile Val Ala Asp Pro Ala Leu Ala Gly Val Arg Val65 70 75 80Val Ile Leu Thr Asn Tyr Gly Leu Asp Glu Tyr Val Phe His Ala Leu 85 90 95Arg Ala Gly Ala Thr Gly Phe Leu Val Lys Asp Ile Glu Pro Asp Asp 100 105 110Leu Leu His Ala Val Arg Val Ala Ala Arg Gly Asp Ala Leu Leu Ala 115 120 125Pro Ser Ile Thr Arg Met Leu Ile Asn Arg Tyr Val Ser Glu Pro Leu 130 135 140Cys Ala Asp Val Thr Pro Gly Met Glu Glu Leu Thr Asn Arg Glu Arg145 150 155 160Glu Ala Val Ala Leu Ala Ala Arg Gly Leu Ser Asn Asp Glu Ile Ala 165 170 175Asp Arg Met Val Ile Ser Pro Leu Thr Ala Lys Thr His Val Asn Arg 180 185 190Ala Met Thr Lys Leu Gln Ala Arg Asp Arg Ala Gln Leu Val Val Phe 195 200 205Ala Tyr Glu Ser Gly Leu Val Ser Pro Gly Asn Arg 210 215 22027663DNAMicromonospora sp. strain 046-ECO11 27atgatcagga tcatgctgct cgacgaccag ccgctgctgc gcagcgggtt ccgcgcgctc 60ctcgacgccg aggacgacat cgaggtggtg gccgagggcg ggaacggccg ggagggcctg 120gcgctggccc ggcagcacct gcccgatctc gccctgatcg acatccagat gccggtcatg 180gacggcgtcg agacgacccg gcagatcgtc gcggatccgg cgctggccgg ggtacgcgtc 240gtcatcctca ccaactacgg cctcgacgag tacgtcttcc acgcgctgcg cgccggcgcc 300accggcttcc tggtcaagga catcgagccg gacgacctgc tgcacgccgt gcgggtcgcc 360gcgcgcggtg acgcgctgct cgcgccgtcg atcacccgga tgctgatcaa caggtacgtg 420tcggagccgc tctgcgcgga cgtcacgccc ggcatggagg agctgaccaa ccgggaacgc 480gaggcggtcg ccctggccgc ccggggcctg tccaacgacg agatcgccga tcgcatggtg 540atcagcccgc tgaccgcgaa gacccacgtc aaccgcgcca tgaccaagct gcaggcccgc 600gaccgcgccc agctggtggt gttcgcctac gagtccggcc tggtgtcacc cggcaatcgc 660tga 66328131PRTMicromonospora sp. strain 046-ECO11 28Met Phe Ile Arg Arg Leu Leu Thr Ala Ala Ala Ala Gly Val Leu Gly1 5 10 15Gly Leu Ala Leu Val Ala Pro Ala Ala Ala Gln Val Thr Ala Ala Asp 20 25 30Gly Asp Gly Gly Ser Gly Arg Ala Gly Ser Val Leu Ala Leu Ala Leu 35 40 45Ala Leu Leu Gly Leu Val Leu Gly Gly Trp Ala Leu Arg Ser Ala Gly 50 55 60Arg Gly Gly Gly Arg Gly Asn Ala Ile Ala Ala Leu Val Leu Ala Val65 70 75 80Ala Gly Leu Ile Ala Gly Val Val Ala Leu Ala Gly Ser Asp Gly Gly 85 90 95Val Gly Ser Gly Asn Gly Arg Gly Gly Ala Ile Val Ala Val Val Leu 100 105 110Ala Leu Ile Gly Ile Ala Val Gly Gly Leu Ala Phe Thr Arg Ser Arg 115 120 125Arg Ala Ala 13029396DNAMicromonospora sp. strain 046-ECO11 29atgttcatcc gtcgtttgct caccgccgcc gcagccggcg tcctcggtgg gctcgcactc 60gtcgcaccgg cggccgcgca ggtgacggcc gccgacggtg acggtggttc cggccgcgcc 120ggatccgtgc tggcgctcgc gctcgcgttg ctcggcctcg tcctgggcgg gtgggcgttg 180cgctccgcgg ggcgcggcgg cggtcgtggc aacgcgatcg ccgcgctggt gctcgcggtg 240gccggcctga tcgccggcgt ggtcgccctg gccggctccg acggtggtgt cggcagcggc 300aacggccgtg gtggcgccat cgtggccgtc gtgctggcgc tgatcgggat cgccgtcggc 360ggcctggcat tcacccgctc ccggcgcgcc gcctga 39630154PRTMicromonospora sp. strain 046-ECO11 30Met Arg Lys Val Phe Ala Gly Leu Ala Ala Phe Leu Leu Leu Val Leu1 5 10 15Val Val Gln Phe Phe Leu Ala Ala Ser Gly Ala Phe Ser Asn Glu Ala 20 25 30Asn Glu Glu Ala Phe Arg Pro His Arg Ile Leu Gly Leu Gly Ser Ile 35 40

45Leu Val Ala Val Val Leu Thr Val Ala Ala Ala Val Met Arg Met Pro 50 55 60Gly Arg Ile Ile Gly Leu Ser Gly Leu Val Ala Gly Leu Gly Ile Leu65 70 75 80Gln Ala Leu Ile Ala Val Ile Ala Lys Ala Phe Gly Asp Ser Ala Gly 85 90 95Asp Ser Ala Val Gly Arg Tyr Val Phe Gly Leu His Ala Val Asn Gly 100 105 110Leu Val Met Val Ala Val Ala Arg Val Ile Leu Arg Ser Val Arg Ala 115 120 125Ala Pro Asp Thr Thr Thr Thr Pro Gly Val Asp Thr Thr Val Thr Gly 130 135 140Pro Ala Ala Asp Ser Ala Arg Thr Ala Ser145 15031465DNAMicromonospora sp. strain 046-ECO11 31atgcgcaaag tgttcgccgg actggcagcg ttcctgctgc tcgtgctcgt ggtgcagttc 60ttcctggccg ccagcggcgc gttcagcaac gaggccaacg aggaggcgtt ccgccctcac 120cggatcctgg gcctggggag catcctcgtc gccgtggtgc tgacggtggc cgccgcggtg 180atgcggatgc ccggccggat catcggcctg tccggcctgg tcgccgggct gggcatcctg 240caggccctga tcgcggtcat cgccaaggcg ttcggcgact cggccggtga ctcggccgtc 300ggccggtacg tgttcggcct gcacgcggtc aacggactgg tgatggtggc cgtcgcccgc 360gtcatcctgc gcagcgtccg ggcggcgccg gacacgacca ccacgcccgg cgtggacacg 420acggtcaccg gtccggcggc cgactcggcg cgaacggcgt catga 46532661PRTMicromonospora sp. strain 046-ECO11 32Met Ser Thr Leu Gln Trp Ile Leu Val Asp His Val Val Ala Leu Leu1 5 10 15Gly Val Ala Thr Trp Phe Ala Thr Gly Val Thr Ala Ala Leu Gly Arg 20 25 30His Arg Ile Ala Leu Ala Leu Leu Gly Ala Ala Val Leu Val Thr Val 35 40 45Ala Arg Leu Gly Thr Val Ala Leu Leu Ala Asp Arg Gly Trp Trp Phe 50 55 60Val Gln Glu Lys Val Leu Leu Gly Leu Pro Met Leu Gly Ala Ala Gly65 70 75 80Leu Val Ala Val Leu Leu Ala Gly Pro Arg Leu Leu Ala Ala Arg Gln 85 90 95Ser Pro Ala Ala Asp Leu Pro Ala Gly Ala Leu Val Ala Val Leu Thr 100 105 110Ala Gly Phe Ala Ala Leu Ala Gly Leu Val Val Thr Phe Thr Ala Gly 115 120 125Tyr Pro Leu Thr Trp Ser Thr Ala Leu Ile Ala Val Ala Leu Val Cys 130 135 140Ala Ala Ala Leu Leu Thr Ala Arg Val Val Gly Arg Pro Ala Ala Pro145 150 155 160Ala Ala Glu Ala Gly Ser Pro Glu His Thr Pro Ala Ala Ala Gly Pro 165 170 175Thr Ala Leu Ser Arg Arg Arg Phe Leu Gly Val Ala Gly Gly Val Val 180 185 190Ala Ala Gly Ala Gly Ala Thr Gly Val Gly Leu Leu Phe Arg Asp Pro 195 200 205Glu Ala Met Val Thr Gly Gly Gly Pro Gly His Ala Gly Gly Ala Arg 210 215 220Pro Lys Val Ser Val Ala Asp Leu Arg Gly Pro Gly Ala Pro Ala Ala225 230 235 240Gly Gly Thr Ala Arg Arg His Val Leu Thr Ala Arg Thr Gly Thr Val 245 250 255Thr Ile Pro Ser Gly Arg Pro Ile Asp Ala Trp Ser Tyr Glu Gly Arg 260 265 270Leu Pro Gly Pro Ala Ile Thr Ala Thr Glu Gly Asp Leu Ile Glu Val 275 280 285Thr Leu Arg Asn Ala Asp Ile Glu Asp Gly Val Thr Val His Trp His 290 295 300Gly Tyr Asp Val Pro Cys Gly Glu Asp Gly Ala Pro Gly Ala Thr Gln305 310 315 320His Ala Val Gln Pro Gly Gly Glu Phe Val Tyr Arg Phe Gln Ala Asp 325 330 335Gln Val Gly Thr Tyr Trp Tyr His Thr His Gln Ala Ser His Pro Ala 340 345 350Val Arg Lys Gly Leu Tyr Gly Thr Leu Val Val Thr Pro Arg Glu Asp 355 360 365Arg Pro Glu Ala Glu Arg Gly Leu Asp Leu Thr Leu Pro Val His Thr 370 375 380Phe Asp Asp Val Thr Ile Leu Gly Asp Gln Glu Gly Arg Ala Val His385 390 395 400Asp Val Arg Pro Gly Gln Pro Val Arg Leu Arg Leu Ile Asn Thr Asp 405 410 415Ser Asn Pro His Trp Phe Ala Val Val Gly Ser Pro Phe Arg Val Val 420 425 430Ala Val Asp Gly Arg Asp Leu Asn Gln Pro Gly Glu Val Arg Glu Val 435 440 445Gly Leu Arg Leu Pro Ala Gly Gly Arg Tyr Asp Leu Thr Leu Ala Met 450 455 460Pro Asp Ala Lys Val Thr Leu Leu Leu Asp Asn Asp Ser Asp Gln Gly465 470 475 480Val Leu Leu Arg Pro Pro Gly Val Gly Gly Gly Asp Arg Pro Leu Pro 485 490 495Asp Thr Ala Asp Trp Pro Glu Phe Asp Leu Leu Gly Tyr Gly Glu Pro 500 505 510Ala Pro Val Pro Phe Asp Ala Asp Asp Ala Asp Arg His Phe Thr Ile 515 520 525Val Leu Asp Arg Ala Leu Ala Met Val Asp Gly Lys Pro Ala Tyr Ala 530 535 540Gln Thr Val Asp Gly Arg Ala His Pro Ser Val Pro Asp Gln Leu Val545 550 555 560Arg Glu Gly Asp Val Val Arg Phe Thr Val Val Asn Arg Ser Leu Glu 565 570 575Thr His Pro Trp His Leu His Gly His Pro Val Leu Ile Leu Ser Arg 580 585 590Asp Gly Arg Pro Tyr Ser Gly Ser Pro Leu Trp Met Asp Thr Phe Asp 595 600 605Val Arg Pro Gly Glu Val Trp Glu Val Ala Phe Arg Ala Asp Asn Pro 610 615 620Gly Val Trp Met Asn His Cys His Asn Leu Pro His Gln Glu Gln Gly625 630 635 640Met Met Leu Arg Leu Val Tyr Asp Gly Val Thr Thr Pro Phe Ala Ser 645 650 655Thr Ser His Ala His 660331986DNAMicromonospora sp. strain 046-ECO11 33atgagcacgc tccaatggat cctcgtggac cacgtcgtgg cgctgctcgg tgtcgcgacg 60tggttcgcaa cgggtgtcac ggcagctctc ggccgccacc ggatcgcgtt ggcgctcctc 120ggcgccgcgg tgctggtgac agtcgcccgc ctgggcaccg tggcgctgct ggccgaccgc 180ggctggtggt tcgtccagga gaaggttctg ctggggctgc cgatgctcgg cgccgcgggg 240ctcgtcgcgg tgctcctggc cggcccgcgc ctgctcgcgg cccggcagtc accggcggcg 300gacctgccgg ccggcgcgct ggtcgcggtg ctgaccgccg gcttcgccgc gctggccggc 360ctggtggtga cgttcaccgc cgggtacccg ctgacgtgga gcaccgcgct gatcgccgtc 420gccctcgtct gcgccgccgc gctgctcacc gcgcgggtgg tcggacgacc cgccgccccg 480gccgcggagg ccggctcccc ggagcacacg ccggcggcgg ccgggcccac ggcgctgtcc 540cgccgccggt tcctcggcgt ggccggggga gtggtcgcgg cgggcgccgg cgccaccggc 600gtcggcctgc tcttccgcga cccggaggcg atggtcaccg gaggcggccc cggacacgcc 660ggtggcgccc gccccaaggt ctccgtggcg gacctgcgcg gccccggcgc tccggcggcg 720ggcggcacgg cgcgacgcca cgtgctcacc gcccggacgg gcaccgtcac gattccgtcc 780ggacgtccga tcgacgcctg gagctacgag ggccgcctgc ccgggccggc catcaccgcg 840accgagggcg acctgatcga ggtgacgctc cgcaacgccg acatcgagga cggcgtcacc 900gtgcactggc acgggtacga cgtgccgtgc ggcgaggacg gcgcgccggg cgccacgcag 960cacgcggtgc agcccggcgg cgagttcgtc taccggttcc aggcggacca ggtggggacg 1020tactggtacc acacccacca ggcgtcgcac cccgccgtgc gcaaagggct gtacgggacg 1080ctcgtcgtga cgccgcgcga ggaccggccg gaagcggagc gcgggctgga cctgacgctg 1140ccggtgcaca cgttcgacga cgtcacgatc ctcggcgacc aggagggacg cgccgtccac 1200gacgtccgcc ccggccagcc ggtgcgactg cgtctgatca acaccgactc caacccgcac 1260tggttcgccg tcgtcggctc gcccttccgc gtggtggccg tcgacggccg cgacctcaac 1320cagccgggcg aggtacgcga ggtcgggctc cgcctgcccg ccggaggccg gtacgacctg 1380accctggcca tgccggacgc caaggtcacg ctgctgctcg acaacgactc cgaccagggc 1440gtcctgctgc gcccgccggg cgtcggcggt ggtgaccgcc cgctgccgga caccgccgac 1500tggcccgagt tcgacctgct gggctacggc gagccggcgc ccgtgccgtt cgacgccgac 1560gacgccgacc gccacttcac catcgtcctc gaccgggccc tggccatggt cgacggcaag 1620cccgcgtacg cccagaccgt cgacggtcgc gcacatccct ccgtccccga ccagctcgtc 1680cgggaggggg acgtcgtgcg cttcacggtg gtcaaccgga gcctcgaaac ccacccgtgg 1740cacctgcacg gccatccggt gctgatcctg tcccgcgacg gccggccgta ctccggcagc 1800ccgctgtgga tggacacctt cgacgtgcgg ccgggagagg tgtgggaggt ggcgttccgg 1860gcggacaatc cgggtgtctg gatgaaccac tgccacaacc tgccgcacca ggagcagggc 1920atgatgctgc ggctcgtcta cgacggtgtc accacgccct tcgccagcac gagccacgca 1980cactga 198634129PRTMicromonospora sp. strain 046-ECO11 34Met Thr Ala Asp Leu His Gly Leu Ala Ser Val Arg Tyr Ile Val Asp1 5 10 15Asp Val Ser Ala Ala Ile Glu Phe Tyr Thr Thr His Leu Gly Phe Thr 20 25 30 Val Ser Thr Ala Phe Pro Pro Ala Phe Ala Asp Val Val Arg Gly Pro 35 40 45Leu Arg Leu Leu Leu Ser Gly Pro Thr Ser Ser Gly Ala Arg Val Thr 50 55 60Pro Ala Asp Ala Ala Gly Cys Gly Arg Asn Arg Ile His Leu Ile Val65 70 75 80Asp Asp Leu Asp Ala Glu Arg Glu Arg Leu Glu Arg Ala Gly Val Thr 85 90 95Leu Arg Ser Asp Val Val Ala Gly Pro Gly Gly Arg Gln Phe Leu Ile 100 105 110Ala Asp Pro Ala Gly Asn Leu Val Glu Val Phe Glu Pro Ala Ala Arg 115 120 125Gly35390DNAMicromonospora sp. strain 046-ECO11 35atgaccgcag acctgcacgg cctggccagc gtccgctaca tcgtcgacga cgtgtcggcg 60gcgatcgagt tctacaccac ccacctgggt ttcacggtgt cgaccgcgtt cccgccggcc 120ttcgccgacg tggtgcgcgg gccgctgcgg ctcctgctgt ccgggccgac cagctcgggc 180gcccgggtca ccccggcgga cgcggccggg tgcgggcgca accgcatcca cctgatcgtc 240gacgatctcg acgccgaacg ggagcggctg gagcgcgccg gggtgacgtt gcgcagcgac 300gtcgtggccg ggccgggcgg ccgtcagttc ctgatcgccg acccggcggg caacctggtc 360gaggtgttcg agccggcagc ccgcggctga 39036178PRTMicromonospora sp. strain 046-ECO11 36Met Leu Thr Ala Val Val Ala Ser Pro His Ser Pro Glu Asn Thr Ser1 5 10 15Arg His Pro Thr Gly Gly Asp Ala Val Asp Glu Ala Thr Pro Arg Thr 20 25 30Pro Val Ala Ala Arg Pro Thr Trp Ser Pro Ala Thr Ala Pro Val Trp 35 40 45Leu Val Gly Val Leu Ala Thr Leu Ala Gly Ala Val Ala Ala Glu Ala 50 55 60Phe Thr Leu Ala Ala Arg Gly Phe Gly Val Pro Met Glu Ala Ala Gly65 70 75 80Val Trp Glu Glu Gln Ala Gln Ala Ile Pro Val Gly Ala Ile Ala Arg 85 90 95Ser Val Val Leu Trp Ser Ile Gly Gly Ile Val Leu Ala Val Val Val 100 105 110Ala Arg Arg Ala Arg Arg Pro Val Arg Ala Phe Val Ala Gly Thr Val 115 120 125Ala Phe Thr Val Leu Ser Leu Ala Ala Pro Ala Phe Ala Arg Asp Thr 130 135 140Pro Val Ser Thr Gln Leu Val Leu Ala Gly Thr His Val Ile Ala Gly145 150 155 160Ala Val Ile Ile Ser Ile Leu Ala Ala Arg Leu Ala Ala Pro Thr Pro 165 170 175Pro Arg37537DNAMicromonospora sp. strain 046-ECO11 37atgttgactg ccgtcgtggc gtccccgcat tctcccgaga acacatcgag gcacccgacc 60ggaggcgacg ccgtggatga ggccactccc cgaactcccg tcgcggcacg gcccacctgg 120tcgccggcca ccgctccggt gtggctggtc ggcgtgctgg ccaccctcgc cggggccgtg 180gccgcggagg cgttcacgct cgccgcccgg ggcttcggcg taccgatgga ggcggccggc 240gtctgggagg agcaggcgca ggcgatcccg gtgggggcca tcgcccgcag cgtcgtgctc 300tggtcgatcg gcggaatcgt cctggcggtg gtcgtggcgc ggcgggcccg gcggcccgtg 360cgtgccttcg tggccggcac cgtcgcgttc accgtgctgt ccctcgccgc gcccgccttc 420gcccgggaca ccccggtgtc gacgcagctc gtcctcgccg gcacccacgt gatcgccggc 480gccgtgatca tctccatcct ggccgcgcgg ctcgccgcgc ccaccccgcc ccggtaa 53738661PRTMicromonospora sp. strain 046-ECO11 38Met Asp Gly Thr Glu Ser Asn Val Thr Gly Phe Pro Asp Leu Leu Ser1 5 10 15Gly Leu Gly Gly Asp Gly Arg Ala Phe Ala Leu Leu His Arg Pro Gly 20 25 30Ala Ala Gly Cys Ala Tyr Val Glu Val Leu Thr Gly Glu Val Cys Asp 35 40 45Val Asp Thr Leu Gly Glu Leu Pro Leu Pro Thr Glu Pro Ala Thr Gly 50 55 60Ala Arg His Asp Leu Leu Val Ala Val Pro Tyr Arg Gln Val Thr Glu65 70 75 80Arg Gly Phe Asp Cys His Asp Asp Gly Ala Pro Leu Leu Ala Met Arg 85 90 95Val His Glu Gln Phe Gly Leu Asp Arg Gly Gln Ala Leu Ala Gly Leu 100 105 110Pro Glu Arg Gly Val Pro Val Thr Asp Ala Asp Phe Asp Leu Ser Asp 115 120 125Glu Asp Tyr Ala Ala Ile Val Lys Arg Val Val Gly Asp Glu Ile Gly 130 135 140Leu Gly Ala Gly Ser Asn Phe Val Ile Arg Arg Thr Phe Thr Ala Arg145 150 155 160Leu Ala Asp Tyr Ser Ile Ala Thr Glu Leu Ala Leu Phe Arg Arg Leu 165 170 175Leu Thr Gly Glu Leu Gly Ser Tyr Trp Thr Phe Leu Phe His Ser Gly 180 185 190Ala Gly Thr Phe Ile Gly Ala Ser Pro Glu Arg His Val Ser Met Ile 195 200 205Asp Gly Thr Val Ser Met Asn Pro Ile Ser Gly Thr Tyr Arg His Pro 210 215 220Pro Asn Gly Pro Ala Val Ser Gly Leu Leu Glu Phe Leu Asn Asp Pro225 230 235 240Lys Glu Ala Asn Glu Leu Tyr Met Val Val Asp Glu Glu Leu Lys Met 245 250 255Met Ala Arg Met Cys Ala Ser Gly Gly Gln Val His Gly Pro Phe Leu 260 265 270Lys Glu Met Ala Arg Val Thr His Ser Glu Tyr Ile Leu Thr Gly Arg 275 280 285Ser Asp Leu Asp Val Arg Asp Val Leu Arg Glu Thr Leu Leu Ala Pro 290 295 300Thr Val Thr Gly Ser Pro Ile Glu Asn Ala Phe Arg Val Ile Thr Arg305 310 315 320His Glu Thr Thr Gly Arg Gly Tyr Tyr Gly Gly Val Leu Ala Leu Met 325 330 335Gly Arg Asp Ser Ala Gly Ser Arg Thr Leu Asp Ser Ala Ile Met Ile 340 345 350Arg Thr Ala Glu Ile Asp Asp Ala Gly Thr Leu Arg Leu Gly Val Gly 355 360 365Ala Thr Leu Val Arg Asp Ser Lys Pro Glu Ser Glu Val Ala Glu Thr 370 375 380Arg Ala Lys Ala Gly Ala Met Arg Ala Ala Leu Gly Leu Gly Val Asp385 390 395 400Pro Asp Gly Pro Asp Gly Gly Arg Thr Thr Ala Ala Arg Ala Arg Ser 405 410 415Ser Leu Ala Thr Asp Pro Arg Val Arg Arg Ala Leu Arg Glu Arg Asn 420 425 430Thr Thr Leu Ser Arg Phe Trp Leu Asp Gly Ala Glu Arg Arg Thr Pro 435 440 445Asn Pro Ala Leu Thr Gly Arg Arg Val Leu Val Val Asp Asn Glu Asp 450 455 460Thr Phe Met Ala Met Leu Asp His Gln Leu Arg Ala Leu Gly Leu Arg465 470 475 480Ser Ser Ile Ala Arg Phe Asp Ser Arg Leu Arg Pro Asp Gly His Asp 485 490 495Leu Val Val Val Gly Pro Gly Pro Gly Asp Pro Gly Asp Leu Thr Asp 500 505 510Pro Arg Met Arg Thr Leu Arg Gly Leu Thr Arg Asp Leu Leu Ala Gly 515 520 525Thr Val Pro Phe Leu Ser Ile Cys Leu Gly His Gln Val Leu Ala Ala 530 535 540Glu Leu Gly Phe Pro Leu Ala Arg Arg Ala Val Pro Asn Gln Gly Val545 550 555 560Gln Lys Arg Ile Asp Leu Phe Gly Arg Pro Glu Leu Val Gly Phe Tyr 565 570 575Asn Thr Tyr Thr Ala Arg Ser Ala His Asp Val Val Ala Gly Gly Arg 580 585 590Arg Gly Pro Ile Glu Ile Ser Arg Ser Pro Asp Ser Gly Asp Val His 595 600 605Ala Leu Arg Gly Pro Gly Phe Arg Ser Val Gln Phe His Leu Glu Ser 610 615 620Val Leu Thr Gln His Gly Pro Arg Ile Leu Gly Asp Leu Leu Val Ser625 630 635 640Leu Leu Ala Asp Gly Thr Ala Ala Ala Ala Ala Glu Ala Ala Gly Arg 645 650 655Arg Gly Asn Arg Pro 660391986DNAMicromonospora sp. strain 046-ECO11 39atggacggga cggaatcgaa cgtgaccgga ttccccgatc tgctgtccgg tctcggcggc 60gacgggcgcg ccttcgccct gctgcaccgg cccggcgcgg ccgggtgcgc gtacgtggag 120gttctgaccg gcgaggtgtg cgacgtggac actctcggcg agctgcccct gcccaccgag 180ccggcgaccg gcgcgcggca cgacctgctc gtggcggtgc cgtaccggca ggtcaccgaa 240cgggggttcg actgccacga cgacggcgcg ccgctgctcg cgatgcgcgt ccacgagcag 300ttcgggctcg accgcggaca ggcgctggcg ggcctgcccg aacgcggtgt gccggtgacc 360gacgccgact tcgacctcag cgacgaggac tacgccgcga tcgtcaagcg ggtggtgggt 420gacgagatcg ggctgggcgc cggatccaac ttcgtcatcc ggcgcacctt caccgcgcgg 480ctggccgact actcgatcgc cacggaactg gcgctcttcc gccggttgct gaccggcgaa 540ctgggttcct actggacgtt tctgttccac tccggcgccg gcacgttcat cggcgcgtca 600ccggaacgac acgtcagcat gatcgacgga accgtctcga tgaatcccat cagcgggacc 660taccggcacc ccccgaacgg

cccggccgtt tccggtctgc tggaattcct gaacgacccg 720aaagaggcta acgaactcta catggtcgtc gacgaggaac tgaaaatgat ggcgcggatg 780tgcgcctccg gcggccaggt gcacggcccg ttcctcaagg aaatggcgcg ggtgacgcac 840tccgagtaca tcctgaccgg ccgcagcgac ctggacgtgc gcgacgtgct gcgggagacc 900ctgctcgcgc cgacggtcac cggcagcccg atcgagaacg cgttccgggt catcacccgc 960cacgagacga ccggccgcgg ctactacggc ggcgtgctcg cgttgatggg ccgtgactcg 1020gccggcagcc gtacgctcga ctcggccatc atgatccgca ccgccgagat cgacgacgcg 1080ggcacgctgc gcctgggcgt cggcgccacc ctcgtgcggg actccaagcc ggagtcggag 1140gtggccgaga cgcgggccaa ggcgggcgcc atgcgcgcgg cgctcggcct cggcgtcgac 1200ccggacggcc cggacggcgg gcggaccacg gccgcgcggg ctcgttcgtc cctggccacc 1260gacccccggg tacggcgggc gttgcgcgag cgcaacacca cactgtcgag gttctggctc 1320gacggcgcgg agcggcgcac cccgaacccg gcgctgaccg gacgccgcgt gctcgtcgtc 1380gacaacgagg acacgttcat ggccatgctc gaccaccagt tgcgggccct cgggctgcgg 1440tcgagcatcg cccggttcga cagccggctg cggccggacg gacacgacct cgtcgtcgtc 1500ggtcccggcc ccggcgaccc gggcgacctg accgacccgc gtatgcggac cctgcgcggg 1560ctcacccgcg acctgctcgc cggaacggtg ccgttcctgt ccatctgcct gggccaccag 1620gtgctcgccg ccgaactggg gttccccctc gcccggcgcg cggtgcccaa ccagggtgtg 1680cagaagcgga tcgacctgtt cggccggccg gaactcgtgg ggttctacaa cacctacacc 1740gcccgctccg cgcacgacgt ggtggccggt ggccggcggg gcccgatcga gatcagccgc 1800agcccggaca gcggggacgt gcacgcgctg cgcggcccgg gattccgttc cgtccagttc 1860cacctggagt ccgtcctcac ccagcacggc ccacggatcc tgggcgacct gctggtctcc 1920ctgctcgccg acggcacggc cgccgccgcg gccgaggcgg cgggccggcg cgggaaccgc 1980ccgtga 198640427PRTMicromonospora sp. strain 046-ECO11 40Val Lys Thr Thr Val Asp Val Leu Val Gln Lys Tyr Gly Gly Thr Ser1 5 10 15Leu Gln Thr Leu Asp Arg Val Arg His Ala Ala Leu Arg Ile Ala Glu 20 25 30Ala Arg Arg His Gly Ser Ala Val Thr Val Val Val Ser Ala Arg Gly 35 40 45Ser Arg Thr Asp Asp Leu Leu Arg Leu Ala Ala Asp Val Gly Ala Ala 50 55 60Gly Pro Ser Arg Glu Leu Asp Gln Leu Leu Ala Val Gly Glu Ser Glu65 70 75 80Ser Ala Ala Leu Met Ala Leu Ala Leu Thr Gly Leu Gly Val Pro Ala 85 90 95Val Ser Leu Thr Gly His Gln Ala Glu Ile His Thr Thr Asp Arg His 100 105 110Gly Asp Ala Leu Ile Ser Arg Ile Gly Ala Ala Arg Val Glu Ala Ala 115 120 125Leu Gly Arg Gly Glu Val Ala Val Val Thr Gly Phe Gln Gly Ile Asp 130 135 140Arg Ala Gly Asp Val Ala Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr145 150 155 160Ala Val Ala Leu Ala Ala Arg Leu Arg Ala Ser Ala Cys Glu Ile Tyr 165 170 175Thr Asp Val Asp Gly Val Phe Ser Ala Asp Pro Arg Ile Leu Pro Ala 180 185 190Ala Arg Cys Leu Pro Trp Val Glu Pro Gly Val Met Ala Glu Met Ala 195 200 205Phe Ala Gly Ala Arg Val Leu His Thr Arg Cys Ile Glu Leu Ala Ala 210 215 220Met Glu Gly Val Glu Val Arg Val Arg Asn Ala Ser Ser Gln Ala Pro225 230 235 240Gly Thr Ile Val Val Asp Arg Pro Asp Asp Arg Pro Leu Glu Thr Arg 245 250 255Arg Ala Val Val Ala Val Thr His Asp Thr Asp Val Val Arg Val Leu 260 265 270Val His Cys Arg Asp Gly Arg Arg Asp Met Ala Pro Asp Val Phe Glu 275 280 285Val Leu Ala Ala His Gly Ala Val Ala Asp Leu Val Ala Arg Ser Gly 290 295 300Pro Tyr Glu Ser Glu Phe Arg Met Gly Phe Thr Ile Arg Arg Ser Gln305 310 315 320Ala Glu Ala Val Arg Thr Ala Leu His Asp Leu Thr Ala Ser Phe Asp 325 330 335Gly Gly Val His Phe Asp Glu Asn Val Gly Lys Val Ser Val Val Gly 340 345 350Met Gly Leu Leu Ser Arg Pro Glu His Thr Ala Arg Leu Met Ala Ala 355 360 365Leu Ala Ala Ala Gly Ile Ser Thr Ser Trp Ile Ser Thr Ser Gln Met 370 375 380Arg Leu Ser Val Ile Val Ser Arg Asp Arg Thr Val Asp Ala Val Glu385 390 395 400Ala Leu His Arg Ala Phe Arg Leu Asp Arg Ser Glu Pro Ala Asp Ala 405 410 415Thr Ser Leu Thr Ser Arg Arg Ser Ala Thr Ala 420 425411284DNAMicromonospora sp. strain 046-ECO11 41gtgaagacga ctgtggacgt gctggtccag aaatacgggg gcacctcgct gcagaccctc 60gaccgcgttc ggcacgccgc gctgcggatc gccgaggcgc ggcggcacgg ctccgccgtg 120acagtggtcg tgtcggcgcg cggcagccgg accgacgacc tgctgcggct ggcggccgac 180gtcggcgccg cgggtccgtc ccgggaactc gaccagttgc tcgcagtcgg cgagtccgag 240tcggcggcgc tgatggcgct ggcgttgacc gggctgggag tgccggccgt ctcgctgacc 300gggcaccagg cggagatcca caccaccgac cggcacggcg acgcgctgat ctcgcggatc 360ggggcggcgc gggtggaagc ggcgctgggc cgtggcgagg tcgccgtggt caccggattc 420cagggcatcg accgggccgg tgacgtcgcc acgctggggc gcggcggctc cgacacgaca 480gcggtggcgc tcgcggcccg gctccgcgcg tcggcgtgcg agatctacac cgacgtggac 540ggcgtcttca gcgccgaccc ccgcatcctt ccggcggcgc gttgcctgcc gtgggtggag 600cccggcgtca tggcggagat ggcgttcgcc ggcgcgcggg tcctgcacac ccgatgcatc 660gagctggccg ccatggaagg ggtcgaagtg cgcgtgcgca acgcgtcgtc gcaggcgccc 720ggaacgatag tcgtggaccg gcccgacgac cggccgctgg agacccggcg ggccgtggtg 780gcggtcaccc acgacaccga tgtcgtccgc gtgctggtgc actgccgcga cggccgccgg 840gacatggcac ccgacgtgtt cgaggtgctg gccgcccatg gggcggtggc ggacctggtg 900gcccggtccg ggccctacga gagcgagttc cggatggggt tcaccatccg ccgcagccag 960gccgaagcgg tgcggaccgc gctgcacgac ctcaccgcgt ccttcgacgg cggggtccac 1020ttcgacgaga acgtcggcaa ggtgtccgtg gtcggcatgg gcctgctcag ccgccccgag 1080cacacggccc ggctgatggc ggcgctggcc gcggcgggga tctcgacgag ctggatctcc 1140acctcccaga tgcggctgtc ggtgatcgtg tcgcgggacc gcaccgtcga cgccgtcgaa 1200gccctgcacc gcgcgttccg cctggaccgg tccgagccgg cggacgccac gtccctgacc 1260tcccgccgtt ccgccaccgc ctga 128442274PRTMicromonospora sp. strain 046-ECO11 42Val Ala Val Leu Asn Ala Ser Phe Ala Arg Gly Leu Arg Leu Arg Arg1 5 10 15Leu Phe Arg Arg Gly Asp Gly Arg Leu Leu Val Val Pro Leu Asp His 20 25 30Ser Val Thr Asp Gly Pro Leu Arg Arg Gly Asp Leu Asn Ser Leu Leu 35 40 45Gly Glu Leu Ala Gly Thr Gly Val Asp Ala Val Val Leu His Lys Gly 50 55 60Ser Leu Arg His Val Asp His Gly Trp Phe Gly Asp Met Ser Leu Ile65 70 75 80Val His Leu Ser Val Ser Thr Arg His Ala Pro Asp Pro Asp Ala Lys 85 90 95Tyr Leu Val Ala His Val Glu Glu Ala Leu Arg Leu Gly Ala Asp Ala 100 105 110Val Ser Val His Val Asn Leu Gly Ser Pro Gln Glu Ala Arg Gln Ile 115 120 125Ala Asp Leu Ala Ala Val Ala Gly Glu Cys Asp Arg Trp Asn Val Pro 130 135 140Leu Leu Ala Met Val Tyr Ala Arg Gly Pro Gln Ile Thr Asp Ser Arg145 150 155 160Ala Pro Glu Leu Val Ala His Ala Ala Thr Leu Ala Ala Asp Leu Gly 165 170 175Ala Asp Ile Val Lys Thr Asp Tyr Val Gly Thr Pro Glu Gln Met Ala 180 185 190Glu Val Val Arg Gly Cys Pro Ile Pro Leu Ile Val Ala Gly Gly Pro 195 200 205Arg Ser Ala Asp Thr Pro Thr Val Leu Ala Tyr Val Ser Asp Ala Leu 210 215 220Arg Gly Gly Val Ala Gly Met Ala Met Gly Arg Asn Val Phe Gln Ala225 230 235 240Glu Gln Pro Gly Leu Met Ala Ala Ala Val Ala Arg Leu Val His Glu 245 250 255Pro Arg His Val Pro Asp Arg Tyr Asp Val Asp Asp Arg Leu Ala Leu 260 265 270Thr Ser 43825DNAMicromonospora sp. strain 046-ECO11 43gtggccgtac tcaacgcttc gttcgctcgt ggcctgcgtc tgcgccgact gttccgacgc 60ggcgacggac gcctgctcgt cgtcccgctc gaccactccg tcaccgacgg gccgctgcgc 120cgcggcgacc tgaactcgct gctcggtgag ctcgccggca ccggcgtgga cgccgtggtg 180ctgcacaagg gcagcctgcg gcacgtcgac cacggctggt tcggcgacat gtcgctgatc 240gtgcatctga gcgtgagcac ccggcacgcc ccggacccgg acgcgaagta cctggtcgcg 300cacgtggagg aggcgctgcg gctgggcgcc gacgcggtca gcgtgcacgt caacctcggc 360tcaccgcagg aggcgcggca gatcgccgac ctggcggcgg tggcggggga gtgcgaccgc 420tggaacgtcc cgctgctggc catggtgtac gcccgcgggc cgcagatcac cgactcccgg 480gcaccggagc tggtggcgca cgccgcgacg ctcgccgcgg acctcggcgc cgacatcgtc 540aagaccgact acgtgggcac gcccgagcag atggccgagg tggtgcgcgg ctgcccgatc 600ccgctgatcg tggccggcgg cccgcgctcg gccgacactc cgacggtgct cgcctacgtc 660tcggacgcgc tgcgcggcgg cgtggccggg atggccatgg gccgcaacgt gttccaggcc 720gagcagcccg gcctgatggc cgccgccgtg gcacggctgg tgcacgagcc acggcacgtg 780ccggaccggt acgacgtcga cgaccggctc gcccttacgt cctga 82544367PRTMicromonospora sp. strain 046-ECO11 44Val Lys Leu Cys Trp Leu Asp Ile Arg Asn Val Asn Gly Ala Lys Glu1 5 10 15Ala Ile Val Glu Glu Ala Val His Gln Arg Val Asp Ala Val Val Ala 20 25 30Ala Asp Pro Ala Asp Leu Glu Thr Leu Pro Pro Thr Val Lys Lys Val 35 40 45Leu Phe Pro Gln Gly Gly Pro Leu Pro Glu Lys Leu Glu Pro Ala Asp 50 55 60Leu Val Ile Val Glu Pro Ala Arg His Gly Glu Pro Ala Glu Leu Ala65 70 75 80Ala Arg Tyr Pro Glu Val Glu Phe Gly Arg Phe Val Glu Ile Val Asp 85 90 95Ala Asp Ser Leu Glu Asp Ala Cys Arg Ser Ala Arg His Asp Arg Trp 100 105 110Ser Leu Leu Tyr Phe Arg Asp Pro Thr Lys Ile Pro Leu Glu Ile Val 115 120 125Leu Ala Ala Ala Ala Gly Ala Glu Gly Ser Ile Ile Thr Gln Val Ala 130 135 140Asp Val Glu Glu Ala Glu Ile Val Phe Gly Val Leu Glu His Gly Ser145 150 155 160Asp Gly Val Met Leu Ala Pro Arg Ala Val Gly Glu Ala Thr Glu Leu 165 170 175Arg Thr Ala Ala Val Ser Thr Ala Ala Asp Leu Ser Leu Val Glu Leu 180 185 190Glu Val Thr Gly Ile Arg Arg Val Gly Met Gly Glu Arg Ala Cys Val 195 200 205Asp Thr Cys Thr Asn Phe Arg Leu Asp Glu Gly Ile Leu Val Gly Ser 210 215 220His Ser Thr Gly Met Ile Leu Cys Cys Ser Glu Thr His Pro Leu Pro225 230 235 240Tyr Met Pro Thr Arg Pro Phe Arg Val Asn Ala Gly Ala Leu His Ser 245 250 255Tyr Thr Leu Ser Ala Gly Gly Arg Thr Asn Tyr Leu Ser Glu Leu Val 260 265 270Ser Gly Gly Arg Val Leu Ala Val Asp Ser Gln Gly Lys Ser Arg Val 275 280 285Val Thr Val Gly Arg Val Lys Ile Glu Thr Arg Pro Leu Leu Ala Ile 290 295 300Asp Ala Val Ser Pro Ser Gly Thr Arg Val Asn Leu Ile Val Gln Asp305 310 315 320Asp Trp His Val Arg Val Leu Gly Pro Gly Gly Thr Val Leu Asn Val 325 330 335Thr Glu Leu Thr Ala Gly Thr Lys Val Leu Gly Tyr Leu Pro Val Glu 340 345 350Lys Arg His Val Gly Tyr Pro Ile Asp Glu Phe Cys Ile Glu Lys 355 360 365451104DNAMicromonospora sp. strain 046-ECO11 45gtgaagctgt gctggctgga catccgtaac gtcaacggcg ccaaggaggc aatcgtcgag 60gaggcggtcc accagcgggt ggacgccgtc gtggcggccg atccggccga cctggagacg 120cttcccccga cggtgaagaa ggtgctgttc ccgcagggcg ggccgctgcc ggagaagctg 180gaaccggccg acctggtgat cgtcgagccg gcccggcacg gcgagcccgc cgagctggcg 240gcccggtacc cggaggtgga gttcggccgg ttcgtcgaga tcgtcgacgc ggacagcctg 300gaggacgcct gccggtccgc gcgccacgac cggtggagcc tgctgtactt ccgcgacccc 360accaagatcc cgctggagat cgtgctggcg gccgcggcgg gcgcggaggg cagcatcatc 420acccaggtcg ccgacgtcga ggaggcggag atcgtcttcg gcgtcctgga gcacggctcg 480gacggagtga tgctggcgcc ccgcgccgtg ggggaggcca ccgagctgcg gaccgccgcg 540gtgagcacgg cggcggacct gtcgctcgtg gagctggagg tcaccggcat ccggcgggtg 600ggcatgggcg agcgcgcctg cgtcgacacg tgcacgaact tccgtctgga cgagggcatc 660ctggtcggct cgcactccac cggcatgatc ctgtgctgca gcgagacgca tccgctgccg 720tacatgccga cccggccgtt ccgggtcaac gccggcgcgc tgcactcgta cacgctctcc 780gccggcgggc ggaccaacta cctcagcgag ctggtctccg gcggccgggt gctcgccgtg 840gactcgcagg ggaagtcccg cgtcgtcaca gtgggacggg tcaagatcga gacgcgtccg 900ctgctggcga tcgacgcggt ctccccctcc gggacacgcg tcaacctcat cgtccaggac 960gactggcacg tgcgcgtgct cgggccgggc ggcaccgtgc tcaacgtgac cgagctgacc 1020gccggcacga aggtgctcgg ttacctgccg gtggagaagc ggcacgtcgg ctacccgatc 1080gacgagttct gcatcgagaa gtga 110446253PRTMicromonospora sp. strain 046-ECO11 46Met Thr Ala Gln Pro Val Leu Asp Phe His Val Arg Leu Ala Pro Arg1 5 10 15Pro Gly Ala Arg Glu Arg Leu Leu Ala Ala Leu Arg Glu Cys Gly Leu 20 25 30Ala Arg Ala Val Val Cys Ala Gly Gly Thr Ile Asp Leu Asp Arg Leu 35 40 45Ser Arg Gln Leu Val Thr Gly Gly His Val Glu Thr Asp Ala Asp Asn 50 55 60Asp Ala Val Ala Ala Ala Cys Ala Gly Thr Asp Gly Arg Leu Val Pro65 70 75 80Phe Phe Phe Ala Asn Pro His Arg Pro Ala Glu Ala Tyr Arg Ala Arg 85 90 95Ala Ala Glu Phe Arg Gly Leu Glu Ile Ser Pro Ala Val His Gly Val 100 105 110Ala Leu Thr Asp Pro Arg Val Ala Asp Leu Val Ala Val Ala Ala Glu 115 120 125Phe Asp His Pro Val Tyr Val Val Cys Leu Asp Arg Pro Gly Ala Gly 130 135 140Val Ala Asp Leu Val Gly Leu Ser Arg Arg Phe Pro Gln Val Ser Phe145 150 155 160Val Leu Gly His Ser Gly Val Gly Asn Ile Asp Leu Tyr Ala Leu Thr 165 170 175Leu Ile Gln Asp Glu Pro Asn Ile Ser Leu Glu Thr Ser Gly Gly Tyr 180 185 190Thr Cys Val Ala Glu Ala Ala Leu Arg Arg Leu Gly Asp Asp Arg Val 195 200 205Val Phe Gly Ser Glu Tyr Pro Leu Gln His Pro Ala Val Glu Leu Ala 210 215 220Lys Phe Gln Ala Leu Arg Leu Pro Pro Glu Arg Trp Arg Arg Ile Ala225 230 235 240Trp Asp Asn Ala His Arg Leu Leu Gly Glu Glu Lys Arg 245 25047762DNAMicromonospora sp. strain 046-ECO11 47atgaccgcgc agccggtgct ggacttccac gtacgcctgg cgccccggcc cggggcgcgg 60gagcggctgc tcgccgcgct gcgcgagtgc gggctggcgc gggcggtggt gtgcgcgggc 120ggcaccatcg acctggaccg gctgtcccgc cagctcgtca ccggcggcca cgtcgagacc 180gacgccgaca acgacgcggt ggcggcggcc tgcgccggca ccgacggccg gctggtgccg 240ttcttcttcg ccaacccgca ccggccggcc gaggcgtacc gggcccgcgc cgccgagttc 300cgcggcctgg agatctcacc cgccgtccac ggcgtcgccc tgaccgaccc gcgggtcgcc 360gacctcgtgg ccgtggcggc ggagttcgac catccggtgt acgtggtctg cctggaccga 420cccggcgcgg gcgtggccga cctggtcggc ctgagccgcc ggttcccgca ggtgagcttc 480gtgctcgggc acagcggcgt cggcaacatc gacctctacg ccctgaccct gatccaggac 540gagccgaaca tctcgctgga gacctccggc ggctacacct gcgtggccga ggcggcgcta 600cgccgcctcg gcgacgaccg ggtggtgttc ggctccgagt acccgctgca gcacccggcc 660gtggaactgg ccaagttcca ggcgttgcga ctgccgccgg agcggtggcg gcggatcgcc 720tgggacaacg cgcatcgact gctaggagag gagaagcggt ga 76248438PRTMicromonospora sp. strain 046-ECO11 48Val Ser Glu Pro Ser Ser Ser Leu Pro Arg Leu Gly Gln Trp His Gly1 5 10 15Leu Glu Asp Leu Arg Arg Leu Gln Glu Lys Gln Leu Ala Glu Thr Phe 20 25 30Thr Trp Ala Ala Arg Ser Pro Phe Tyr Arg Ala Arg Leu Ala Ser Gly 35 40 45Ala Pro Pro Val Thr Pro Ala Asp Leu Ala Asp Leu Pro Leu Thr Thr 50 55 60Lys Gln Asp Leu Arg Asp Asn Tyr Pro Phe Gly Met Leu Ala Val Pro65 70 75 80Arg Glu Arg Leu Ala Thr Tyr His Glu Ser Ser Gly Thr Ala Gly Lys 85 90 95Pro Thr Pro Ser Tyr Tyr Thr Ala Glu Asp Trp Thr Asp Leu Ala Glu 100 105 110Arg Phe Ala Arg Lys Trp Ile Gly Met Ser Ala Asp Asp Val Phe Leu 115 120 125Val Arg Thr Pro Tyr Ala Leu Leu Leu Thr Gly His Leu Ala His Ala 130 135 140Ala Ala Arg Leu Arg Gly Ala Thr Val Val Pro Gly Asp Asn Arg Ser145 150 155 160Leu Ala Met Pro Tyr Ala Arg Val Val Arg Val Met His Asp Leu Asp 165 170 175Val Thr Leu Thr Trp Ser Val Pro Thr Glu Cys Leu Ile Trp Ala Ala 180 185 190Ala Ala Ile Ala Ala Gly His Arg Pro Asp Ile Asp Phe Pro Ala Leu 195

200 205Arg Ala Leu Phe Val Gly Gly Glu Pro Met Thr Asp Ala Arg Arg Arg 210 215 220Arg Ile Ser Arg Leu Trp Gly Val Pro Val Ile Glu Glu Tyr Gly Ser225 230 235 240Thr Glu Thr Gly Ser Leu Ala Gly Glu Cys Pro Glu Gly Arg Leu His 245 250 255Leu Trp Ala Asp Arg Ala Leu Phe Glu Val Tyr Asp Pro Asp Thr Gly 260 265 270Ala Val Arg Ala Asp Gly Asp Gly Gln Leu Val Val Thr Pro Leu Phe 275 280 285Arg Glu Ala Met Pro Leu Leu Arg Tyr Asn Leu Glu Asp Asn Val Ser 290 295 300Val Ser Tyr Asp Asp Cys Gly Cys Gly Trp Lys Leu Pro Thr Val Arg305 310 315 320Val Leu Gly Arg Ser Ala Phe Gly Tyr Arg Val Gly Gly Thr Thr Ile 325 330 335Thr Gln His Gln Leu Glu Glu Leu Val Phe Ser Leu Pro Glu Ala His 340 345 350Arg Val Met Phe Trp Arg Ala Lys Ala Glu Pro Ala Leu Leu Arg Val 355 360 365Glu Ile Glu Val Ala Ala Ala His Arg Val Ala Ala Glu Ala Glu Leu 370 375 380Thr Ala Ala Ile Arg Ala Ala Phe Gly Val Asp Ser Glu Val Thr Gly385 390 395 400Leu Ala Pro Gly Thr Leu Ile Pro Leu Asp Ala Leu Thr Ser Met Pro 405 410 415Asp Val Val Lys Pro Arg Ser Leu Phe Gly Pro Asp Glu Asp Trp Ser 420 425 430Lys Ala Leu Leu Tyr Tyr 435491317DNAMicromonospora sp. strain 046-ECO11 49gtgagcgagc caagttcgag cctgccccgg ctcggccagt ggcacggcct cgaggacctg 60cggcgcctcc aggagaagca actggcggag acgttcacct gggcggcccg gtcgccgttc 120taccgggcgc ggctggcctc cggcgcgccg ccggtgacgc ccgccgacct ggccgacctg 180ccgctgacca ccaagcagga cctgcgggac aactacccct tcggcatgct cgccgtgccc 240cgcgaacggc tggcgaccta ccacgagtcg agcgggaccg ccgggaagcc caccccctcc 300tactacaccg cggaggactg gaccgacctg gcggagcgct tcgcccgcaa gtggatcggc 360atgtccgccg acgacgtctt cctggtccgc acgccgtacg cgctgctgct gaccgggcat 420ctcgcccacg ccgcagcccg gctgcgtggg gccacggtgg tacctggcga caaccggtcg 480ctggcgatgc cgtacgcccg ggtggtccgg gtgatgcacg acctggacgt cacgctcacc 540tggtcggtgc cgacggagtg cctgatctgg gccgccgcgg cgatcgcggc cgggcaccgg 600cccgacatcg acttcccggc gctgcgcgcg ctgttcgtcg gcggcgagcc gatgaccgac 660gcccgccggc ggcggatcag ccgcctgtgg ggggtgccgg tcatcgagga gtacggctcg 720acggagaccg gcagcctggc cggggagtgc cccgagggac gcctgcacct gtgggccgac 780cgggcgctgt tcgaggtgta cgacccggac accggcgccg tccgcgcgga cggcgacggc 840cagctcgtgg tcacgccgct gttccgggag gcgatgccgc tgctgcggta caacctggag 900gacaacgtgt cggtctccta cgacgactgc ggatgcggct ggaagctgcc caccgtgcgg 960gtgctcggcc ggtcggcgtt cggctaccgg gtcggcggca ccaccatcac ccagcaccag 1020ctggaggaac tggtcttctc cctgccggag gcgcaccggg tgatgttctg gcgggccaag 1080gcggagccgg cgctgttgcg ggtcgagatc gaggtggccg ccgcgcaccg ggtcgccgcc 1140gaggcggagc tgaccgccgc gatccgggcc gccttcggcg tggacagcga ggtcaccggc 1200ctggcgccgg gaaccctgat cccgctcgac gcgctgacca gcatgccgga cgtggtgaag 1260ccacgcagcc tgttcggtcc ggacgaggac tggagcaaag cgctcctcta ctactga 131750396PRTMicromonospora sp. strain 046-ECO11 50Met Pro Gln Met Arg Val Ala Val Ala Gly Ala Gly Ile Ala Gly Leu1 5 10 15Ala Phe Ala Ala Ala Leu Arg Arg Thr Gly Ile Asp Cys His Val Tyr 20 25 30Glu Gln Ala Asp Gln Leu Met Glu Val Gly Ala Gly Val Gln Val Ala 35 40 45Pro Asn Ala Thr Arg Leu Leu His Arg Leu Gly Leu Arg Asp Arg Leu 50 55 60Arg Thr Val Ala Val Ala Pro Gln Ala Ile Glu Met Arg Arg Trp Asp65 70 75 80Asp Gly Thr Leu Leu Gln Arg Thr Gln Leu Gly Ser Val Cys Gly Arg 85 90 95Arg Phe Gly Ala Pro Tyr Tyr Val Val His Arg Ala Asp Leu His Ser 100 105 110Ser Leu Leu Ser Leu Val Pro Pro Asp Arg Val His Leu Gly Ala Arg 115 120 125Leu Thr Ala Val Thr Gln Thr Ala Asp Glu Ala Tyr Leu His Leu Ser 130 135 140Asn Gly Thr Thr Val Ala Ala Asp Leu Val Val Gly Ala Asp Gly Ile145 150 155 160His Ser Val Ala Arg Glu Gln Ile Val Ala Asp Arg Pro Arg Phe Ser 165 170 175Gly Gln Ser Ile Tyr Arg Gly Leu Val Pro Ala Glu Arg Val Pro Phe 180 185 190Leu Leu Thr Glu Pro Arg Val Gln Leu Trp Phe Gly Pro Asp Gln His 195 200 205Cys Val Cys Tyr Pro Val Ser Ala Gly Arg Gln Val Ser Phe Gly Ala 210 215 220Thr Val Pro Ala Thr Asp Trp Arg Gln Glu Ser Trp Ser Gly Arg Gly225 230 235 240Asp Val Thr Gln Leu Ala Ala Ala Tyr Ala Gly Trp His Pro Asp Val 245 250 255Thr Arg Leu Ile Ala Ala Ala Asp Arg Val Gly Arg Trp Ala Leu His 260 265 270Asp Arg Asp Ser Ile Asp Arg Leu Ser Ala Gly Arg Val Thr Leu Ile 275 280 285Gly Asp Ala Ala His Pro Met Leu Pro Phe Gln Ala Gln Gly Ala Asn 290 295 300Gln Ala Val Glu Asp Ala Val Val Leu Ala Val Cys Leu Ala Gly Val305 310 315 320Glu Pro Ala Gly Leu Gly Ala Ala Leu Arg Arg Tyr Glu Arg Ile Arg 325 330 335Leu Pro Arg Thr Thr Arg Ile Gln Arg Gln Ser Arg Ala Asn Ala Glu 340 345 350Met Phe His Leu Ala Asp Gly Ala Asp Gln Arg Arg Arg Asp Val Ala 355 360 365Ala Gln Ser Ser Ser Gly Leu Asp Arg His Glu Trp Leu Phe Gly Tyr 370 375 380Asp Ala Glu Lys Ala Thr Thr Thr Ser Gly Ser Ala385 390 395511191DNAMicromonospora sp. strain 046-ECO11 51atgccgcaga tgagggtcgc cgtggccggc gccggcatcg ccgggctcgc cttcgccgcc 60gccctgcgcc ggaccgggat cgactgccac gtgtacgaac aggccgacca gctcatggag 120gtgggcgcgg gcgtgcaggt cgcgccgaac gccacccggc tgctgcaccg gctgggcctg 180cgtgaccgcc tgcgtacggt ggctgtcgcg ccgcaggcga tcgagatgcg ccgctgggac 240gacggcacgc tgctgcaacg cacccagctg ggcagcgtgt gcggacgccg cttcggcgcg 300ccgtactacg tggtgcaccg cgcggacctg cacagcagcc tgctgtcgct ggtgccgccg 360gaccgggtgc acctgggcgc ccgcctcacc gccgtgacgc agaccgccga cgaggcgtac 420ctgcacctgt ccaacggcac cacggtcgcg gcggatctcg tcgtgggcgc cgacggcatc 480cactcggtcg cgcgggagca gatcgtggcg gaccggccgc gcttctccgg acagtccatc 540taccgcgggc tggtgccggc cgagcgggtg ccgttcctgc tcaccgaacc ccgggtgcag 600ttgtggttcg ggccggacca gcactgcgtc tgctacccgg tgtccgccgg ccggcaggtg 660agcttcggcg cgacggtgcc cgccaccgac tggcggcagg agtcgtggtc gggccggggc 720gacgtgacgc aactcgcggc cgcgtacgcg ggctggcacc cggacgtcac ccggctgatc 780gccgcggccg accgggtcgg caggtgggcg ctgcacgacc gggacagcat cgaccggctc 840agcgcgggac gggtgaccct gatcggcgac gccgcgcacc cgatgctgcc gttccaggcg 900cagggcgcga accaggccgt cgaggacgcg gtggtgctcg cggtctgcct ggccggcgtg 960gaaccggcgg gcctgggcgc cgcgctgcgc cgctacgaac ggatccgcct gccccggacc 1020acccggatcc agcggcagtc ccgggccaac gccgagatgt tccacctggc cgacggcgcc 1080gaccagcgcc gccgggacgt cgccgcacaa tcctcgtccg gcctggaccg ccacgaatgg 1140ctcttcgggt acgacgccga gaaagccacc acgaccagcg ggagcgcctg a 119152261PRTMicromonospora sp. strain 046-ECO11 52Met Glu Leu Thr Gly Ile Glu Ser Lys Val Ala Leu Val Thr Gly Ala1 5 10 15Gly Gln Gly Ile Gly Ala Ala Val Ala Gly Val Leu Ala Arg Ala Gly 20 25 30Ala Gln Val Ala Ala Val Asp Arg Asn Ala Glu Ala Leu Thr Thr Val 35 40 45Val Thr Lys Leu Ala Ala Glu Gly Asp Ser Ala Arg Ala Tyr Cys Val 50 55 60Asp Val Cys Asp Ser Glu Ala Val Asp Ala Leu Val Arg Arg Val Glu65 70 75 80Asp Glu Met Gly Pro Val Ala Ile Leu Val Asn Ala Ala Gly Val Leu 85 90 95His Thr Gly Arg Val Val Glu Leu Ser Asp Arg Gln Trp Arg Arg Thr 100 105 110Phe Ser Val Asn Ala Asp Gly Val Phe His Val Ser Arg Ala Val Ala 115 120 125Arg Arg Met Val Gly Arg Arg Arg Gly Ala Ile Val Thr Val Ala Ser 130 135 140Asn Ala Ala Gly Val Pro Arg Thr Glu Met Ala Ala Tyr Ala Ala Ser145 150 155 160Lys Ala Ala Ser Ala Gln Phe Thr Arg Cys Leu Gly Leu Glu Leu Ser 165 170 175Gly Tyr Gly Ile Arg Cys Asn Val Val Ser Pro Gly Ser Thr Asp Thr 180 185 190Pro Met Leu Arg Ala Met Leu Gly Glu Gly Ala Asp Pro Ser Ala Val 195 200 205Ile Glu Gly Thr Pro Gly Ala Tyr Arg Val Gly Ile Pro Leu Arg Lys 210 215 220Leu Ala Gln Pro Arg Asp Val Ala Glu Ala Val Ala Tyr Leu Val Ser225 230 235 240Asp Gln Ala Gly His Val Thr Met His Asp Leu Tyr Val Asp Gly Gly 245 250 255Ala Ala Leu His Val 26053786DNAMicromonospora sp. strain 046-ECO11 53atggaactga ccggaatcga gtcgaaggtc gccctggtca cgggcgcggg gcagggcatc 60ggcgccgccg tggccggtgt cctggcgagg gcgggcgcgc aggtggcggc ggtggaccgc 120aacgccgagg cgctgaccac cgtcgtgacg aagctcgccg ccgagggcga ctcggcgcgc 180gcctactgcg tcgacgtgtg cgacagcgag gcggtggacg cgctggtgcg ccgggtcgag 240gacgagatgg ggccggtcgc catcctggtc aacgccgccg gcgtgctgca caccggacgg 300gtcgtcgagc tgtcggaccg gcagtggcgc cggaccttct cggtgaacgc cgacggcgtg 360ttccacgtgt cccgggcggt ggcgcggcgg atggtgggcc gccgtcgtgg cgcgatcgtc 420accgtggcgt cgaacgccgc cggggtgccg cgtaccgaga tggccgcgta cgccgcctcc 480aaggccgcgt ccgcgcagtt cacccgctgc ctggggcttg agctgtccgg ctacggcatc 540cggtgcaacg tggtctcgcc cggctccacc gacaccccca tgctgcgggc catgctcggc 600gagggcgccg acccgagcgc ggtgatcgag ggcacgccgg gcgcgtaccg cgtcggcatc 660ccgctgcgca agctggccca gccgcgcgac gtggccgagg cggtcgccta tctggtgtcc 720gaccaggcgg gccacgtgac catgcacgac ctgtacgtcg acggcggcgc ggccctgcac 780gtgtga 78654224PRTMicromonospora sp. strain 046-ECO11 54Met Ala Met Thr Pro Ile Ala Pro Tyr Arg Met Pro Gly Asp Gly Asp1 5 10 15Leu Pro Gly Thr Ala Leu Pro Trp Arg Pro His Pro Asp Arg Ala Ala 20 25 30Val Leu Val His Asp Leu Gln Arg Tyr Phe Leu Arg Pro Phe Glu Ala 35 40 45Gly Glu Ser Pro Met Ala Glu Leu Leu Pro Asn Val Ala Lys Leu Leu 50 55 60Ala Thr Ala Arg Ala Ala Gly Val Pro Val Leu Tyr Thr Ala Gln Pro65 70 75 80Gly Gly Met Ser Arg Gln Asp Arg Gly Leu Leu His Asp Leu Trp Gly 85 90 95Pro Gly Met Ser Ser Ala Glu Asp Asp Arg Gly Ile Val Asp Asp Val 100 105 110Ala Pro Gln Pro Gly Asp Thr Val Leu Thr Lys Trp Arg Tyr Ser Ala 115 120 125Phe Phe Arg Ser Asp Leu Glu Glu Arg Leu Arg Gly Ala Gly Arg Asp 130 135 140Gln Leu Val Val Cys Gly Val Tyr Ala His Met Gly Cys Leu Ile Thr145 150 155 160Ala Cys Asp Ala Phe Ser Arg Asp Ile Glu Ala Phe Leu Val Ala Asp 165 170 175Ala Leu Ala Asp Leu Ser Arg Glu Asp His Leu Met Ala Leu Arg Tyr 180 185 190Ala Ala Asp Arg Cys Ala Val Pro Leu Trp Thr Ala Asp Val Leu Asp 195 200 205Gly Leu Ala Asp Ala Ala Gly Arg Pro Asp Gln Ser Ser Thr Gln Arg 210 215 22055675DNAMicromonospora sp. strain 046-ECO11 55atggccatga ccccgatcgc gccgtaccgc atgcccggcg acggcgacct gcccggcacc 60gcgctgccct ggcgtccgca cccggaccgg gccgccgtgc tggtgcacga cctgcaacgc 120tacttcctgc gcccgttcga ggccggggag tccccgatgg ccgaactgct ccccaacgtc 180gcgaagctgc tcgccacggc gcgggcggcc ggcgtgccgg tgctgtacac cgcgcagccc 240ggcggcatga gccggcagga ccgcgggttg ctgcacgacc tgtggggccc cggcatgagc 300agcgccgagg acgaccgggg catcgtcgac gacgtcgccc cgcagccggg cgacacggtg 360ctgaccaagt ggcgctacag cgcgttcttc cgcagcgacc tggaggagcg actgcgcggt 420gcgggacggg accagctcgt ggtctgcggc gtgtacgcgc acatggggtg cctgatcacc 480gcctgcgacg cgttcagccg cgacatcgag gcgttcctgg tggcggacgc gctggccgac 540ctatcgcgcg aggaccacct gatggcgctg cgctacgccg cggaccgctg cgcggtgccg 600ttgtggacgg cggatgtgct ggacgggctg gcggacgccg ccgggcgtcc ggatcagagc 660agcacccaac gatga 67556233PRTMicromonospora sp. strain 046-ECO11 56Met Ser Asp Arg Thr Arg Val Val Val Val Gly Gly Thr Ser Gly Ile1 5 10 15Gly Arg His Phe Ala Arg Phe Cys Ala Glu Arg Gly Asp Asp Val Val 20 25 30Ile Thr Gly Arg Ser Ala Ala Arg Thr Lys Thr Val Ala Asp Glu Ile 35 40 45Gly Gly Arg Thr Arg Gly Leu Ala Leu Asp Leu Ala Glu Pro Glu Thr 50 55 60Ile Ala Asp Ala Leu Ala Asp Val Pro His Val Asp Arg Leu Val Val65 70 75 80Ala Ala Leu Asp Arg Asp Tyr Asn Thr Val Arg Ala Tyr Arg Pro Gly 85 90 95Asp Ala Ala Arg Leu Leu Thr Val Lys Leu Val Gly Tyr Thr Ala Val 100 105 110Leu His Ala Leu Ala Pro Arg Met Thr Asp Glu Ser Ala Val Val Leu 115 120 125Leu Gly Gly Leu Ala Ser His Arg Pro Tyr Pro Gly Ser Thr Ser Val 130 135 140Thr Thr Ala Asn Gly Gly Ile Ser Ala Leu Val Arg Thr Leu Ala Val145 150 155 160Glu Leu Ser Pro Val Arg Val Asn Ala Leu His Pro Ser Ile Val Ser 165 170 175Asp Thr Pro Phe Trp Ser Asp Lys Pro Ala Ala Arg Glu Ala Ala Ala 180 185 190Thr Arg Ala Leu Ser Arg Arg Pro Val Thr Met Gln Asp Cys Ala Glu 195 200 205Ala Ile Asp Phe Leu Leu Thr Asn Arg Ser Ile Asn Gly Val Asn Leu 210 215 220Asn Ile Asp Gly Gly Asp Val Leu Ile225 23057702DNAMicromonospora sp. strain 046-ECO11 57atgtcggatc ggacccgggt cgtggtcgtc ggcggaacct cggggatcgg gcggcacttc 60gcccgattct gcgccgaacg cggagacgac gtggtgatca ccggccgttc ggcggcccgg 120accaagaccg tggcggacga gatcggcggg cggacccgtg ggctcgctct cgacctggcc 180gagccggaga cgatcgcgga cgcgctcgcc gacgtgccgc acgtcgaccg gctcgtggtc 240gcggcgctgg accgcgacta caacaccgtc cgcgcgtacc ggccgggcga cgcggcgcgg 300ctgctgaccg tcaagctggt cggctacacg gcggtcctgc acgccctcgc cccgcggatg 360accgacgaga gcgcagtcgt gctgctcggc ggcctggcca gccaccggcc gtatcccggc 420tccacctccg tcacgaccgc caacggcggg atcagcgcgc tggtgcggac cctggctgtg 480gaactctcgc cggtccgggt caacgccctg cacccgagca tcgtctccga cacgccgttc 540tggagcgaca agcccgccgc gcgggaggcc gccgcgaccc gcgcgctcag ccgacggccg 600gtcaccatgc aggactgcgc cgaggcgatc gacttcctgc tgacgaaccg ctcgataaac 660ggggtcaacc tgaacatcga cggcggggac gtgctcatct ga 70258246PRTMicromonospora sp. strain 046-ECO11 58Met Thr Ser Ala Leu Arg Thr Ser Ala Trp Thr Tyr Asp Asp Phe Thr1 5 10 15Ser Arg Glu Leu Asp Pro Ala Arg Trp Ala Ile Met Ser Ile Ala Gly 20 25 30Ala Asp Gly Gln Thr His Arg Tyr Gln Asp Arg Asn Ala Gln Val Arg 35 40 45Thr Gly Asp Gly Arg Leu Glu Leu Thr Val Asp Pro Phe Thr Arg Phe 50 55 60His Asp Thr Asp Pro Arg Gln Asn Asn Ala Lys Gln Met Tyr Arg Ser65 70 75 80Val Arg Arg Phe Ala Val Pro Ala Glu Gly Ser Leu Thr Val Glu Val 85 90 95Glu Met Gly Val Arg Thr Tyr Arg Gln Ile Pro His Asp Leu Leu Asp 100 105 110Ala Phe Gly Thr Val Asn Leu Phe Asp Leu Glu Thr Gly Val Val Phe 115 120 125Asn Ala Ala Ala Thr Asn Asp Thr Val Tyr Ala Thr Val Glu Arg Leu 130 135 140Val Leu Pro Gly Val Thr Gln Pro His Glu His Tyr Ile His Arg Val145 150 155 160Val Leu Asp Val Pro Thr Glu Pro Gly Arg Ala His Gly Tyr Ala Ile 165 170 175Thr Tyr Arg Ala Pro Thr Ser Glu Val Glu Phe His Val Asp Gly Arg 180 185 190Leu Ala Tyr Trp Ala Arg Val Pro Val Pro Val Thr Gly Phe His Ala 195 200 205Gly Met Ala Leu Phe Ser Ala Arg Asp Leu Ala Arg Tyr Pro Arg Glu 210 215 220Gln Arg Glu His Gly Gln Gly Ala Thr Gly Trp Trp Gly Pro Trp Arg225 230 235 240Ile Ala Ser Gly Val Arg 24559741DNAMicromonospora sp. strain 046-ECO11 59atgacgtcgg cactgagaac cagcgcgtgg acgtacgacg acttcaccag ccgcgagctg 60gaccccgccc gctgggcgat catgtcgatc gccggcgcgg acgggcagac ccacaggtac 120caggaccgca acgcccaggt ccgcaccggc gacgggcggc tggagctgac

cgtcgacccg 180ttcacccgct tccacgacac cgatccccgg cagaacaacg ccaagcagat gtaccggtcg 240gtgcggcgct tcgccgtgcc ggcggagggc tcgctgaccg tcgaggtgga gatgggcgtg 300cggacgtacc ggcagatccc gcacgacctg ctggacgcgt tcggcacggt gaacctgttc 360gacctggaga ccggcgtcgt gttcaacgcc gccgccacga acgacaccgt gtacgcgacg 420gtcgagcgcc tggtgctgcc cggcgtgacc cagccgcacg agcactacat ccaccgggtg 480gtcctggacg tgccgacgga gccgggccgg gcgcacggat acgccatcac ctaccgggcg 540ccgacgtcgg aggtggagtt ccacgtcgac ggccggctcg cctactgggc gcgggtcccg 600gtgccggtga ccggattcca cgccggcatg gcgctcttct ccgcccgcga cctggcccgg 660tacccccgcg agcagcggga gcacgggcag ggcgcgaccg ggtggtgggg gccgtggcgg 720atcgcctccg gcgtcagatg a 74160111PRTMicromonospora sp. strain 046-ECO11 60Met Asp Thr Ala Ala Pro Ala Thr Asp Gly Gly Arg Tyr Leu Ala Val1 5 10 15His His Ser Ala Glu Phe Arg Glu Leu Arg Arg Arg Ser Ser Thr Phe 20 25 30Thr Leu Trp Ala Ser Val Ala Phe Phe Gly Trp Trp Phe Leu Gly Ser 35 40 45Leu Leu Ala Thr Tyr Ala Pro Asp Phe Phe Arg Glu Lys Val Ala Gly 50 55 60Pro Val Asn Val Gly Leu Leu Phe Val Phe Leu Ser Phe Ala Phe Val65 70 75 80Val Thr Leu Ala Ala Phe Tyr Leu Arg Tyr Ala Arg Thr His Leu Asp 85 90 95Pro Leu Ser Glu Lys Ile Arg Ala Asp Leu Glu Gly Ala Ser Arg 100 105 11061336DNAMicromonospora sp. strain 046-ECO11 61atggacacgg cagctccggc aacggacggc ggtcgctacc tcgccgtcca tcacagcgca 60gagttcaggg aactacggcg acgatcgagc acgttcacgc tctgggccag cgtcgccttc 120ttcggctggt ggttcctcgg cagcctgctc gccacctacg cgccggactt cttccgggag 180aaggtggccg gcccggtcaa cgtgggtctg ctcttcgtct tcctgtcgtt cgccttcgtg 240gtgacgctcg ccgccttcta cctgcgttac gcccgcacgc atctcgatcc gctcagcgag 300aagatccgtg ccgacctgga aggagcgtcc cgatga 33662559PRTMicromonospora sp. strain 046-ECO11 62Met Ser Val Ile Leu Ala Asp Pro Pro Pro Pro Val Asp Asn Thr Trp1 5 10 15Ala Thr Pro Ala Ile Ala Val Pro Val Thr Ile Val Leu Ala Leu Ala 20 25 30Val Leu Tyr Leu Val Arg Ser Ala Arg Ala Ser Thr Thr Thr Ala Asp 35 40 45Gly Phe Leu Leu Ala Asp Arg Arg Ile Gly Pro Val Gln Asn Ala Leu 50 55 60Ala Val Ala Ser Ala Pro Leu Met Tyr Ser Thr Met Tyr Ile Ile Thr65 70 75 80Gly His Ile Ala Leu Ser Gly Tyr Asp Ala Ile Leu Leu Met Thr Ala 85 90 95Phe Thr Met Gly Thr Met Leu Ala Leu Phe Leu Phe Ala Gly Pro Val 100 105 110Arg Asn Val Gly Gly Tyr Thr Leu Gly Asp Leu Leu Ala Val Arg Thr 115 120 125Arg Glu Arg Pro Ala Arg Ile Ala Ser Ala Val Leu Thr Leu Leu Thr 130 135 140Tyr Val Met Leu Thr Val Ile Met Met Ala Ala Ile Ala Phe Ile Phe145 150 155 160Asn Arg Trp Phe Gly Val Asp Ala Leu Val Gly Leu Val Leu Pro Val 165 170 175Phe Val Val Gly Leu Ile Thr Val Gly Tyr Val Tyr Leu Gly Gly Met 180 185 190Leu Gly Val Thr Arg Ile Leu Val Phe Lys Leu Val Leu Ser Val Val 195 200 205Val Val Gly Val Leu Thr Ala Trp Val Leu Ala Arg Phe Asp Leu Asn 210 215 220Leu Phe Ser Leu Leu Glu Arg Ala Glu Ala Asn Ala Ala Pro Val Pro225 230 235 240Ser Gly Ser Asp Leu Leu Gly Pro Gly Arg Leu Phe Gly Glu Gly Ala 245 250 255Thr Thr Leu Val His Leu Ser Lys Leu Phe Ala Ile Ala Val Gly Val 260 265 270Ala Ala Ile Pro Phe Leu Phe Met Arg Asn Phe Ala Val Thr Ser Gly 275 280 285Arg Asp Ala Arg Arg Ser Thr Gly Trp Ala Ser Met Ile Ile Val Gly 290 295 300Phe Tyr Leu Cys Leu Ser Val Val Gly Leu Gly Ala Val Ala Ile Leu305 310 315 320Gly Arg Asp Asn Ile Gly Val Ile Lys Ala His Arg Asp Ile Ser Phe 325 330 335Pro Lys Leu Ala Asp Glu Leu Gly Gly Pro Val Met Val Gly Ser Leu 340 345 350Ala Gly Val Ala Val Leu Thr Ile Val Gly Val Phe Ala Pro Leu Leu 355 360 365His Ser Ala Val Thr Thr Val Thr Lys Asp Leu Asn Val Ile Arg Gly 370 375 380Arg Arg Leu Asp Pro Ala Ala Glu Leu Arg Asp Ile Lys Arg Asn Thr385 390 395 400Leu Ile Ile Gly Val Gly Ser Val Leu Leu Ala Val Val Met Leu Pro 405 410 415Val Arg Thr His Ile Phe Ile Pro Thr Ser Ile Asp Ile Ala Gly Ala 420 425 430Val Val Leu Pro Ile Val Val Tyr Ala Leu Phe Trp Arg Arg Phe Asn 435 440 445Thr Arg Gly Leu Gln Trp Thr Val Tyr Gly Gly Leu Ala Leu Thr Ala 450 455 460Phe Leu Val Leu Phe Ser Asn Gly Val Ser Gly Glu Pro Asp Ala Ile465 470 475 480Phe Pro Asp Arg Asn Phe Lys Phe Val Asp Val Glu Pro Ala Leu Ile 485 490 495Thr Val Pro Val Gly Phe Leu Leu Gly Tyr Leu Gly Ser Ile Thr Ser 500 505 510Arg Glu Arg Asp Asp Ala Ala Phe Ala Glu Met Gln Val Arg Ser Leu 515 520 525Thr Gly Ala Val Val Thr Gly Pro Pro Arg Pro Ala Ala Val Asp Asp 530 535 540Glu Asp Arg Asp Gly Arg Gln Asp Arg Ala Pro Ser Pro Val Ser545 550 555631680DNAMicromonospora sp. strain 046-ECO11 63atgagcgtca tcctcgccga cccgccaccc ccggtcgaca acacgtgggc gacgcccgcg 60atcgccgtgc cggtcaccat cgtcctcgcg ctcgcggtgc tctacctggt ccggtcggcg 120cgcgccagca ccaccaccgc ggacggcttc ctgctggccg accggcggat cgggccggtg 180cagaacgcgc tggcggtggc ctccgcgccg ctgatgtact cgacgatgta catcatcacc 240ggccacatcg cgctcagcgg ctacgacgcc atcctgctga tgaccgcctt caccatgggc 300accatgctcg cgctgttcct cttcgccggg ccggtgcgca acgtgggcgg ctacacgctc 360ggtgacctgc tcgcggtccg tacccgggag cggccggcgc ggatcgcgtc ggcggtgctc 420acgctgctga cgtacgtcat gctgacggtg atcatgatgg ccgccatcgc gttcatcttc 480aaccgctggt tcggcgtcga cgccctcgtc ggcctggtcc tcccggtgtt cgtcgtcggt 540ctgatcacgg tggggtacgt gtacctcggc gggatgctcg gggtcacccg catcctggtg 600ttcaagctgg tgctgtcggt ggtcgtcgtg ggcgtgctga ccgcctgggt gctggcccgc 660ttcgacctga acctcttcag cctgctggag cgggccgagg cgaacgcggc gccggtgccc 720agcggcagcg acctgctggg cccgggccgg ctgttcggcg agggcgcgac cacgctcgtg 780cacctgtcga agctgttcgc catcgccgtc ggagtggcgg ccattccgtt cctgttcatg 840cgcaacttcg cggtgaccag cgggcgggac gcgcgccggt cgaccgggtg ggcgtcgatg 900atcatcgtcg ggttctacct gtgcctgtcc gtcgtcgggc tcggtgccgt cgcgatcctc 960ggccgggaca acatcggcgt catcaaggcc caccgcgaca tcagcttccc caagctcgcc 1020gacgagctcg gcggtccggt gatggtcggc tccctggccg gcgtcgcggt cctgacgatc 1080gtcggcgtct tcgcgccgct gctgcacagc gccgtgacga cggtgaccaa ggacctgaac 1140gtgatccgcg gccggcggct ggatccggcc gccgagctgc gggacatcaa gcgcaacacc 1200ctgatcatcg gcgtcggctc cgtgctgctg gcggtcgtga tgctgccggt acggacccac 1260atcttcatcc cgacctcgat cgacattgcc ggcgcggtgg tcctgccgat cgtcgtctac 1320gcgttgttct ggcggcgttt caacacccgc ggactgcagt ggacggtcta cggcggcctc 1380gcgctcaccg cgttcctggt gctgttctcc aacggtgtct cgggcgagcc ggacgccatc 1440ttcccggacc gcaacttcaa gttcgtggac gtcgagcccg cgctgatcac ggtgccggtc 1500ggcttcctgc tcggctacct cggctcgatc accagccggg agcgcgacga cgccgcgttc 1560gccgagatgc aggtccggtc cctcaccgga gctgtcgtca cgggaccgcc gcggccggcc 1620gccgtggacg acgaggaccg cgacggccgc caggaccggg cgcccagccc ggtgagctga 1680645960DNAMicromonospora sp. strain 046-ECO11 64ccacacccct cgggaggcaa ctgtggatcc ggtaccggtt ctggtcgtgg gcgcgggccc 60ggtcggcatg gtcaccgcgc tggcgctcgc ccgtcacggc gtcgcctgcg tcctcgtcga 120ccagggcttc gagacgtcgg tccatcccaa gctggactac gtcaacgccc gcagcatgga 180gttcctccgc cagttcggcc tcgccgacga cgtccgtgcc gccggcgtcg cgcccgagca 240ccgggccgac gtcatctggt cgaccggcct ggccggtgag ccgatcacca ggtgggggct 300gccctcggtg acgcaggagt ggcgccgcat cgccgagcac aacgacggca cccagccggc 360cgagcccggc cagcggatct cccagatcga cctggaaccg gtcctgcggg cccgctgccg 420gcgggagccc cttgtcgacc tgcgcctcgg cgtacggttc gactcgctga cccaggacga 480cgcgggggtc accagcgtcc tcgccgacga caccggcggc gaggtccggg tgcggtcgga 540gtacgtggtc gggtgcgacg gcgcgtcgag ccaggtccgc cgggccgtgg gcatcggtga 600ggaggggttc gacgtgcccg gcctgccggg cgccttcatg gtgcacttca ccagccggga 660cctggacagc ctgcaccggc acggccggtt ctggcactac ttcgcgttcc ggtacgtgat 720catcgcccag gacgaggtcg acacctggac cgcgcacgtc aacggcgtcg acccgaacga 780gttcgacgag ccgccggccg acccggaggc gttcctgctc gacacgatcc gcaccgagct 840gcggatcgac aaggtgctgc tcacctcgcg ctggcgtccc ggcttcatgc tcgccgacag 900gtaccgcgcc ggccgggtgc tgctcgccgg tgactcggcc caccggatgt tccccaccgg 960cgcgtacggc atgaacaccg gcatcggcga cgccgtcgac gtggcctgga agctggccgc 1020tgtcgtccgg ggcttcggcg gccccgggct gctcgacagc tacgacgccg aacgccgccc 1080ggtggggcgg cgcaacatgc gcacctcgca ccggcacctg ggcgtgcacc tgcgggcggg 1140cgagctcctg cgcggcggcg ccccgctgcc gtccgtcgcg gccttcctcg acgccgagcg 1200gggcgagaac gagtaccggg ggatcgagct cggctaccgc tactccggct cgccggtgct 1260ctggccggag ggcccggggg agccctcgga cgacccgcgg gcgtacgccc cgacgacctg 1320gcccggcgcc cgtccgccca gcctcctgct gagcgacggg cagcagatct tcgaccggtt 1380cgacccggcc tcgttcaccc tcgtggactt caccggtgac ggcgccgccg gtccgctgct 1440ggcggcggcg gccgcgcggg ggctcccggt cacccacacc gtggtgaccg acccccgggc 1500tcgtgagctg tgggaacgcg acctcgtcct gctgcggccg gaccaccacg tcgcctggcg 1560gggaaacacc gtgccgccgg accccgacgc cgtggtccag cgcgtgcggg gtggcggata 1620ggcgcgacgt gccgtcaccg gcggcccggg tcacgcgcac acgcgaccgg ccggtccggc 1680tgactctcga ctggaggaca gatgcagcaa tccggttcaa cggcggaacg cagcccactc 1740gggccgtggg agggcatgcc ggcggtccag caaccggact ggcaggacca cccggcgtac 1800gcggagacct gtcaggcgtt ggcgtcggcc ccgccgctgg tcccacccgg ggaggtacgg 1860gggttccggc agctgttgtc ggagctggcg tcgaccgacg ggctcctgct gcagttgggc 1920gactgcgccg agagcctcta cgagtgcacc ccccggcaca cctcggacaa gatcgaggtc 1980atcgaccggc tgggggaccg gctcagcgag ctcaccgggc gcaacgtgct gcgggtgggc 2040cggatggccg ggcagttcgc caagccccgg tcgcaggcga cggagtggca cgacgcgctg 2100agcatcccct ccttccgcgg ccacatgatc aattccgagc tggccgcgcc cggtacgcgc 2160aaggccgacc ctcgccgcat gtggtgggcg tacgaggcga gcgaccgggt gcagcgggtc 2220ctgcgcgccc accgggaggg caaccggcgt gccgcgcgga ccgaggggcc gtggtcgagc 2280cacgaggccc tggtcgtcga ctacgagtcc cgcctgatcc gccgggaccc ggacacgggc 2340gagcactacc tggcgtcgac ccacctgccg tgggtggggg agcggacccg ccggtccgcc 2400gaggcgcacg tggccatgct gtccacggtg gtgaacccgg tcggctgcaa gatcgggccg 2460gacgccgacc cggacgacgt cctgcgggtg tgcgaggcgc tcgacccgcg gcgcgatccg 2520ggccgtctcg tcctgatccc gcggatgggc cgggaccgga tccgggagtc cctgccgccg 2580atcgtccgcg cggtggtgaa cgcggggcac cccgtgctct ggctgagcga tcccatgcac 2640ggcaacaccg tcaaggcctc ggtcggcctg aagacgcgcc acctctccga cgtggtcacc 2700gaggcgctgt ggttccgcga catcctcgac cagcagcggc agcacgccgc cgggctgcac 2760atcgaggtcg ccgccaccga cgtgaccgag tgcgtcggcg gttcggtggc cggcgaggag 2820gacctggcgc ggcactacac ctcgctgtgc gacccgcggc tcaacccggg tcaggccacc 2880gagctgatcg aagcgtgggc caaggacacc gcgacggtcg gcccgggacc gcggcgctcc 2940ggcccttcgg cgcggccgga ggtcgccgcc tgacgtcgcc ggtctttgcg ccggccgttt 3000ccgaactgcg ggaaaattga cagaaggaga cctgccggag caaattcggc caggctagcc 3060gcgccgtagt tcgtcgtcca ctacttgcgt gggtagtgtc aactacccgt gccgggaccg 3120tcggtggtgt tgctcagcag gaatcccatc gcaatgatgt gtgagaaggc gtaatccttc 3180gatcggtgac gcgcgtacct catcctatcc gcactgaatc ctgtctcagc tgaagcgagt 3240gtttccaatg tggggcagct caaacacgct ggaagtgaag ggcaacgacg agagattccc 3300cctgcccgat gcagctacgg aggatcggtc tgtgcttggc gagacggttc cggtttccgc 3360gctgctgccc ggtgactccc cgcggctggc gggcgagaac gtcgagcaca tccggctgct 3420ggccgcgatg cacgacctcc cgccgatcct ggtgcaacgc ggcacgatgc gggtgatcga 3480cggcatgcac cggctgcggg ccgccaagct gcgcggcgac gagaccgtgc gggtgacgtt 3540cttcgacggg gacgacgccg cggcgttcct gctctcggtc gacgccaaca tcaaacacgg 3600gctgccgttg tcccgcgccg accgggaggc cgccgccacc cgcatcctgc ggttgtatcc 3660gcagtggtcg gaccgcgccg tcgccgcggc ggccgggctg tcaccgacca cggcgagcgg 3720catccggcgc cgcctgctgc aaccggcggc gcgggagggc agccgggtgg gacgggacgg 3780gcgggtgcgc ccgctggacg gctcggcggg ccgacggcgg gccagcgcgg tcatcgcgct 3840ccggccggac gcgcccctgc gtgccatcgc gcaggaggcc ggggtgtcgg tgggcacggc 3900gcgggacgtg cgcgcccggt tgcaggcggg ccgggacccc gtcctgacct cgcagcgacc 3960ggcggccgag cccgagccgg ccgccgacga cgggccggag gcgcgcagac gccggctcgg 4020ccagccctcc gtgccgcctg tcgactggcc ggcggtacgg ggcaacctga tccgggaccc 4080cgcggtgaag tacgccgagc tgggccgggc cttcgtccgc tgggccgacg ggcacgtggt 4140ggatccggcg gcctggcgcg agttcgtcga cgccgtgccg ccgtactggc gcaaatcggt 4200ggccgagctg gcccgttcgt gcgccagcgc ctggctggcg ttcgcccagg aactggagga 4260ccgggcgtga aaatggcggc cggcatattt acggtggttg ccgacagcgc gtcgcattcc 4320actgtcgcgg ccactacccg atcgagtagt ggaccggctt gaataacgcg cgttaatgtt 4380ccttcgatcc gctgccctca tttttcggtg agcacatttt tgcggcggtc caatggagag 4440gagaattccc ggtgaacatt ctgaggcggc cgcggaaacg gcatctcggg ggtgtcgcgg 4500ccgtcgccgc ggcgatcgcc ctggtggcgt cgctgacaaa cggtgtggcg gctgccccgc 4560aggcgccgac cttcgacctc gacaacggga acgccctgac cgacgtcatc tacccggccc 4620tcaacaccga gccgcgggtc gagtacagcg gccggcccgg gtcctgggcc gcggaccgcg 4680ccatgctcat cgaactgccg tggttcgacg ccctggcggc gtaccacccc accgcggtcg 4740gcatcttctc caccatcggc cgccgtcccg ccgaggagca cacgacgcgc aacaagaaca 4800tcgccgtcat ctactcggcc tacacctcgc tcagcaagct ctacccccag cacgaggcga 4860cctggcagcg gatgatggcc accgcgggcc tggacccggc cgtcaccgcg gaggaccgga 4920ccaccgccag cggcatcggc atcctcgcct cgaagaacgc gatggcggcg cgccggaacg 4980acggcacgaa ccgcgacggc gacgcgggcg gccgtcgcta caaccgtgag ccgtacgccg 5040accacaccgg ctaccggccg gtcaacagcc cgtacgagct gcgcttcccg tcgcgctggc 5100agccgaacac catctccaag cgcgaggtcg tcctgacgca ggagttcgcg acgccccagt 5160tcggccgggt caagccgatc accttcgagc ggcccgagca gttccggctc accccgccgc 5220cgaaccacca cctgttgaac ccgaagggct accggaagca ggccgacgag gtgctgcgcg 5280cctcggcggg cctggacgac cgcaagaaga tgagcgcgga gatcttcagc gacaacatca 5340cgccgtacgg cgccatcgcg cacacgctcc tgcggggccg gtacaacacc gaggactccg 5400tccggttcat cgtgatgact gacgtcgccg ggttcgacgt ggcgatcgcg tcctggtact 5460acatgcgcaa gtacgactcg gtgcagccgt tcagcgcgat ccgccacctg tacccgaaca 5520agaagctgac cgcgtggggc ggcccgggcc ggggcaccgt caacgacatc accggcaccc 5580agtggcgcag ctacctcagc tcggtcgcca tcgcggctcc ggattacccg tcggtcaacg 5640cggcggtctg cgtcgcctac gcccaggtcg cgcgccggtt caccggcacg gacaagctga 5700ccgtcgtgat cccggtccgc aagggctcct cgatcgtgga accgggcgtg accccggccg 5760ccgacatgat gctcacctgg aacagctact cggagtgggc cgccgagtgc gggcagagcc 5820gggtctgggc cggcgagaac ttccccgcct cggtcgcggc cgccgaccag tacgcgccgc 5880agatcggcga ccgtgccttc gacttcgtcc agagcaagct gaacgggcgc tgacgcccgc 5940gtaccggtcc gtgctgccgg 596065532PRTMicromonospora sp. strain 046-ECO11 65Val Asp Pro Val Pro Val Leu Val Val Gly Ala Gly Pro Val Gly Met1 5 10 15Val Thr Ala Leu Ala Leu Ala Arg His Gly Val Ala Cys Val Leu Val 20 25 30Asp Gln Gly Phe Glu Thr Ser Val His Pro Lys Leu Asp Tyr Val Asn 35 40 45Ala Arg Ser Met Glu Phe Leu Arg Gln Phe Gly Leu Ala Asp Asp Val 50 55 60Arg Ala Ala Gly Val Ala Pro Glu His Arg Ala Asp Val Ile Trp Ser65 70 75 80Thr Gly Leu Ala Gly Glu Pro Ile Thr Arg Trp Gly Leu Pro Ser Val 85 90 95Thr Gln Glu Trp Arg Arg Ile Ala Glu His Asn Asp Gly Thr Gln Pro 100 105 110Ala Glu Pro Gly Gln Arg Ile Ser Gln Ile Asp Leu Glu Pro Val Leu 115 120 125Arg Ala Arg Cys Arg Arg Glu Pro Leu Val Asp Leu Arg Leu Gly Val 130 135 140Arg Phe Asp Ser Leu Thr Gln Asp Asp Ala Gly Val Thr Ser Val Leu145 150 155 160Ala Asp Asp Thr Gly Gly Glu Val Arg Val Arg Ser Glu Tyr Val Val 165 170 175Gly Cys Asp Gly Ala Ser Ser Gln Val Arg Arg Ala Val Gly Ile Gly 180 185 190Glu Glu Gly Phe Asp Val Pro Gly Leu Pro Gly Ala Phe Met Val His 195 200 205Phe Thr Ser Arg Asp Leu Asp Ser Leu His Arg His Gly Arg Phe Trp 210 215 220His Tyr Phe Ala Phe Arg Tyr Val Ile Ile Ala Gln Asp Glu Val Asp225 230 235 240Thr Trp Thr Ala His Val Asn Gly Val Asp Pro Asn Glu Phe Asp Glu 245 250 255Pro Pro Ala Asp Pro Glu Ala Phe Leu Leu Asp Thr Ile Arg Thr Glu 260 265 270Leu Arg Ile Asp Lys Val Leu Leu Thr Ser Arg Trp Arg Pro Gly Phe 275 280 285Met Leu Ala Asp Arg Tyr Arg Ala Gly Arg Val Leu Leu Ala Gly Asp 290 295 300Ser Ala His Arg Met Phe Pro Thr Gly Ala Tyr Gly Met Asn Thr Gly305 310 315 320Ile Gly Asp Ala Val Asp Val Ala Trp Lys Leu Ala Ala Val Val Arg 325 330 335Gly Phe Gly Gly Pro Gly Leu Leu Asp Ser Tyr Asp Ala Glu Arg Arg

340 345 350Pro Val Gly Arg Arg Asn Met Arg Thr Ser His Arg His Leu Gly Val 355 360 365His Leu Arg Ala Gly Glu Leu Leu Arg Gly Gly Ala Pro Leu Pro Ser 370 375 380Val Ala Ala Phe Leu Asp Ala Glu Arg Gly Glu Asn Glu Tyr Arg Gly385 390 395 400Ile Glu Leu Gly Tyr Arg Tyr Ser Gly Ser Pro Val Leu Trp Pro Glu 405 410 415Gly Pro Gly Glu Pro Ser Asp Asp Pro Arg Ala Tyr Ala Pro Thr Thr 420 425 430Trp Pro Gly Ala Arg Pro Pro Ser Leu Leu Leu Ser Asp Gly Gln Gln 435 440 445Ile Phe Asp Arg Phe Asp Pro Ala Ser Phe Thr Leu Val Asp Phe Thr 450 455 460Gly Asp Gly Ala Ala Gly Pro Leu Leu Ala Ala Ala Ala Ala Arg Gly465 470 475 480Leu Pro Val Thr His Thr Val Val Thr Asp Pro Arg Ala Arg Glu Leu 485 490 495Trp Glu Arg Asp Leu Val Leu Leu Arg Pro Asp His His Val Ala Trp 500 505 510Arg Gly Asn Thr Val Pro Pro Asp Pro Asp Ala Val Val Gln Arg Val 515 520 525Arg Gly Gly Gly 530661599DNAMicromonospora sp. strain 046-ECO11 66gtggatccgg taccggttct ggtcgtgggc gcgggcccgg tcggcatggt caccgcgctg 60gcgctcgccc gtcacggcgt cgcctgcgtc ctcgtcgacc agggcttcga gacgtcggtc 120catcccaagc tggactacgt caacgcccgc agcatggagt tcctccgcca gttcggcctc 180gccgacgacg tccgtgccgc cggcgtcgcg cccgagcacc gggccgacgt catctggtcg 240accggcctgg ccggtgagcc gatcaccagg tgggggctgc cctcggtgac gcaggagtgg 300cgccgcatcg ccgagcacaa cgacggcacc cagccggccg agcccggcca gcggatctcc 360cagatcgacc tggaaccggt cctgcgggcc cgctgccggc gggagcccct tgtcgacctg 420cgcctcggcg tacggttcga ctcgctgacc caggacgacg cgggggtcac cagcgtcctc 480gccgacgaca ccggcggcga ggtccgggtg cggtcggagt acgtggtcgg gtgcgacggc 540gcgtcgagcc aggtccgccg ggccgtgggc atcggtgagg aggggttcga cgtgcccggc 600ctgccgggcg ccttcatggt gcacttcacc agccgggacc tggacagcct gcaccggcac 660ggccggttct ggcactactt cgcgttccgg tacgtgatca tcgcccagga cgaggtcgac 720acctggaccg cgcacgtcaa cggcgtcgac ccgaacgagt tcgacgagcc gccggccgac 780ccggaggcgt tcctgctcga cacgatccgc accgagctgc ggatcgacaa ggtgctgctc 840acctcgcgct ggcgtcccgg cttcatgctc gccgacaggt accgcgccgg ccgggtgctg 900ctcgccggtg actcggccca ccggatgttc cccaccggcg cgtacggcat gaacaccggc 960atcggcgacg ccgtcgacgt ggcctggaag ctggccgctg tcgtccgggg cttcggcggc 1020cccgggctgc tcgacagcta cgacgccgaa cgccgcccgg tggggcggcg caacatgcgc 1080acctcgcacc ggcacctggg cgtgcacctg cgggcgggcg agctcctgcg cggcggcgcc 1140ccgctgccgt ccgtcgcggc cttcctcgac gccgagcggg gcgagaacga gtaccggggg 1200atcgagctcg gctaccgcta ctccggctcg ccggtgctct ggccggaggg cccgggggag 1260ccctcggacg acccgcgggc gtacgccccg acgacctggc ccggcgcccg tccgcccagc 1320ctcctgctga gcgacgggca gcagatcttc gaccggttcg acccggcctc gttcaccctc 1380gtggacttca ccggtgacgg cgccgccggt ccgctgctgg cggcggcggc cgcgcggggg 1440ctcccggtca cccacaccgt ggtgaccgac ccccgggctc gtgagctgtg ggaacgcgac 1500ctcgtcctgc tgcggccgga ccaccacgtc gcctggcggg gaaacaccgt gccgccggac 1560cccgacgccg tggtccagcg cgtgcggggt ggcggatag 159967423PRTMicromonospora sp. strain 046-ECO11 67Met Gln Gln Ser Gly Ser Thr Ala Glu Arg Ser Pro Leu Gly Pro Trp1 5 10 15Glu Gly Met Pro Ala Val Gln Gln Pro Asp Trp Gln Asp His Pro Ala 20 25 30Tyr Ala Glu Thr Cys Gln Ala Leu Ala Ser Ala Pro Pro Leu Val Pro 35 40 45Pro Gly Glu Val Arg Gly Phe Arg Gln Leu Leu Ser Glu Leu Ala Ser 50 55 60Thr Asp Gly Leu Leu Leu Gln Leu Gly Asp Cys Ala Glu Ser Leu Tyr65 70 75 80Glu Cys Thr Pro Arg His Thr Ser Asp Lys Ile Glu Val Ile Asp Arg 85 90 95Leu Gly Asp Arg Leu Ser Glu Leu Thr Gly Arg Asn Val Leu Arg Val 100 105 110Gly Arg Met Ala Gly Gln Phe Ala Lys Pro Arg Ser Gln Ala Thr Glu 115 120 125Trp His Asp Ala Leu Ser Ile Pro Ser Phe Arg Gly His Met Ile Asn 130 135 140Ser Glu Leu Ala Ala Pro Gly Thr Arg Lys Ala Asp Pro Arg Arg Met145 150 155 160Trp Trp Ala Tyr Glu Ala Ser Asp Arg Val Gln Arg Val Leu Arg Ala 165 170 175His Arg Glu Gly Asn Arg Arg Ala Ala Arg Thr Glu Gly Pro Trp Ser 180 185 190Ser His Glu Ala Leu Val Val Asp Tyr Glu Ser Arg Leu Ile Arg Arg 195 200 205Asp Pro Asp Thr Gly Glu His Tyr Leu Ala Ser Thr His Leu Pro Trp 210 215 220Val Gly Glu Arg Thr Arg Arg Ser Ala Glu Ala His Val Ala Met Leu225 230 235 240Ser Thr Val Val Asn Pro Val Gly Cys Lys Ile Gly Pro Asp Ala Asp 245 250 255Pro Asp Asp Val Leu Arg Val Cys Glu Ala Leu Asp Pro Arg Arg Asp 260 265 270Pro Gly Arg Leu Val Leu Ile Pro Arg Met Gly Arg Asp Arg Ile Arg 275 280 285Glu Ser Leu Pro Pro Ile Val Arg Ala Val Val Asn Ala Gly His Pro 290 295 300Val Leu Trp Leu Ser Asp Pro Met His Gly Asn Thr Val Lys Ala Ser305 310 315 320Val Gly Leu Lys Thr Arg His Leu Ser Asp Val Val Thr Glu Ala Leu 325 330 335Trp Phe Arg Asp Ile Leu Asp Gln Gln Arg Gln His Ala Ala Gly Leu 340 345 350His Ile Glu Val Ala Ala Thr Asp Val Thr Glu Cys Val Gly Gly Ser 355 360 365Val Ala Gly Glu Glu Asp Leu Ala Arg His Tyr Thr Ser Leu Cys Asp 370 375 380Pro Arg Leu Asn Pro Gly Gln Ala Thr Glu Leu Ile Glu Ala Trp Ala385 390 395 400Lys Asp Thr Ala Thr Val Gly Pro Gly Pro Arg Arg Ser Gly Pro Ser 405 410 415Ala Arg Pro Glu Val Ala Ala 420681272DNAMicromonospora sp. strain 046-ECO11 68atgcagcaat ccggttcaac ggcggaacgc agcccactcg ggccgtggga gggcatgccg 60gcggtccagc aaccggactg gcaggaccac ccggcgtacg cggagacctg tcaggcgttg 120gcgtcggccc cgccgctggt cccacccggg gaggtacggg ggttccggca gctgttgtcg 180gagctggcgt cgaccgacgg gctcctgctg cagttgggcg actgcgccga gagcctctac 240gagtgcaccc cccggcacac ctcggacaag atcgaggtca tcgaccggct gggggaccgg 300ctcagcgagc tcaccgggcg caacgtgctg cgggtgggcc ggatggccgg gcagttcgcc 360aagccccggt cgcaggcgac ggagtggcac gacgcgctga gcatcccctc cttccgcggc 420cacatgatca attccgagct ggccgcgccc ggtacgcgca aggccgaccc tcgccgcatg 480tggtgggcgt acgaggcgag cgaccgggtg cagcgggtcc tgcgcgccca ccgggagggc 540aaccggcgtg ccgcgcggac cgaggggccg tggtcgagcc acgaggccct ggtcgtcgac 600tacgagtccc gcctgatccg ccgggacccg gacacgggcg agcactacct ggcgtcgacc 660cacctgccgt gggtggggga gcggacccgc cggtccgccg aggcgcacgt ggccatgctg 720tccacggtgg tgaacccggt cggctgcaag atcgggccgg acgccgaccc ggacgacgtc 780ctgcgggtgt gcgaggcgct cgacccgcgg cgcgatccgg gccgtctcgt cctgatcccg 840cggatgggcc gggaccggat ccgggagtcc ctgccgccga tcgtccgcgc ggtggtgaac 900gcggggcacc ccgtgctctg gctgagcgat cccatgcacg gcaacaccgt caaggcctcg 960gtcggcctga agacgcgcca cctctccgac gtggtcaccg aggcgctgtg gttccgcgac 1020atcctcgacc agcagcggca gcacgccgcc gggctgcaca tcgaggtcgc cgccaccgac 1080gtgaccgagt gcgtcggcgg ttcggtggcc ggcgaggagg acctggcgcg gcactacacc 1140tcgctgtgcg acccgcggct caacccgggt caggccaccg agctgatcga agcgtgggcc 1200aaggacaccg cgacggtcgg cccgggaccg cggcgctccg gcccttcggc gcggccggag 1260gtcgccgcct ga 127269340PRTMicromonospora sp. strain 046-ECO11 69Met Trp Gly Ser Ser Asn Thr Leu Glu Val Lys Gly Asn Asp Glu Arg1 5 10 15Phe Pro Leu Pro Asp Ala Ala Thr Glu Asp Arg Ser Val Leu Gly Glu 20 25 30Thr Val Pro Val Ser Ala Leu Leu Pro Gly Asp Ser Pro Arg Leu Ala 35 40 45Gly Glu Asn Val Glu His Ile Arg Leu Leu Ala Ala Met His Asp Leu 50 55 60Pro Pro Ile Leu Val Gln Arg Gly Thr Met Arg Val Ile Asp Gly Met65 70 75 80His Arg Leu Arg Ala Ala Lys Leu Arg Gly Asp Glu Thr Val Arg Val 85 90 95Thr Phe Phe Asp Gly Asp Asp Ala Ala Ala Phe Leu Leu Ser Val Asp 100 105 110Ala Asn Ile Lys His Gly Leu Pro Leu Ser Arg Ala Asp Arg Glu Ala 115 120 125Ala Ala Thr Arg Ile Leu Arg Leu Tyr Pro Gln Trp Ser Asp Arg Ala 130 135 140Val Ala Ala Ala Ala Gly Leu Ser Pro Thr Thr Ala Ser Gly Ile Arg145 150 155 160Arg Arg Leu Leu Gln Pro Ala Ala Arg Glu Gly Ser Arg Val Gly Arg 165 170 175Asp Gly Arg Val Arg Pro Leu Asp Gly Ser Ala Gly Arg Arg Arg Ala 180 185 190Ser Ala Val Ile Ala Leu Arg Pro Asp Ala Pro Leu Arg Ala Ile Ala 195 200 205Gln Glu Ala Gly Val Ser Val Gly Thr Ala Arg Asp Val Arg Ala Arg 210 215 220Leu Gln Ala Gly Arg Asp Pro Val Leu Thr Ser Gln Arg Pro Ala Ala225 230 235 240Glu Pro Glu Pro Ala Ala Asp Asp Gly Pro Glu Ala Arg Arg Arg Arg 245 250 255Leu Gly Gln Pro Ser Val Pro Pro Val Asp Trp Pro Ala Val Arg Gly 260 265 270Asn Leu Ile Arg Asp Pro Ala Val Lys Tyr Ala Glu Leu Gly Arg Ala 275 280 285Phe Val Arg Trp Ala Asp Gly His Val Val Asp Pro Ala Ala Trp Arg 290 295 300Glu Phe Val Asp Ala Val Pro Pro Tyr Trp Arg Lys Ser Val Ala Glu305 310 315 320Leu Ala Arg Ser Cys Ala Ser Ala Trp Leu Ala Phe Ala Gln Glu Leu 325 330 335Glu Asp Arg Ala 340701023DNAMicromonospora sp. strain 046-ECO11 70atgtggggca gctcaaacac gctggaagtg aagggcaacg acgagagatt ccccctgccc 60gatgcagcta cggaggatcg gtctgtgctt ggcgagacgg ttccggtttc cgcgctgctg 120cccggtgact ccccgcggct ggcgggcgag aacgtcgagc acatccggct gctggccgcg 180atgcacgacc tcccgccgat cctggtgcaa cgcggcacga tgcgggtgat cgacggcatg 240caccggctgc gggccgccaa gctgcgcggc gacgagaccg tgcgggtgac gttcttcgac 300ggggacgacg ccgcggcgtt cctgctctcg gtcgacgcca acatcaaaca cgggctgccg 360ttgtcccgcg ccgaccggga ggccgccgcc acccgcatcc tgcggttgta tccgcagtgg 420tcggaccgcg ccgtcgccgc ggcggccggg ctgtcaccga ccacggcgag cggcatccgg 480cgccgcctgc tgcaaccggc ggcgcgggag ggcagccggg tgggacggga cgggcgggtg 540cgcccgctgg acggctcggc gggccgacgg cgggccagcg cggtcatcgc gctccggccg 600gacgcgcccc tgcgtgccat cgcgcaggag gccggggtgt cggtgggcac ggcgcgggac 660gtgcgcgccc ggttgcaggc gggccgggac cccgtcctga cctcgcagcg accggcggcc 720gagcccgagc cggccgccga cgacgggccg gaggcgcgca gacgccggct cggccagccc 780tccgtgccgc ctgtcgactg gccggcggta cggggcaacc tgatccggga ccccgcggtg 840aagtacgccg agctgggccg ggccttcgtc cgctgggccg acgggcacgt ggtggatccg 900gcggcctggc gcgagttcgt cgacgccgtg ccgccgtact ggcgcaaatc ggtggccgag 960ctggcccgtt cgtgcgccag cgcctggctg gcgttcgccc aggaactgga ggaccgggcg 1020tga 102371493PRTMicromonospora sp. strain 046-ECO11 71Val Asn Ile Leu Arg Arg Pro Arg Lys Arg His Leu Gly Gly Val Ala1 5 10 15Ala Val Ala Ala Ala Ile Ala Leu Val Ala Ser Leu Thr Asn Gly Val 20 25 30Ala Ala Ala Pro Gln Ala Pro Thr Phe Asp Leu Asp Asn Gly Asn Ala 35 40 45Leu Thr Asp Val Ile Tyr Pro Ala Leu Asn Thr Glu Pro Arg Val Glu 50 55 60Tyr Ser Gly Arg Pro Gly Ser Trp Ala Ala Asp Arg Ala Met Leu Ile65 70 75 80Glu Leu Pro Trp Phe Asp Ala Leu Ala Ala Tyr His Pro Thr Ala Val 85 90 95Gly Ile Phe Ser Thr Ile Gly Arg Arg Pro Ala Glu Glu His Thr Thr 100 105 110Arg Asn Lys Asn Ile Ala Val Ile Tyr Ser Ala Tyr Thr Ser Leu Ser 115 120 125Lys Leu Tyr Pro Gln His Glu Ala Thr Trp Gln Arg Met Met Ala Thr 130 135 140Ala Gly Leu Asp Pro Ala Val Thr Ala Glu Asp Arg Thr Thr Ala Ser145 150 155 160Gly Ile Gly Ile Leu Ala Ser Lys Asn Ala Met Ala Ala Arg Arg Asn 165 170 175Asp Gly Thr Asn Arg Asp Gly Asp Ala Gly Gly Arg Arg Tyr Asn Arg 180 185 190Glu Pro Tyr Ala Asp His Thr Gly Tyr Arg Pro Val Asn Ser Pro Tyr 195 200 205Glu Leu Arg Phe Pro Ser Arg Trp Gln Pro Asn Thr Ile Ser Lys Arg 210 215 220Glu Val Val Leu Thr Gln Glu Phe Ala Thr Pro Gln Phe Gly Arg Val225 230 235 240Lys Pro Ile Thr Phe Glu Arg Pro Glu Gln Phe Arg Leu Thr Pro Pro 245 250 255Pro Asn His His Leu Leu Asn Pro Lys Gly Tyr Arg Lys Gln Ala Asp 260 265 270Glu Val Leu Arg Ala Ser Ala Gly Leu Asp Asp Arg Lys Lys Met Ser 275 280 285Ala Glu Ile Phe Ser Asp Asn Ile Thr Pro Tyr Gly Ala Ile Ala His 290 295 300Thr Leu Leu Arg Gly Arg Tyr Asn Thr Glu Asp Ser Val Arg Phe Ile305 310 315 320Val Met Thr Asp Val Ala Gly Phe Asp Val Ala Ile Ala Ser Trp Tyr 325 330 335Tyr Met Arg Lys Tyr Asp Ser Val Gln Pro Phe Ser Ala Ile Arg His 340 345 350Leu Tyr Pro Asn Lys Lys Leu Thr Ala Trp Gly Gly Pro Gly Arg Gly 355 360 365Thr Val Asn Asp Ile Thr Gly Thr Gln Trp Arg Ser Tyr Leu Ser Ser 370 375 380Val Ala Ile Ala Ala Pro Asp Tyr Pro Ser Val Asn Ala Ala Val Cys385 390 395 400Val Ala Tyr Ala Gln Val Ala Arg Arg Phe Thr Gly Thr Asp Lys Leu 405 410 415Thr Val Val Ile Pro Val Arg Lys Gly Ser Ser Ile Val Glu Pro Gly 420 425 430Val Thr Pro Ala Ala Asp Met Met Leu Thr Trp Asn Ser Tyr Ser Glu 435 440 445Trp Ala Ala Glu Cys Gly Gln Ser Arg Val Trp Ala Gly Glu Asn Phe 450 455 460Pro Ala Ser Val Ala Ala Ala Asp Gln Tyr Ala Pro Gln Ile Gly Asp465 470 475 480Arg Ala Phe Asp Phe Val Gln Ser Lys Leu Asn Gly Arg 485 490721482DNAMicromonospora sp. strain 046-ECO11 72gtgaacattc tgaggcggcc gcggaaacgg catctcgggg gtgtcgcggc cgtcgccgcg 60gcgatcgccc tggtggcgtc gctgacaaac ggtgtggcgg ctgccccgca ggcgccgacc 120ttcgacctcg acaacgggaa cgccctgacc gacgtcatct acccggccct caacaccgag 180ccgcgggtcg agtacagcgg ccggcccggg tcctgggccg cggaccgcgc catgctcatc 240gaactgccgt ggttcgacgc cctggcggcg taccacccca ccgcggtcgg catcttctcc 300accatcggcc gccgtcccgc cgaggagcac acgacgcgca acaagaacat cgccgtcatc 360tactcggcct acacctcgct cagcaagctc tacccccagc acgaggcgac ctggcagcgg 420atgatggcca ccgcgggcct ggacccggcc gtcaccgcgg aggaccggac caccgccagc 480ggcatcggca tcctcgcctc gaagaacgcg atggcggcgc gccggaacga cggcacgaac 540cgcgacggcg acgcgggcgg ccgtcgctac aaccgtgagc cgtacgccga ccacaccggc 600taccggccgg tcaacagccc gtacgagctg cgcttcccgt cgcgctggca gccgaacacc 660atctccaagc gcgaggtcgt cctgacgcag gagttcgcga cgccccagtt cggccgggtc 720aagccgatca ccttcgagcg gcccgagcag ttccggctca ccccgccgcc gaaccaccac 780ctgttgaacc cgaagggcta ccggaagcag gccgacgagg tgctgcgcgc ctcggcgggc 840ctggacgacc gcaagaagat gagcgcggag atcttcagcg acaacatcac gccgtacggc 900gccatcgcgc acacgctcct gcggggccgg tacaacaccg aggactccgt ccggttcatc 960gtgatgactg acgtcgccgg gttcgacgtg gcgatcgcgt cctggtacta catgcgcaag 1020tacgactcgg tgcagccgtt cagcgcgatc cgccacctgt acccgaacaa gaagctgacc 1080gcgtggggcg gcccgggccg gggcaccgtc aacgacatca ccggcaccca gtggcgcagc 1140tacctcagct cggtcgccat cgcggctccg gattacccgt cggtcaacgc ggcggtctgc 1200gtcgcctacg cccaggtcgc gcgccggttc accggcacgg acaagctgac cgtcgtgatc 1260ccggtccgca agggctcctc gatcgtggaa ccgggcgtga ccccggccgc cgacatgatg 1320ctcacctgga acagctactc ggagtgggcc gccgagtgcg ggcagagccg ggtctgggcc 1380ggcgagaact tccccgcctc ggtcgcggcc gccgaccagt acgcgccgca gatcggcgac 1440cgtgccttcg acttcgtcca gagcaagctg aacgggcgct ga 1482739762DNAMicromonospora sp. strain 046-ECO11 73cagccacggc gttccgaccc cccgcaagat ggcttgtata gcaaggtatc ttgcgatgca 60tggacggggc acgtgagcgg atcactacga acatccgcaa gggcgtgctg gagtactgcg 120tgctcgccct gctctcgcgg cgcgacatgt acggcctgga actggccgac tggctcgccg 180tccgcggtct gaccgcgagc gagggcagcc tgtatccgct gctcgcccgc atgcggcagg 240ccggctccgt gcagacccgg tgggtggccc ccgagcaggg gcacgcccgg cggtactacg 300cgatcaccga ccaggggcgg gcgcacctgc gggtgttcgc ggcggtgtgg caggagatcc 360agccgcacgt ggacgacctg atgggggagg aagcatgagc gacgacggcc tcccggaggc 420ggcgtggacc

tatctgcgcg cgctcgacgc ggagttgtcc gacgtcccgt ccggcacggc 480ggaggagatc gtcgcggatg tccgcgcgca catcgccgac gccctcgaca gcggacggag 540cgcccacgag atcctcgccg gcctcggcgc cgcgcgggac gtggcccggc aggcgcgcga 600ggagctgggg ctgccggccc aggaccgccc ggcccgggcc ggccggaccc tgtccctggc 660cgcggtggcg gtcggcgtgc tgatcgccgt gtgcgtgagc ttcctgctgc cgtccgcagt 720gccggtggag ccgatccagg ccggccccgg cgagcagggc gtcctccgcc ggctcggccc 780cggaatcgcg ctgctcacgc tgctgccggc gctcgtcgcg gccgcgccgc tcgtggcgcc 840cgcccgggca cgtgccgggg tacggttcgc cggcgcggcg gtcctgacga tgttcgcctg 900cgcggccggc gagacgggcc tgtactactt cccgctcgcg ctgatggcct gggcggcggc 960gatcgtgccg tgggccctgc ggcgcggagc cggtggacgg tggtggcgct atctgaccgg 1020tggattcgtg gcgatgcccg gcgtgctggt ggcggtcgcg tcggccggtg gctcggtcgg 1080cgtcggctgg gtcggcgcgg cgctgtggat cgccgggccg ctcgcggccg gcgcgctgtg 1140cgcctacggg atccgggccg gctacgccgt gaccgcgctg gccggcgcgc tggccatagc 1200gctctcgatg gccgagcgcg gcttcctgtt cgccgccttc tggctgttcg gcgggctgta 1260cctggcgctc ggcgccgctg cgtacaccgc ctcgcgggcc gtcgacggcg acgccgccgc 1320gacgcccggc ccgccggccc ggccggaacc cgcgccggcc cccggaggct gacccggggg 1380ccgtggcgcc ggccggctag gcggggacgg cctgcgggtc gccggcggcg tcgtgcgcgg 1440ccatcgtctc ctgccggacg ggctcctcgc gcaggatcgc cgcgtgcagc cacgcgtccg 1500ggatggcgaa gccgtccacg agcgtgcgca tgtccgggcg cagctccttg agcagcccgt 1560tcaccacgct ggtgatggtc ttcgagcggg ccggggtgag ccggccgtgc tcgagcagcc 1620agcccttgtt cgcctcgatc acggtgagcg cgtacaggtc gcagacccgg gacagcagtt 1680ccttgaccgc cgggtcggcg atggcgtcga tcccggcgac gaacgcctcc agcgtcaccc 1740ggtcgatgtg cgccgcggcg acggcgagga cgtggtcctg gacgtcgttg aagatgtcga 1800aggggcggtc cttcttggtg gacgcgccac cgcgcaggcg gcggaccgcg ctgtcgagca 1860ggtgctcctc gcggtcctcg aagagcttga gctgccagcc ccggtcggtg acggcgacct 1920cgtcgtcgcg cccgggcacg gcgctgacca gacgtgcgat cagcgcccgc gcggcggtgc 1980gttccagcac catctcgcgt acctgctcgg ccacgaagga ggcgcgtccc cagccgtcga 2040gcgagccgaa ctcgtcccgg tagccggtca gcagcccctt ggcgaccagt tgcagcagca 2100ccgtgttgtc gccctcgaag gtggtgaaga catcggtgtc ggccttgagg ctgggcaggc 2160ggttctcgga caggtagccg gcgccgccac acgcctcccg gcagatctgg atggtgcggg 2220tggcgtgcca ggtctgcgcc gccttcagac cggcggcccg ggactccagc tcccgctgcc 2280ggtgctcgtc gaccggcccg tcgccgccct ggatgtcgtc gagcgccgcg accagctccg 2340cctgggcgaa ggtcagcgcg tacgtggtgg ccagcgcggg cagcagcttg cgctggtgcg 2400ccaggtagtc gttgagcagc acctcgcggt cgccgtcggc gtcggcgaac tgccggcgga 2460tgtcgccgta gcgcaccgcg atggccagcg ccgacttggt ggccgccgac gcggcgccgc 2520ccacgctcac ccggccccgg accagggtgc ccagcatggt gaagaagcgc cgggagtcgt 2580tctcgatcgg gctggagtac gtgccgtcct cggcgacctg cgcgtactgg tccagcagca 2640tctcccgcgg cacccgcacg tggtcgaagc tgagccgccc gttgtccacg ccgagcaggc 2700cggccttggg cccggcgtcg ccgatggtca cgccgggcat cggcttgccg tgctcgtcgc 2760ggatcggcac cagccaggcg tgcaccccgt ggcggcgccc gccggtgacg agctgggcga 2820acaccacagc catccgcccg tcccgggccg cgttgccgat gtagtccttg cgcgcggcct 2880cgtgcggggt gtgcaggtcg aaggtctgcg tctgcgggtc gtagacgcag gtggtgcgca 2940gttgctgcac gtccgagccg tggccggtct cggtcatcgc gaagcagccg aagagccggc 3000ccgcgacgat gtcccgcagg taggcgtcgt ggtgccgctt cgtgccgagg gcggcgaccg 3060cgccgccgaa caggccccac tgcacgccgg ccttcaccat cagtgacagg tccacctggg 3120ccagcatctc ggtggcgacg atcgaggcgc ccacgtcgcc gcggccgccg tactcggcgg 3180ggaaaccgga ggcgatgccc agctcgacgg ggagttcgga cagcagccgg gtgatgcgct 3240cgcgggcctg gtcaccggtc tcgccgtaca ccgggaggaa gcgttcgtcg aggtgttcgc 3300ggtgcgcccg gcggacctcg gcccaccggc cgtcgagcgc ttcccgcagg cgtgtgacgt 3360cgatgcggcc ggatgcgtga tcgagcattg tcactcctcg gggcagcgga catttgcgta 3420tactctcggc ctgatcaaca ttaccggcgg tgatcgcacc ccgctggcgg agcgcgtggt 3480gagcccggcc acccccggcg gttcggccac ccgtgaagct gaggttaggc tgtcctcact 3540tcacagcact ggaggcatcc cctcgtgtcc ccgcttcccc ccggcagcgc cgtcaccgcc 3600cggcacgtgc tccgccaggc gctgcgccgc cagcgccgcc cggtgctgat cggcgtgacc 3660ctgctcgggc tgcaccaggt caccgaggcg ctcgtgccgg tggcgatcgg cgtcatcatc 3720gaccgggccg tggtgaccgg cgacccgtgg gcgctcgcgt actccgtcgc cggcctcgcc 3780gccctgttca ccgtgctggc gttcgcctac cgcaacggcg cccgccaggc gttcgcggcg 3840gtggaacggg aggcgcacct gctgcgggtc gagctggccg agcgcgcgct cgacccgcgc 3900gggcaccgct ccggcctgcg cgacggcgag ctgctctcgg tcgccgcctc cgacgccgaa 3960ctctccgcgt acgtggtccg ggtggccggc ttcggcgtcg ccgcggtgag cgcgctgacc 4020gtcgcggcgg tcgcgctgct ggtcatcgac gtcccgctcg gactcggcgt gctcatcggc 4080gtaccggtgc tggtcctggc gctgcaacgg atggcgccgc tgctgtcccg gcgcagcgcc 4140tcccagcagg aggccctcgc ggagaccacg gcgctcgccg tggacctcgt ctccggcctg 4200cgcgtgctgc gcggcatcgg cgcccagcac cacgccgccg gccggtacgc cgaggccagc 4260cgacgcgccc tcgccgtgac gctgcgcgcc gccaacacca agggcctgca cctcgggctc 4320accaccgccg cgaacggcct cttcctcgcc gccgtcgccg gggtcgccgg ctggctcgcg 4380ctgcgcggcc ggctcaccat cggcgagctg gtcaccgtgg tcgggctcgc gcagttcgtc 4440gccgagccgg tgcagacgct gggctactgc gtgcagctgt tcgcgatggc ccgcgcctcc 4500gccgcccggg tcgggcgcgt gctcggcgcc gagccgctga cccggccggg cagcgcgccc 4560cggccggacc gcacggacgg gccgcggctc gtcctcgacc acgtcggcca cgccgcgctg 4620gacggggtgt gcctgcgcgt cgacccggga gagatcgtcg gcgtcctggc gtacgacccg 4680gccgacgcgg acgcgctggt ggcgctgctg tccgggcggg tgcccgcgga ccggcgccgg 4740ggcacggtac gcgtcgacgg ggtacccgcc gacgacctgg acgtcgacgc gctgcgcggc 4800gccgtcctgg tcgagccgca cgacgtgacg ctgttcgagg gaaccgtggc cgccaacctc 4860gccgccggga gcaggaccga ggaggggcgc ctgcgcgccg cggtccgggc ggccgcggcg 4920gacgacgtgg tggacgcgca ccccggcggc ctcggccacc ggctcgtcga gcggggcgcc 4980aacctctccg gcgggcagcg ccagcggctc gggctggcgc gggcgctgca cgccgacccg 5040ccggtgctgg tgctgcacga ccccaccacc gccgtggacg cggccaccga ggcccaactc 5100gccgacggac tggccggcgc gcgccgcgaa gcgccccggg gcacgctgct ggtcaccagc 5160agccccgccc tgctgcggat caccgaccgg gtggtggtga tcgccgacgg ccgggtgacc 5220gccgagggga cgcacgagca cctgctggcc accgacgccc gctaccgcga ggagacactg 5280cggtgaccgc tgacccgcgt accgccgaac ccacccgggt gttgctgccc accgcgaccg 5340cccggcggac ctggacgacg ctcggcgcgg agttccgccg gcggcccggc ctcagcgccg 5400ccgcgaccgc cgtgctcgtc gccgccgcca ccggcgggct ggtcgcgccc tgggtgctcg 5460gccgcctcgt cgacgacgtc atcgccgacg ccccggtctc ccggatcgcc ggccgggtgg 5520cggtgatcgc cggcgcggca gtgctcaccg gactgctcac cgccgccggg gccgcgctcg 5580cgtcccgcct gggggagacg gtgctggccc ggctgcgcga gcgggtcctc gaccgggcgc 5640tgcacctgcc ctcggcgacg ctggaacggg ccggcaccgg cgacctgctg gcccgggtcg 5700gcgacgacgt ggcggtggtg acgaacgtga tcgcggtcag cggcccggcg ttcgtcggcg 5760cgctgctgtc cgtggtgctg accgtgttcg ggctggtcgc gctcgactgg cggctcggcc 5820tcgccgggct ggtcgccgcg cccgcctacg cgctggcgct gcgctggtac ctgcgccggt 5880cggcgccgta ctacgcccgc gagcgcgtcg ccaccggcga gcggacgcag gcgatggccg 5940gcgcgctgcg tggcgcggcc accgtgcgcg cgtaccggac cgaggacgcg cacgtcgcgg 6000cgatcgccga gcgctccggc gtggcgcgcg acctgtcgct ggagatcttc aacctgcaca 6060cccggttcgg gctgcggatc aacaggtcgg agttcctcgg cctggccgcg gtgctcgtcg 6120ccgggttctt cctggtccgc gccgacctgg tcacagtggg cgcggcgacc accgccgcgc 6180tctacttcca ccggctgttc aacccgatcg gcctgctgct gatggagtcc gactcggtgc 6240tgcaggccgg cgcgagcctc gcccggctgg tcggcgtggc cacgctgccc gacaccgccc 6300cgtccgggcc cgcgccgtcg gcggccgggc ggcgcggccc ggcggcgctg gacgtcacgg 6360tccgccggca ccgctacgac gacgacggcc ctctggtcct ggccgacgtc gacctgcgcc 6420tggccccggg cgagcgggtc gcgctcgtgg gcgccagcgg cgcgggcaag agcacgctcg 6480ccggcatcgc cgccgggatc atcgcgccca ccgacgggtc ggtacgcctg ggcggcgtgc 6540cgctgaccga gcggggcgag cacgccgtgc ggcgcgacgt cgcgctggtc agccaggagg 6600tgcacgtctt cgctggaccg ctcgccgagg atctgcgcct ggctgccccg gacgccaccg 6660acgccgaact gctcgacgcg ctggaccggg tcggcgccac cacctggctg cgcgcgctgc 6720cggacgggct ggccacagcg gtcggcgagg gcggccaccg gctcaccgcc gcgcaggccc 6780agcaggtcgc cctggcccgg ctggtgctgg ccgcgcccgc cgtcgccgtg ctggacgagg 6840ccaccgccga ggccggcagc gccggagcgc gtgacctgga ccgggcggcg ctggccgcca 6900ccgagggacg gaccacgctg atcgtggcgc accggctcag ccaggcggtc gccgccgacc 6960ggatcgtcct gctcgaccac gggcggatcg tggagcaggg cacgcactcg gaactgctcg 7020ccgccgacgg ccggtacggg catctgtggc gctcctggag cgtcccggta tgatcgcgca 7080ccgcccatcg gcccaggtga ggggaacatg accgacgcgc cggcccgctt cgtgctcttc 7140ccggggcggc accacctgct gacccggttc caggccgact acctgcggcg gctggccggg 7200gacgacgcca cagtggtctg ggcggtgacg tcggccaacc acgagaacac caggcgcaac 7260ccggtgccct accaccggcg ggaggccgcg atcgaacgat tcagcgtgct gagcgggctg 7320cgctcggtgg tggtgccgat cttcgacacc gcgtacaccg acgcgttcgc cgaggtgacg 7380ctgaagtcca tcgcggtggc caccgggctc gaactcaccc ccgccgacac cgtgctggcc 7440tgctccacgc cggaggtcgc gaagctgtac gagcagctcg gcttttcgat cgcgccggtc 7500gaggcggacc cggacctgcc cgagccgccc gaacggccgt gggacgtgct gctgcgcctg 7560gccgccgggg acgagacctg gcgcgcgctc acccacccgg ccaccatcga cgtgttcgag 7620cgctaccgcc tggtcgagtc gatccggtcg gtggtgaacg acccgctcgt cggcgacgag 7680ggcggtctca cagtgacccg cgactaccgg acctacgtcg aggcgttcgc cacggccgcg 7740cagcgcaagt gggactcggt acgccggtac gtgcagcccg gccgcatcgt ggacatcggc 7800tgcggcgcgg gcgccgtcct ggaactcgcc gaccgggagg ccgcgctgcg tgagagcgac 7860ctgatcggcg tggaggtcgc ccgccacctc taccaggagt gcctgcacaa gaaggcgcag 7920ggcgtgttcc gcaacgccaa cgtctacttc ttccaccgca acgtcctcgg cggcgcggtg 7980ttcaaggacc gctcggtcga caccacgctc acgttcgcgc tgacccacga gatctggtcg 8040tacgggcggc ggcgggagtc gctgctgcag ttcgcccgcc gcatccacga ccacacggtg 8100cccggcggcg tctggatcaa cagcgacgtg tgcggtccgg acgacccccg gcggcaggtg 8160ctcctgcgac tgtccaccga cgacggcgac aacccggccg cgccccgccc cgacctcgcc 8220gagctgacct cggcggaggt ccggcgttac gtcggcgggc tgtcgacgcg ggcgcggctg 8280gaccagttcg ccgtcgactt cgcgttcgac ttcgactacg agccgctccc cgacggcgcg 8340gtacgcctga cgctgggcgc cgcgatggac tacctgaccc gcaaggacta cacggacaac 8400tggctgtcgg agacgcagga gcagttctgc ggcctgagct tcgccgactg gacggacctg 8460ctcaccgagg cggggttcga gatcggcccg gcgtcggcgc cggtgcgcaa cgagtgggtg 8520atcgacaacc ggatcgcgcc agtcgcgtcc ctcaccgacc tcgacggccg gccgctggac 8580tggccgacca cccacgtcct caccgtcgcc caccgccccc gcaaccagtg agaccgacgg 8640cgcccgccgc gttcggcggg cgccgtcgtc gctcaccggc tcagcgcgat ccggatcgcc 8700aggacgatca ggatgagccc ggtcagccgt tcgatcacca gcagcacgga cggccgggtc 8760agccagggct gcaacctgtc gatgagcatg atgtagcagg cccaccagag caccgcgagg 8820ccgatgaacg tggcggcgag caccgccgta cgggccgccg ccccctcgcc gggcttgacg 8880aactgcggca cgaacgagac gtagaagacg accaccttga cgttcagcag ctggctggtg 8940acgcccatga cgaacgagcg gcgggccacg tgcggctcgt cggcggccgg ggtgtccggc 9000accggcgcgg ggccggtgtc cgtgtccggc ccggcgccgc ccgcgccgac agtgaccggc 9060tgcgccgccg ggaccgtccg gcgcggccgg gtcgcccaga ggatcgtgcc gcccaggtag 9120agcaggtaca gcgcgccggc gacgcgcagc accgtgtaga gcgtcggcga ggagaccagc 9180agggcggaca ggccggcggt cgcgaacgac gcgtgcacca gcgcggcgac gaacagcccg 9240gccagcacca cgaacccggc ccgccggccg tacctgacgg tctgccgggt gacgagcgcg 9300aagtcgacgc ccggcacgat gatgatgagc aggctggcgg cgacgaaact gatgatctgg 9360atgtcagaca cgacgccggc tctcctgtcc tccggcgagc gccggcactg cctcctcgat 9420gacggagacg ccgctgtcct ggcgtggtcc gtgccggcgc cactgttccc gcagccggat 9480ccggccgtcc ggcagccgtt cgggccggga ctcgcactcg ccgatgacta tggtgccgtc 9540ggtgagcacc tccaggtagg cgaagcgcac gacgccctgc gcgtcgcagg tgccggccag 9600ccggccgtgc cggaccgggc cgccggtgat ctccgcccag accaggtcgc cacgctggtg 9660gtagtgcccc cgcagcggct cggcgccgtc accggcgtcg tggtccaccg agacgaagac 9720gcggccgtcg tagtcgaatg tcgtcatcgc gctcacgccc ac 976274112PRTMicromonospora sp. strain 046-ECO11 74Met Asp Gly Ala Arg Glu Arg Ile Thr Thr Asn Ile Arg Lys Gly Val1 5 10 15Leu Glu Tyr Cys Val Leu Ala Leu Leu Ser Arg Arg Asp Met Tyr Gly 20 25 30Leu Glu Leu Ala Asp Trp Leu Ala Val Arg Gly Leu Thr Ala Ser Glu 35 40 45Gly Ser Leu Tyr Pro Leu Leu Ala Arg Met Arg Gln Ala Gly Ser Val 50 55 60Gln Thr Arg Trp Val Ala Pro Glu Gln Gly His Ala Arg Arg Tyr Tyr65 70 75 80Ala Ile Thr Asp Gln Gly Arg Ala His Leu Arg Val Phe Ala Ala Val 85 90 95Trp Gln Glu Ile Gln Pro His Val Asp Asp Leu Met Gly Glu Glu Ala 100 105 11075339DNAMicromonospora sp. strain 046-ECO11 75atggacgggg cacgtgagcg gatcactacg aacatccgca agggcgtgct ggagtactgc 60gtgctcgccc tgctctcgcg gcgcgacatg tacggcctgg aactggccga ctggctcgcc 120gtccgcggtc tgaccgcgag cgagggcagc ctgtatccgc tgctcgcccg catgcggcag 180gccggctccg tgcagacccg gtgggtggcc cccgagcagg ggcacgcccg gcggtactac 240gcgatcaccg accaggggcg ggcgcacctg cgggtgttcg cggcggtgtg gcaggagatc 300cagccgcacg tggacgacct gatgggggag gaagcatga 33976325PRTMicromonospora sp. strain 046-ECO11 76Met Ser Asp Asp Gly Leu Pro Glu Ala Ala Trp Thr Tyr Leu Arg Ala1 5 10 15Leu Asp Ala Glu Leu Ser Asp Val Pro Ser Gly Thr Ala Glu Glu Ile 20 25 30Val Ala Asp Val Arg Ala His Ile Ala Asp Ala Leu Asp Ser Gly Arg 35 40 45Ser Ala His Glu Ile Leu Ala Gly Leu Gly Ala Ala Arg Asp Val Ala 50 55 60Arg Gln Ala Arg Glu Glu Leu Gly Leu Pro Ala Gln Asp Arg Pro Ala65 70 75 80Arg Ala Gly Arg Thr Leu Ser Leu Ala Ala Val Ala Val Gly Val Leu 85 90 95Ile Ala Val Cys Val Ser Phe Leu Leu Pro Ser Ala Val Pro Val Glu 100 105 110Pro Ile Gln Ala Gly Pro Gly Glu Gln Gly Val Leu Arg Arg Leu Gly 115 120 125Pro Gly Ile Ala Leu Leu Thr Leu Leu Pro Ala Leu Val Ala Ala Ala 130 135 140Pro Leu Val Ala Pro Ala Arg Ala Arg Ala Gly Val Arg Phe Ala Gly145 150 155 160Ala Ala Val Leu Thr Met Phe Ala Cys Ala Ala Gly Glu Thr Gly Leu 165 170 175Tyr Tyr Phe Pro Leu Ala Leu Met Ala Trp Ala Ala Ala Ile Val Pro 180 185 190Trp Ala Leu Arg Arg Gly Ala Gly Gly Arg Trp Trp Arg Tyr Leu Thr 195 200 205Gly Gly Phe Val Ala Met Pro Gly Val Leu Val Ala Val Ala Ser Ala 210 215 220Gly Gly Ser Val Gly Val Gly Trp Val Gly Ala Ala Leu Trp Ile Ala225 230 235 240Gly Pro Leu Ala Ala Gly Ala Leu Cys Ala Tyr Gly Ile Arg Ala Gly 245 250 255Tyr Ala Val Thr Ala Leu Ala Gly Ala Leu Ala Ile Ala Leu Ser Met 260 265 270Ala Glu Arg Gly Phe Leu Phe Ala Ala Phe Trp Leu Phe Gly Gly Leu 275 280 285Tyr Leu Ala Leu Gly Ala Ala Ala Tyr Thr Ala Ser Arg Ala Val Asp 290 295 300Gly Asp Ala Ala Ala Thr Pro Gly Pro Pro Ala Arg Pro Glu Pro Ala305 310 315 320Pro Ala Pro Gly Gly 32577978DNAMicromonospora sp. strain 046-ECO11 77atgagcgacg acggcctccc ggaggcggcg tggacctatc tgcgcgcgct cgacgcggag 60ttgtccgacg tcccgtccgg cacggcggag gagatcgtcg cggatgtccg cgcgcacatc 120gccgacgccc tcgacagcgg acggagcgcc cacgagatcc tcgccggcct cggcgccgcg 180cgggacgtgg cccggcaggc gcgcgaggag ctggggctgc cggcccagga ccgcccggcc 240cgggccggcc ggaccctgtc cctggccgcg gtggcggtcg gcgtgctgat cgccgtgtgc 300gtgagcttcc tgctgccgtc cgcagtgccg gtggagccga tccaggccgg ccccggcgag 360cagggcgtcc tccgccggct cggccccgga atcgcgctgc tcacgctgct gccggcgctc 420gtcgcggccg cgccgctcgt ggcgcccgcc cgggcacgtg ccggggtacg gttcgccggc 480gcggcggtcc tgacgatgtt cgcctgcgcg gccggcgaga cgggcctgta ctacttcccg 540ctcgcgctga tggcctgggc ggcggcgatc gtgccgtggg ccctgcggcg cggagccggt 600ggacggtggt ggcgctatct gaccggtgga ttcgtggcga tgcccggcgt gctggtggcg 660gtcgcgtcgg ccggtggctc ggtcggcgtc ggctgggtcg gcgcggcgct gtggatcgcc 720gggccgctcg cggccggcgc gctgtgcgcc tacgggatcc gggccggcta cgccgtgacc 780gcgctggccg gcgcgctggc catagcgctc tcgatggccg agcgcggctt cctgttcgcc 840gccttctggc tgttcggcgg gctgtacctg gcgctcggcg ccgctgcgta caccgcctcg 900cgggccgtcg acggcgacgc cgccgcgacg cccggcccgc cggcccggcc ggaacccgcg 960ccggcccccg gaggctga 97878663PRTMicromonospora sp. strain 046-ECO11 78Met Leu Asp His Ala Ser Gly Arg Ile Asp Val Thr Arg Leu Arg Glu1 5 10 15Ala Leu Asp Gly Arg Trp Ala Glu Val Arg Arg Ala His Arg Glu His 20 25 30Leu Asp Glu Arg Phe Leu Pro Val Tyr Gly Glu Thr Gly Asp Gln Ala 35 40 45Arg Glu Arg Ile Thr Arg Leu Leu Ser Glu Leu Pro Val Glu Leu Gly 50 55 60Ile Ala Ser Gly Phe Pro Ala Glu Tyr Gly Gly Arg Gly Asp Val Gly65 70 75 80Ala Ser Ile Val Ala Thr Glu Met Leu Ala Gln Val Asp Leu Ser Leu 85 90 95Met Val Lys Ala Gly Val Gln Trp Gly Leu Phe Gly Gly Ala Val Ala 100 105 110Ala Leu Gly Thr Lys Arg His His Asp Ala Tyr Leu Arg Asp Ile Val 115 120 125Ala Gly Arg Leu Phe Gly Cys Phe Ala Met Thr Glu Thr Gly His Gly 130 135 140Ser Asp Val Gln Gln Leu Arg Thr Thr Cys Val Tyr Asp Pro Gln Thr145 150 155 160Gln Thr Phe Asp Leu His Thr Pro His Glu Ala Ala Arg Lys Asp Tyr 165 170 175Ile Gly Asn Ala Ala Arg Asp Gly Arg Met Ala Val Val Phe Ala Gln 180 185 190Leu Val Thr Gly Gly Arg Arg His Gly Val His Ala Trp Leu Val Pro 195 200 205Ile Arg Asp Glu His Gly Lys Pro Met Pro Gly Val Thr Ile Gly Asp 210 215 220Ala Gly Pro Lys Ala Gly Leu Leu Gly Val Asp Asn Gly Arg Leu Ser225 230 235

240Phe Asp His Val Arg Val Pro Arg Glu Met Leu Leu Asp Gln Tyr Ala 245 250 255Gln Val Ala Glu Asp Gly Thr Tyr Ser Ser Pro Ile Glu Asn Asp Ser 260 265 270Arg Arg Phe Phe Thr Met Leu Gly Thr Leu Val Arg Gly Arg Val Ser 275 280 285Val Gly Gly Ala Ala Ser Ala Ala Thr Lys Ser Ala Leu Ala Ile Ala 290 295 300Val Arg Tyr Gly Asp Ile Arg Arg Gln Phe Ala Asp Ala Asp Gly Asp305 310 315 320Arg Glu Val Leu Leu Asn Asp Tyr Leu Ala His Gln Arg Lys Leu Leu 325 330 335Pro Ala Leu Ala Thr Thr Tyr Ala Leu Thr Phe Ala Gln Ala Glu Leu 340 345 350Val Ala Ala Leu Asp Asp Ile Gln Gly Gly Asp Gly Pro Val Asp Glu 355 360 365His Arg Gln Arg Glu Leu Glu Ser Arg Ala Ala Gly Leu Lys Ala Ala 370 375 380Gln Thr Trp His Ala Thr Arg Thr Ile Gln Ile Cys Arg Glu Ala Cys385 390 395 400Gly Gly Ala Gly Tyr Leu Ser Glu Asn Arg Leu Pro Ser Leu Lys Ala 405 410 415Asp Thr Asp Val Phe Thr Thr Phe Glu Gly Asp Asn Thr Val Leu Leu 420 425 430Gln Leu Val Ala Lys Gly Leu Leu Thr Gly Tyr Arg Asp Glu Phe Gly 435 440 445Ser Leu Asp Gly Trp Gly Arg Ala Ser Phe Val Ala Glu Gln Val Arg 450 455 460Glu Met Val Leu Glu Arg Thr Ala Ala Arg Ala Leu Ile Ala Arg Leu465 470 475 480Val Ser Ala Val Pro Gly Arg Asp Asp Glu Val Ala Val Thr Asp Arg 485 490 495Gly Trp Gln Leu Lys Leu Phe Glu Asp Arg Glu Glu His Leu Leu Asp 500 505 510Ser Ala Val Arg Arg Leu Arg Gly Gly Ala Ser Thr Lys Lys Asp Arg 515 520 525Pro Phe Asp Ile Phe Asn Asp Val Gln Asp His Val Leu Ala Val Ala 530 535 540Ala Ala His Ile Asp Arg Val Thr Leu Glu Ala Phe Val Ala Gly Ile545 550 555 560Asp Ala Ile Ala Asp Pro Ala Val Lys Glu Leu Leu Ser Arg Val Cys 565 570 575Asp Leu Tyr Ala Leu Thr Val Ile Glu Ala Asn Lys Gly Trp Leu Leu 580 585 590Glu His Gly Arg Leu Thr Pro Ala Arg Ser Lys Thr Ile Thr Ser Val 595 600 605Val Asn Gly Leu Leu Lys Glu Leu Arg Pro Asp Met Arg Thr Leu Val 610 615 620Asp Gly Phe Ala Ile Pro Asp Ala Trp Leu His Ala Ala Ile Leu Arg625 630 635 640Glu Glu Pro Val Arg Gln Glu Thr Met Ala Ala His Asp Ala Ala Gly 645 650 655Asp Pro Gln Ala Val Pro Ala 660791992DNAMicromonospora sp. strain 046-ECO11 79atgctcgatc acgcatccgg ccgcatcgac gtcacacgcc tgcgggaagc gctcgacggc 60cggtgggccg aggtccgccg ggcgcaccgc gaacacctcg acgaacgctt cctcccggtg 120tacggcgaga ccggtgacca ggcccgcgag cgcatcaccc ggctgctgtc cgaactcccc 180gtcgagctgg gcatcgcctc cggtttcccc gccgagtacg gcggccgcgg cgacgtgggc 240gcctcgatcg tcgccaccga gatgctggcc caggtggacc tgtcactgat ggtgaaggcc 300ggcgtgcagt ggggcctgtt cggcggcgcg gtcgccgccc tcggcacgaa gcggcaccac 360gacgcctacc tgcgggacat cgtcgcgggc cggctcttcg gctgcttcgc gatgaccgag 420accggccacg gctcggacgt gcagcaactg cgcaccacct gcgtctacga cccgcagacg 480cagaccttcg acctgcacac cccgcacgag gccgcgcgca aggactacat cggcaacgcg 540gcccgggacg ggcggatggc tgtggtgttc gcccagctcg tcaccggcgg gcgccgccac 600ggggtgcacg cctggctggt gccgatccgc gacgagcacg gcaagccgat gcccggcgtg 660accatcggcg acgccgggcc caaggccggc ctgctcggcg tggacaacgg gcggctcagc 720ttcgaccacg tgcgggtgcc gcgggagatg ctgctggacc agtacgcgca ggtcgccgag 780gacggcacgt actccagccc gatcgagaac gactcccggc gcttcttcac catgctgggc 840accctggtcc ggggccgggt gagcgtgggc ggcgccgcgt cggcggccac caagtcggcg 900ctggccatcg cggtgcgcta cggcgacatc cgccggcagt tcgccgacgc cgacggcgac 960cgcgaggtgc tgctcaacga ctacctggcg caccagcgca agctgctgcc cgcgctggcc 1020accacgtacg cgctgacctt cgcccaggcg gagctggtcg cggcgctcga cgacatccag 1080ggcggcgacg ggccggtcga cgagcaccgg cagcgggagc tggagtcccg ggccgccggt 1140ctgaaggcgg cgcagacctg gcacgccacc cgcaccatcc agatctgccg ggaggcgtgt 1200ggcggcgccg gctacctgtc cgagaaccgc ctgcccagcc tcaaggccga caccgatgtc 1260ttcaccacct tcgagggcga caacacggtg ctgctgcaac tggtcgccaa ggggctgctg 1320accggctacc gggacgagtt cggctcgctc gacggctggg gacgcgcctc cttcgtggcc 1380gagcaggtac gcgagatggt gctggaacgc accgccgcgc gggcgctgat cgcacgtctg 1440gtcagcgccg tgcccgggcg cgacgacgag gtcgccgtca ccgaccgggg ctggcagctc 1500aagctcttcg aggaccgcga ggagcacctg ctcgacagcg cggtccgccg cctgcgcggt 1560ggcgcgtcca ccaagaagga ccgccccttc gacatcttca acgacgtcca ggaccacgtc 1620ctcgccgtcg ccgcggcgca catcgaccgg gtgacgctgg aggcgttcgt cgccgggatc 1680gacgccatcg ccgacccggc ggtcaaggaa ctgctgtccc gggtctgcga cctgtacgcg 1740ctcaccgtga tcgaggcgaa caagggctgg ctgctcgagc acggccggct caccccggcc 1800cgctcgaaga ccatcaccag cgtggtgaac gggctgctca aggagctgcg cccggacatg 1860cgcacgctcg tggacggctt cgccatcccg gacgcgtggc tgcacgcggc gatcctgcgc 1920gaggagcccg tccggcagga gacgatggcc gcgcacgacg ccgccggcga cccgcaggcc 1980gtccccgcct ag 199280573PRTMicromonospora sp. strain 046-ECO11 80Val Ser Pro Leu Pro Pro Gly Ser Ala Val Thr Ala Arg His Val Leu1 5 10 15Arg Gln Ala Leu Arg Arg Gln Arg Arg Pro Val Leu Ile Gly Val Thr 20 25 30Leu Leu Gly Leu His Gln Val Thr Glu Ala Leu Val Pro Val Ala Ile 35 40 45Gly Val Ile Ile Asp Arg Ala Val Val Thr Gly Asp Pro Trp Ala Leu 50 55 60Ala Tyr Ser Val Ala Gly Leu Ala Ala Leu Phe Thr Val Leu Ala Phe65 70 75 80Ala Tyr Arg Asn Gly Ala Arg Gln Ala Phe Ala Ala Val Glu Arg Glu 85 90 95Ala His Leu Leu Arg Val Glu Leu Ala Glu Arg Ala Leu Asp Pro Arg 100 105 110Gly His Arg Ser Gly Leu Arg Asp Gly Glu Leu Leu Ser Val Ala Ala 115 120 125Ser Asp Ala Glu Leu Ser Ala Tyr Val Val Arg Val Ala Gly Phe Gly 130 135 140Val Ala Ala Val Ser Ala Leu Thr Val Ala Ala Val Ala Leu Leu Val145 150 155 160Ile Asp Val Pro Leu Gly Leu Gly Val Leu Ile Gly Val Pro Val Leu 165 170 175Val Leu Ala Leu Gln Arg Met Ala Pro Leu Leu Ser Arg Arg Ser Ala 180 185 190Ser Gln Gln Glu Ala Leu Ala Glu Thr Thr Ala Leu Ala Val Asp Leu 195 200 205Val Ser Gly Leu Arg Val Leu Arg Gly Ile Gly Ala Gln His His Ala 210 215 220Ala Gly Arg Tyr Ala Glu Ala Ser Arg Arg Ala Leu Ala Val Thr Leu225 230 235 240Arg Ala Ala Asn Thr Lys Gly Leu His Leu Gly Leu Thr Thr Ala Ala 245 250 255Asn Gly Leu Phe Leu Ala Ala Val Ala Gly Val Ala Gly Trp Leu Ala 260 265 270Leu Arg Gly Arg Leu Thr Ile Gly Glu Leu Val Thr Val Val Gly Leu 275 280 285Ala Gln Phe Val Ala Glu Pro Val Gln Thr Leu Gly Tyr Cys Val Gln 290 295 300Leu Phe Ala Met Ala Arg Ala Ser Ala Ala Arg Val Gly Arg Val Leu305 310 315 320Gly Ala Glu Pro Leu Thr Arg Pro Gly Ser Ala Pro Arg Pro Asp Arg 325 330 335Thr Asp Gly Pro Arg Leu Val Leu Asp His Val Gly His Ala Ala Leu 340 345 350Asp Gly Val Cys Leu Arg Val Asp Pro Gly Glu Ile Val Gly Val Leu 355 360 365Ala Tyr Asp Pro Ala Asp Ala Asp Ala Leu Val Ala Leu Leu Ser Gly 370 375 380Arg Val Pro Ala Asp Arg Arg Arg Gly Thr Val Arg Val Asp Gly Val385 390 395 400Pro Ala Asp Asp Leu Asp Val Asp Ala Leu Arg Gly Ala Val Leu Val 405 410 415Glu Pro His Asp Val Thr Leu Phe Glu Gly Thr Val Ala Ala Asn Leu 420 425 430Ala Ala Gly Ser Arg Thr Glu Glu Gly Arg Leu Arg Ala Ala Val Arg 435 440 445Ala Ala Ala Ala Asp Asp Val Val Asp Ala His Pro Gly Gly Leu Gly 450 455 460His Arg Leu Val Glu Arg Gly Ala Asn Leu Ser Gly Gly Gln Arg Gln465 470 475 480Arg Leu Gly Leu Ala Arg Ala Leu His Ala Asp Pro Pro Val Leu Val 485 490 495Leu His Asp Pro Thr Thr Ala Val Asp Ala Ala Thr Glu Ala Gln Leu 500 505 510Ala Asp Gly Leu Ala Gly Ala Arg Arg Glu Ala Pro Arg Gly Thr Leu 515 520 525Leu Val Thr Ser Ser Pro Ala Leu Leu Arg Ile Thr Asp Arg Val Val 530 535 540Val Ile Ala Asp Gly Arg Val Thr Ala Glu Gly Thr His Glu His Leu545 550 555 560Leu Ala Thr Asp Ala Arg Tyr Arg Glu Glu Thr Leu Arg 565 570811722DNAMicromonospora sp. strain 046-ECO11 81gtgtccccgc ttccccccgg cagcgccgtc accgcccggc acgtgctccg ccaggcgctg 60cgccgccagc gccgcccggt gctgatcggc gtgaccctgc tcgggctgca ccaggtcacc 120gaggcgctcg tgccggtggc gatcggcgtc atcatcgacc gggccgtggt gaccggcgac 180ccgtgggcgc tcgcgtactc cgtcgccggc ctcgccgccc tgttcaccgt gctggcgttc 240gcctaccgca acggcgcccg ccaggcgttc gcggcggtgg aacgggaggc gcacctgctg 300cgggtcgagc tggccgagcg cgcgctcgac ccgcgcgggc accgctccgg cctgcgcgac 360ggcgagctgc tctcggtcgc cgcctccgac gccgaactct ccgcgtacgt ggtccgggtg 420gccggcttcg gcgtcgccgc ggtgagcgcg ctgaccgtcg cggcggtcgc gctgctggtc 480atcgacgtcc cgctcggact cggcgtgctc atcggcgtac cggtgctggt cctggcgctg 540caacggatgg cgccgctgct gtcccggcgc agcgcctccc agcaggaggc cctcgcggag 600accacggcgc tcgccgtgga cctcgtctcc ggcctgcgcg tgctgcgcgg catcggcgcc 660cagcaccacg ccgccggccg gtacgccgag gccagccgac gcgccctcgc cgtgacgctg 720cgcgccgcca acaccaaggg cctgcacctc gggctcacca ccgccgcgaa cggcctcttc 780ctcgccgccg tcgccggggt cgccggctgg ctcgcgctgc gcggccggct caccatcggc 840gagctggtca ccgtggtcgg gctcgcgcag ttcgtcgccg agccggtgca gacgctgggc 900tactgcgtgc agctgttcgc gatggcccgc gcctccgccg cccgggtcgg gcgcgtgctc 960ggcgccgagc cgctgacccg gccgggcagc gcgccccggc cggaccgcac ggacgggccg 1020cggctcgtcc tcgaccacgt cggccacgcc gcgctggacg gggtgtgcct gcgcgtcgac 1080ccgggagaga tcgtcggcgt cctggcgtac gacccggccg acgcggacgc gctggtggcg 1140ctgctgtccg ggcgggtgcc cgcggaccgg cgccggggca cggtacgcgt cgacggggta 1200cccgccgacg acctggacgt cgacgcgctg cgcggcgccg tcctggtcga gccgcacgac 1260gtgacgctgt tcgagggaac cgtggccgcc aacctcgccg ccgggagcag gaccgaggag 1320gggcgcctgc gcgccgcggt ccgggcggcc gcggcggacg acgtggtgga cgcgcacccc 1380ggcggcctcg gccaccggct cgtcgagcgg ggcgccaacc tctccggcgg gcagcgccag 1440cggctcgggc tggcgcgggc gctgcacgcc gacccgccgg tgctggtgct gcacgacccc 1500accaccgccg tggacgcggc caccgaggcc caactcgccg acggactggc cggcgcgcgc 1560cgcgaagcgc cccggggcac gctgctggtc accagcagcc ccgccctgct gcggatcacc 1620gaccgggtgg tggtgatcgc cgacggccgg gtgaccgccg aggggacgca cgagcacctg 1680ctggccaccg acgcccgcta ccgcgaggag acactgcggt ga 172282596PRTMicromonospora sp. strain 046-ECO11 82Val Thr Ala Asp Pro Arg Thr Ala Glu Pro Thr Arg Val Leu Leu Pro1 5 10 15Thr Ala Thr Ala Arg Arg Thr Trp Thr Thr Leu Gly Ala Glu Phe Arg 20 25 30Arg Arg Pro Gly Leu Ser Ala Ala Ala Thr Ala Val Leu Val Ala Ala 35 40 45Ala Thr Gly Gly Leu Val Ala Pro Trp Val Leu Gly Arg Leu Val Asp 50 55 60Asp Val Ile Ala Asp Ala Pro Val Ser Arg Ile Ala Gly Arg Val Ala65 70 75 80Val Ile Ala Gly Ala Ala Val Leu Thr Gly Leu Leu Thr Ala Ala Gly 85 90 95Ala Ala Leu Ala Ser Arg Leu Gly Glu Thr Val Leu Ala Arg Leu Arg 100 105 110Glu Arg Val Leu Asp Arg Ala Leu His Leu Pro Ser Ala Thr Leu Glu 115 120 125Arg Ala Gly Thr Gly Asp Leu Leu Ala Arg Val Gly Asp Asp Val Ala 130 135 140Val Val Thr Asn Val Ile Ala Val Ser Gly Pro Ala Phe Val Gly Ala145 150 155 160Leu Leu Ser Val Val Leu Thr Val Phe Gly Leu Val Ala Leu Asp Trp 165 170 175Arg Leu Gly Leu Ala Gly Leu Val Ala Ala Pro Ala Tyr Ala Leu Ala 180 185 190Leu Arg Trp Tyr Leu Arg Arg Ser Ala Pro Tyr Tyr Ala Arg Glu Arg 195 200 205Val Ala Thr Gly Glu Arg Thr Gln Ala Met Ala Gly Ala Leu Arg Gly 210 215 220Ala Ala Thr Val Arg Ala Tyr Arg Thr Glu Asp Ala His Val Ala Ala225 230 235 240Ile Ala Glu Arg Ser Gly Val Ala Arg Asp Leu Ser Leu Glu Ile Phe 245 250 255Asn Leu His Thr Arg Phe Gly Leu Arg Ile Asn Arg Ser Glu Phe Leu 260 265 270Gly Leu Ala Ala Val Leu Val Ala Gly Phe Phe Leu Val Arg Ala Asp 275 280 285Leu Val Thr Val Gly Ala Ala Thr Thr Ala Ala Leu Tyr Phe His Arg 290 295 300Leu Phe Asn Pro Ile Gly Leu Leu Leu Met Glu Ser Asp Ser Val Leu305 310 315 320Gln Ala Gly Ala Ser Leu Ala Arg Leu Val Gly Val Ala Thr Leu Pro 325 330 335Asp Thr Ala Pro Ser Gly Pro Ala Pro Ser Ala Ala Gly Arg Arg Gly 340 345 350Pro Ala Ala Leu Asp Val Thr Val Arg Arg His Arg Tyr Asp Asp Asp 355 360 365Gly Pro Leu Val Leu Ala Asp Val Asp Leu Arg Leu Ala Pro Gly Glu 370 375 380Arg Val Ala Leu Val Gly Ala Ser Gly Ala Gly Lys Ser Thr Leu Ala385 390 395 400Gly Ile Ala Ala Gly Ile Ile Ala Pro Thr Asp Gly Ser Val Arg Leu 405 410 415Gly Gly Val Pro Leu Thr Glu Arg Gly Glu His Ala Val Arg Arg Asp 420 425 430Val Ala Leu Val Ser Gln Glu Val His Val Phe Ala Gly Pro Leu Ala 435 440 445Glu Asp Leu Arg Leu Ala Ala Pro Asp Ala Thr Asp Ala Glu Leu Leu 450 455 460Asp Ala Leu Asp Arg Val Gly Ala Thr Thr Trp Leu Arg Ala Leu Pro465 470 475 480Asp Gly Leu Ala Thr Ala Val Gly Glu Gly Gly His Arg Leu Thr Ala 485 490 495Ala Gln Ala Gln Gln Val Ala Leu Ala Arg Leu Val Leu Ala Ala Pro 500 505 510Ala Val Ala Val Leu Asp Glu Ala Thr Ala Glu Ala Gly Ser Ala Gly 515 520 525Ala Arg Asp Leu Asp Arg Ala Ala Leu Ala Ala Thr Glu Gly Arg Thr 530 535 540Thr Leu Ile Val Ala His Arg Leu Ser Gln Ala Val Ala Ala Asp Arg545 550 555 560Ile Val Leu Leu Asp His Gly Arg Ile Val Glu Gln Gly Thr His Ser 565 570 575Glu Leu Leu Ala Ala Asp Gly Arg Tyr Gly His Leu Trp Arg Ser Trp 580 585 590Ser Val Pro Val 595831791DNAMicromonospora sp. strain 046-ECO11 83gtgaccgctg acccgcgtac cgccgaaccc acccgggtgt tgctgcccac cgcgaccgcc 60cggcggacct ggacgacgct cggcgcggag ttccgccggc ggcccggcct cagcgccgcc 120gcgaccgccg tgctcgtcgc cgccgccacc ggcgggctgg tcgcgccctg ggtgctcggc 180cgcctcgtcg acgacgtcat cgccgacgcc ccggtctccc ggatcgccgg ccgggtggcg 240gtgatcgccg gcgcggcagt gctcaccgga ctgctcaccg ccgccggggc cgcgctcgcg 300tcccgcctgg gggagacggt gctggcccgg ctgcgcgagc gggtcctcga ccgggcgctg 360cacctgccct cggcgacgct ggaacgggcc ggcaccggcg acctgctggc ccgggtcggc 420gacgacgtgg cggtggtgac gaacgtgatc gcggtcagcg gcccggcgtt cgtcggcgcg 480ctgctgtccg tggtgctgac cgtgttcggg ctggtcgcgc tcgactggcg gctcggcctc 540gccgggctgg tcgccgcgcc cgcctacgcg ctggcgctgc gctggtacct gcgccggtcg 600gcgccgtact acgcccgcga gcgcgtcgcc accggcgagc ggacgcaggc gatggccggc 660gcgctgcgtg gcgcggccac cgtgcgcgcg taccggaccg aggacgcgca cgtcgcggcg 720atcgccgagc gctccggcgt ggcgcgcgac ctgtcgctgg agatcttcaa cctgcacacc 780cggttcgggc tgcggatcaa caggtcggag ttcctcggcc tggccgcggt gctcgtcgcc 840gggttcttcc tggtccgcgc cgacctggtc acagtgggcg cggcgaccac cgccgcgctc 900tacttccacc ggctgttcaa cccgatcggc ctgctgctga tggagtccga ctcggtgctg 960caggccggcg cgagcctcgc ccggctggtc ggcgtggcca cgctgcccga caccgccccg 1020tccgggcccg cgccgtcggc ggccgggcgg cgcggcccgg cggcgctgga cgtcacggtc 1080cgccggcacc gctacgacga cgacggccct ctggtcctgg ccgacgtcga cctgcgcctg 1140gccccgggcg agcgggtcgc gctcgtgggc gccagcggcg cgggcaagag cacgctcgcc 1200ggcatcgccg ccgggatcat cgcgcccacc gacgggtcgg tacgcctggg cggcgtgccg 1260ctgaccgagc ggggcgagca cgccgtgcgg cgcgacgtcg cgctggtcag ccaggaggtg 1320cacgtcttcg ctggaccgct cgccgaggat ctgcgcctgg ctgccccgga cgccaccgac 1380gccgaactgc tcgacgcgct

ggaccgggtc ggcgccacca cctggctgcg cgcgctgccg 1440gacgggctgg ccacagcggt cggcgagggc ggccaccggc tcaccgccgc gcaggcccag 1500caggtcgccc tggcccggct ggtgctggcc gcgcccgccg tcgccgtgct ggacgaggcc 1560accgccgagg ccggcagcgc cggagcgcgt gacctggacc gggcggcgct ggccgccacc 1620gagggacgga ccacgctgat cgtggcgcac cggctcagcc aggcggtcgc cgccgaccgg 1680atcgtcctgc tcgaccacgg gcggatcgtg gagcagggca cgcactcgga actgctcgcc 1740gccgacggcc ggtacgggca tctgtggcgc tcctggagcg tcccggtatg a 179184507PRTMicromonospora sp. strain 046-ECO11 84Met Thr Asp Ala Pro Ala Arg Phe Val Leu Phe Pro Gly Arg His His1 5 10 15Leu Leu Thr Arg Phe Gln Ala Asp Tyr Leu Arg Arg Leu Ala Gly Asp 20 25 30Asp Ala Thr Val Val Trp Ala Val Thr Ser Ala Asn His Glu Asn Thr 35 40 45Arg Arg Asn Pro Val Pro Tyr His Arg Arg Glu Ala Ala Ile Glu Arg 50 55 60Phe Ser Val Leu Ser Gly Leu Arg Ser Val Val Val Pro Ile Phe Asp65 70 75 80Thr Ala Tyr Thr Asp Ala Phe Ala Glu Val Thr Leu Lys Ser Ile Ala 85 90 95Val Ala Thr Gly Leu Glu Leu Thr Pro Ala Asp Thr Val Leu Ala Cys 100 105 110Ser Thr Pro Glu Val Ala Lys Leu Tyr Glu Gln Leu Gly Phe Ser Ile 115 120 125Ala Pro Val Glu Ala Asp Pro Asp Leu Pro Glu Pro Pro Glu Arg Pro 130 135 140Trp Asp Val Leu Leu Arg Leu Ala Ala Gly Asp Glu Thr Trp Arg Ala145 150 155 160Leu Thr His Pro Ala Thr Ile Asp Val Phe Glu Arg Tyr Arg Leu Val 165 170 175Glu Ser Ile Arg Ser Val Val Asn Asp Pro Leu Val Gly Asp Glu Gly 180 185 190Gly Leu Thr Val Thr Arg Asp Tyr Arg Thr Tyr Val Glu Ala Phe Ala 195 200 205Thr Ala Ala Gln Arg Lys Trp Asp Ser Val Arg Arg Tyr Val Gln Pro 210 215 220Gly Arg Ile Val Asp Ile Gly Cys Gly Ala Gly Ala Val Leu Glu Leu225 230 235 240Ala Asp Arg Glu Ala Ala Leu Arg Glu Ser Asp Leu Ile Gly Val Glu 245 250 255Val Ala Arg His Leu Tyr Gln Glu Cys Leu His Lys Lys Ala Gln Gly 260 265 270Val Phe Arg Asn Ala Asn Val Tyr Phe Phe His Arg Asn Val Leu Gly 275 280 285Gly Ala Val Phe Lys Asp Arg Ser Val Asp Thr Thr Leu Thr Phe Ala 290 295 300Leu Thr His Glu Ile Trp Ser Tyr Gly Arg Arg Arg Glu Ser Leu Leu305 310 315 320Gln Phe Ala Arg Arg Ile His Asp His Thr Val Pro Gly Gly Val Trp 325 330 335Ile Asn Ser Asp Val Cys Gly Pro Asp Asp Pro Arg Arg Gln Val Leu 340 345 350Leu Arg Leu Ser Thr Asp Asp Gly Asp Asn Pro Ala Ala Pro Arg Pro 355 360 365Asp Leu Ala Glu Leu Thr Ser Ala Glu Val Arg Arg Tyr Val Gly Gly 370 375 380Leu Ser Thr Arg Ala Arg Leu Asp Gln Phe Ala Val Asp Phe Ala Phe385 390 395 400Asp Phe Asp Tyr Glu Pro Leu Pro Asp Gly Ala Val Arg Leu Thr Leu 405 410 415Gly Ala Ala Met Asp Tyr Leu Thr Arg Lys Asp Tyr Thr Asp Asn Trp 420 425 430Leu Ser Glu Thr Gln Glu Gln Phe Cys Gly Leu Ser Phe Ala Asp Trp 435 440 445Thr Asp Leu Leu Thr Glu Ala Gly Phe Glu Ile Gly Pro Ala Ser Ala 450 455 460Pro Val Arg Asn Glu Trp Val Ile Asp Asn Arg Ile Ala Pro Val Ala465 470 475 480Ser Leu Thr Asp Leu Asp Gly Arg Pro Leu Asp Trp Pro Thr Thr His 485 490 495Val Leu Thr Val Ala His Arg Pro Arg Asn Gln 500 505851524DNAMicromonospora sp. strain 046-ECO11 85atgaccgacg cgccggcccg cttcgtgctc ttcccggggc ggcaccacct gctgacccgg 60ttccaggccg actacctgcg gcggctggcc ggggacgacg ccacagtggt ctgggcggtg 120acgtcggcca accacgagaa caccaggcgc aacccggtgc cctaccaccg gcgggaggcc 180gcgatcgaac gattcagcgt gctgagcggg ctgcgctcgg tggtggtgcc gatcttcgac 240accgcgtaca ccgacgcgtt cgccgaggtg acgctgaagt ccatcgcggt ggccaccggg 300ctcgaactca cccccgccga caccgtgctg gcctgctcca cgccggaggt cgcgaagctg 360tacgagcagc tcggcttttc gatcgcgccg gtcgaggcgg acccggacct gcccgagccg 420cccgaacggc cgtgggacgt gctgctgcgc ctggccgccg gggacgagac ctggcgcgcg 480ctcacccacc cggccaccat cgacgtgttc gagcgctacc gcctggtcga gtcgatccgg 540tcggtggtga acgacccgct cgtcggcgac gagggcggtc tcacagtgac ccgcgactac 600cggacctacg tcgaggcgtt cgccacggcc gcgcagcgca agtgggactc ggtacgccgg 660tacgtgcagc ccggccgcat cgtggacatc ggctgcggcg cgggcgccgt cctggaactc 720gccgaccggg aggccgcgct gcgtgagagc gacctgatcg gcgtggaggt cgcccgccac 780ctctaccagg agtgcctgca caagaaggcg cagggcgtgt tccgcaacgc caacgtctac 840ttcttccacc gcaacgtcct cggcggcgcg gtgttcaagg accgctcggt cgacaccacg 900ctcacgttcg cgctgaccca cgagatctgg tcgtacgggc ggcggcggga gtcgctgctg 960cagttcgccc gccgcatcca cgaccacacg gtgcccggcg gcgtctggat caacagcgac 1020gtgtgcggtc cggacgaccc ccggcggcag gtgctcctgc gactgtccac cgacgacggc 1080gacaacccgg ccgcgccccg ccccgacctc gccgagctga cctcggcgga ggtccggcgt 1140tacgtcggcg ggctgtcgac gcgggcgcgg ctggaccagt tcgccgtcga cttcgcgttc 1200gacttcgact acgagccgct ccccgacggc gcggtacgcc tgacgctggg cgccgcgatg 1260gactacctga cccgcaagga ctacacggac aactggctgt cggagacgca ggagcagttc 1320tgcggcctga gcttcgccga ctggacggac ctgctcaccg aggcggggtt cgagatcggc 1380ccggcgtcgg cgccggtgcg caacgagtgg gtgatcgaca accggatcgc gccagtcgcg 1440tccctcaccg acctcgacgg ccggccgctg gactggccga ccacccacgt cctcaccgtc 1500gcccaccgcc cccgcaacca gtga 152486232PRTMicromonospora sp. strain 046-ECO11 86Val Ser Asp Ile Gln Ile Ile Ser Phe Val Ala Ala Ser Leu Leu Ile1 5 10 15Ile Ile Val Pro Gly Val Asp Phe Ala Leu Val Thr Arg Gln Thr Val 20 25 30Arg Tyr Gly Arg Arg Ala Gly Phe Val Val Leu Ala Gly Leu Phe Val 35 40 45Ala Ala Leu Val His Ala Ser Phe Ala Thr Ala Gly Leu Ser Ala Leu 50 55 60Leu Val Ser Ser Pro Thr Leu Tyr Thr Val Leu Arg Val Ala Gly Ala65 70 75 80Leu Tyr Leu Leu Tyr Leu Gly Gly Thr Ile Leu Trp Ala Thr Arg Pro 85 90 95Arg Arg Thr Val Pro Ala Ala Gln Pro Val Thr Val Gly Ala Gly Gly 100 105 110Ala Gly Pro Asp Thr Asp Thr Gly Pro Ala Pro Val Pro Asp Thr Pro 115 120 125Ala Ala Asp Glu Pro His Val Ala Arg Arg Ser Phe Val Met Gly Val 130 135 140Thr Ser Gln Leu Leu Asn Val Lys Val Val Val Phe Tyr Val Ser Phe145 150 155 160Val Pro Gln Phe Val Lys Pro Gly Glu Gly Ala Ala Ala Arg Thr Ala 165 170 175Val Leu Ala Ala Thr Phe Ile Gly Leu Ala Val Leu Trp Trp Ala Cys 180 185 190Tyr Ile Met Leu Ile Asp Arg Leu Gln Pro Trp Leu Thr Arg Pro Ser 195 200 205Val Leu Leu Val Ile Glu Arg Leu Thr Gly Leu Ile Leu Ile Val Leu 210 215 220Ala Ile Arg Ile Ala Leu Ser Arg225 23087699DNAMicromonospora sp. strain 046-ECO11 87gtgtctgaca tccagatcat cagtttcgtc gccgccagcc tgctcatcat catcgtgccg 60ggcgtcgact tcgcgctcgt cacccggcag accgtcaggt acggccggcg ggccgggttc 120gtggtgctgg ccgggctgtt cgtcgccgcg ctggtgcacg cgtcgttcgc gaccgccggc 180ctgtccgccc tgctggtctc ctcgccgacg ctctacacgg tgctgcgcgt cgccggcgcg 240ctgtacctgc tctacctggg cggcacgatc ctctgggcga cccggccgcg ccggacggtc 300ccggcggcgc agccggtcac tgtcggcgcg ggcggcgccg ggccggacac ggacaccggc 360cccgcgccgg tgccggacac cccggccgcc gacgagccgc acgtggcccg ccgctcgttc 420gtcatgggcg tcaccagcca gctgctgaac gtcaaggtgg tcgtcttcta cgtctcgttc 480gtgccgcagt tcgtcaagcc cggcgagggg gcggcggccc gtacggcggt gctcgccgcc 540acgttcatcg gcctcgcggt gctctggtgg gcctgctaca tcatgctcat cgacaggttg 600cagccctggc tgacccggcc gtccgtgctg ctggtgatcg aacggctgac cgggctcatc 660ctgatcgtcc tggcgatccg gatcgcgctg agccggtga 69988132PRTMicromonospora sp. strain 046-ECO11 88Val Gly Val Ser Ala Met Thr Thr Phe Asp Tyr Asp Gly Arg Val Phe1 5 10 15Val Ser Val Asp His Asp Ala Gly Asp Gly Ala Glu Pro Leu Arg Gly 20 25 30His Tyr His Gln Arg Gly Asp Leu Val Trp Ala Glu Ile Thr Gly Gly 35 40 45Pro Val Arg His Gly Arg Leu Ala Gly Thr Cys Asp Ala Gln Gly Val 50 55 60Val Arg Phe Ala Tyr Leu Glu Val Leu Thr Asp Gly Thr Ile Val Ile65 70 75 80Gly Glu Cys Glu Ser Arg Pro Glu Arg Leu Pro Asp Gly Arg Ile Arg 85 90 95Leu Arg Glu Gln Trp Arg Arg His Gly Pro Arg Gln Asp Ser Gly Val 100 105 110Ser Val Ile Glu Glu Ala Val Pro Ala Leu Ala Gly Gly Gln Glu Ser 115 120 125Arg Arg Arg Val 13089399DNAMicromonospora sp. strain 046-ECO11 89gtgggcgtga gcgcgatgac gacattcgac tacgacggcc gcgtcttcgt ctcggtggac 60cacgacgccg gtgacggcgc cgagccgctg cgggggcact accaccagcg tggcgacctg 120gtctgggcgg agatcaccgg cggcccggtc cggcacggcc ggctggccgg cacctgcgac 180gcgcagggcg tcgtgcgctt cgcctacctg gaggtgctca ccgacggcac catagtcatc 240ggcgagtgcg agtcccggcc cgaacggctg ccggacggcc ggatccggct gcgggaacag 300tggcgccggc acggaccacg ccaggacagc ggcgtctccg tcatcgagga ggcagtgccg 360gcgctcgccg gaggacagga gagccggcgt cgtgtctga 399

Claims

1-4. (canceled)

5. Cosmid 046KQ deposited under IDAC accession no. 250203-07.

6. A prokaryotic host cell comprising the cosmid of claim 5.

7. The prokaryotic host cell of claim 6, wherein said host cell is selected from the group consisting of E. coli, Streptomyces lividans, Streptomyces griseofuscus, Streptomyces ambofuchsus, Streptomyces ambofaciens, Actinomycetes, Bacillus, Corynebacteria and Thermoactinomyces.

8. An isolated DNA molecule having 95% sequence identity over the entire length of an open reading frame of the cosmid of claim 5.

9-12. (canceled)

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