Genes and Proteins for the Biosynthesis of the Lantibiotic 107891

Abstract

The Invention relates to the field of lantibiotics, and more specifically to the isolation of nucleic acid molecules that code for the enzymes required for the biosynthetic pathway of the lantibiotic 107891 and the homologues thereof.

Description

SUMMARY

[0001] The present invention relates to the field of lantibiotics, and more specifically to the isolation of nucleic acid molecules that code for the enzymes required for the biosynthetic pathway of the lantibiotic 107891 and the homologues thereof. Disclosed are the functions of the gene products involved in 107891 production. The present invention provides novel biosynthetic genes necessary for 107891 production, the encoded polypeptides, the recombinant vectors comprising the nucleic acid sequences that encode said polypeptides, the host cells transformed with said vectors and methods for producing lantibiotics using said transformed host cells, including methods for producing 107891, a precursor thereof, a derivative thereof or a modified lantibiotic different from 107891 or a precursor thereof.

BACKGROUND OF THE INVENTION

[0002] The continuous increase of pathogenic bacteria resistant to the existing antibiotics is a health global concern, and so there is a pressing need to discover and develop new compound that are active against resistant bacteria. This has led to renewed interest in natural products which have been in the past a rich source of antibiotics such as penicillins, macrolides and glycopeptides. Attractive candidates are also antimicrobial peptides and among them those designated as lantibiotics, i.e. lanthionine-containing antibiotic. Lantibiotics form a particular group within the antimicrobial peptides and are distinguished by several features such as primary and spatial structure characteristics, unique biosynthetic pathways and peptide modification reactions and potent antibacterial activity. These are a group of peptide-derived antimicrobial compounds secreted by Gram-positive bacteria and primarily act on Gram-positive bacteria. Lantibiotics are ribosomally synthesised as prepropeptides which are posttranslationally modified to their biologically active forms. The prepeptide consists of an N-terminal leader sequence, that does not undergo any post-translational modification and is cleaved off during or after secretion from the cell, and a C-terminal region (the propeptide), which is post-translationally modified. Lantibiotics are produced by different bacteria: the common feature of these compounds is the presence of one or more lanthionine residues, which consist of two alanine residues covalently cross-linked by a thioether linkage The thioether bridge is formed when a cysteine residue reacts with a dehydroalanine or dehydrobutyrine moiety to form a lanthionine or methyllantionine residue, respectively. The dehydroamino acid residues are in turn formed by dehydration of serine and threonine, respectively. Based on their structural and functional properties, lantibiotics are usually divided in two groups, type-A and type-B. Type-A lantibiotics are elongated, cationic peptides varying in length from 20 to 34 amino acids residues: nisin, subtilin, epidermin and Pep5 are members of this group. Type-B are globular peptides with a net negative charge: examples of this group of lantibiotics are mersacidin, cinnamycin, lacticin 481 and actagardine. These structural differences reflect on the mechanism of action. Type-A compounds exert their antimicrobial activity by blocking cell wall biosynthesis and by forming pores in the cellular membranes, through a mechanism that may or may not be aided by prior docking on the cellular target lipid II. Type-B lantibiotics also exert their antimicrobial activity by inhibiting peptidoglycan biosynthesis, but these compounds do not form pores once bound to lipid II.

[0003] Lantibiotics have been shown to have efficacy and utility as food additives and antibacterial agents. Nisin, the most studied lantibiotic, is produced by Lactococcus lactis and is active at low concentrations (low nanomolar MICs) against many Gram-positive bacteria including drug resistant strains and the food-borne pathogens Clostridium botulinum and Listeria monocytogenes. It has been extensively used as a food preservative without substantial development of bacterial resistance. Other lantibiotics show interesting biological activities: for example, epidermin shows high potency against Propionibacterium acnes; cinnamycin and duramycin inhibit phospholipase A₂ and angiotensin converting enzyme, providing potential applications as anti-inflammatory agents and for blood pressure regulation, respectively; and mersacidin inhibits many Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA).

[0004] The genes responsible for the biosynthesis of lantibiotics are organized in clusters designated by the locus symbol Ian, with a more specific genotypic designation for each lantibiotic (e.g., nis for nisin, gdm for gallidermin, cin for cinnamycin). Many Ian genes have been sequenced demonstrating a high level of similarity in gene organization. Each cluster includes: the structural gene lanA encoding the prepeptide; and the gene(s) required for the dehydration of Ser and Thr residues in the propeptide portion of LanA and for the thioether formation. For Type-A lantibiotics, LanB carries out the dehydration reactions and LanC is devoted to thioether bridging, whereas in Type-B lantibiotics a single LanM enzyme catalyzes both reactions. Additional genes are usually present in lantibiotic clusters: lanT encodes the ABC transporter for secretion of the lantibiotic, often in combination with a second transport system, encoded by the lanEFG genes; lanP encodes the processing protease, but some clusters lack such a gene which may be part of lanT or its function may be provided by a cellular protease; and lanI encodes a protein involved in self-protection; and lanKR are responsible for regulating expression of the lan genes.

[0005] There exists the potential and the utility to obtain improved lantibiotics by manipulation of occurring natural compounds. However, lantibiotics are structurally complex peptides and their accessibility to chemistry is limited to a few positions in the molecule. One of the major limitations for chemistry is to change the type or order of amino acids present in the peptide backbone. In light of the above, it would be desirable to have genes and enzymes useful for redirecting these steps in lantibiotic formation, in order to obtain derivatives that are hard or impossible to make by chemical means. General methods for the design of novel lantibiotic derivatives directly by fermentation processes with precisely engineered strains would thus be highly desirable.

[0006] In fact unusual aminoacids in lantibiotics solely contribute to their biological activity and also enhance their structural stability. Enzymes involved in lantibiotic biosynthesis represent a high potential for peptide engineering by introducing unusual aminoacids into desired peptides.

[0007] The lantibiotic 107891 shows antibacterial activity against Gram-positive bacteria including methycillin- and vancomycin-resistant strains but shows limited activity against Gram-negative bacteria (for examples, some Moraxella, Neisseria and Haemophilus spp.). 107891 was isolated from fermentation of Microbispora sp. PTA-5024 (WO 2005/04628 A1). It consists of a complex of closely related factors A1 and A2, whose structure can be reconducted to a peptide skeleton, 24 amino acids long, containing lanthionine and methyllanthionine as constituents. In addition, a chlorine atom and one or two --OH residues are present on the molecule. The structure of the components of the 107891 complex is represented by the formula 1 of FIG. 3, where R represents [OH] with the factors A₁(R═OH), factor A₂ (R═--(OH)₂). 107891 appears to combine elements of Type-A and --B lantibiotics: rings A and B in 107891 are highly related to the equivalent rings in Type-A compounds; however, as in Type-B lantibiotics, 107891 is rather globular, it lacks a flexible C-terminal tails and is devoid of charged amino acid residues. Consequently, it cannot be predicted whether the lan cluster devoted to 107891 formation would encode a single LanM enzyme or separate LanB and LanC proteins. Furthermore, there are no precedent for chlorine-containing lantibiotics, thus the genes responsible for this post-translational modification cannot be predicted from available data.

[0008] The design of industrial processes for antibiotic production has been relatively successful, resulting in large size fermentations with antibiotic titers reaching levels of several grams per liter. This has been achieved largely by following empirical, trial and error approaches, and lacks a rational basis. Development of new processes and improvement of current technology thus remains time consuming and may result in bacterial cultures that are unstable, perform inconsistently and accumulate unwanted by-products. In recent years, rational methods have been applied successfully to increase the level of antibiotic produced by Streptomyces spp., which have often involved the manipulation of key regulatory elements present within the gene cluster of interest or the overexpression of rate-limiting steps in the pathway. Therefore, the genes encoding such cluster-associated regulators or limiting steps in the synthesis can be effective tools for yield improvement. However, the cluster-associated regulators so far identified in actinomycetes belong to several different protein families. Even within one family, there is considerable variation in sequence identity. Therefore, the existence, nature, number and sequence of cluster-associated regulators cannot be predicted by comparison to other cluster, even those specifying a related antibiotic. As an example, the tylosin gene cluster encodes four distinct regulators, while none has been found in the cluster specifying the related macrolide antibiotic erythromycin. Similarly, the nature and reason for a rate-limiting step in a biosynthetic pathway cannot be established a priori.

[0009] Therefore, tools for increasing the 107891 yield would be highly desirable. However, there are no examples of clusters from other members of the genus Microbispora. Therefore, the mechanism(s) cannot be predicted through which the producer strain protects itself from the action of 107891, governs the expression of the other lan genes, or coordinates expression of Ian genes with its other cellular processes. Information about these will be very be useful for optimizing the production process.

DESCRIPTION OF THE INVENTION

[0010] The present invention provides a set of isolated polynucleotide molecules required for the biosynthesis of the lantibiotic 107891 in microorganisms.

[0011] So, according to one of its aspect, the present invention relates to polynucleotide molecules which are selected from the contiguous DNA sequence (SEQ ID NO: 1), which represents the mlb gene cluster as isolated from Microbispora sp. PTA-5024 and consists of 17 ORFs encoding the polypeptides required for 107891 formation.

[0012] The amino acid sequences of the polypeptide encoded by said 17 ORFs are provided in SEQ ID NOS: 2 to 18.

[0013] The present invention also provides an isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of:

[0014] a) the mlb gene cluster encoding the polypeptides required for the synthesis of 107891 and homologues thereof (SEQ ID NO: 1);

[0015] b) a nucleotide sequence encoding the same polypeptides encoded by the mlb gene cluster (SEQ ID NO. 1), other than the nucleotide sequence of the mlb gene cluster itself;

[0016] c) any nucleotide sequence of mlb ORFs 1 to 17, encoding the polypeptides encoded by SEQ ID NOS: 2 to 18;

[0017] d) a nucleotide sequence encoding the same polypeptide encoded by any of mlb ORFs 1 to 17 (SEQ ID NOS: 2 to 18), other than the nucleotide sequence of said ORF.

[0018] A further subject matter of this invention is to provide an isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of:

[0019] e) a nucleotide sequence encoding a polypeptide that is, over its full length, at least 65%, preferably 86%, more preferably 90%, most preferably 95% or more, identical in amino acid sequence to a polypeptide encoded by any of mlb ORFs 1 to 17 (SEQ ID NOS: 2 to 18).

[0020] In one embodiment the isolated nucleic acids of this invention comprises an ORF selected from ORFs 1 to 17 (SEQ ID NOS: 2 to 18), which encodes a polypeptide required for the synthesis of the prepropetide of 107891.

[0021] In another embodiment, the nucleic acid comprises an ORF selected from ORFs 1 to 17 (SEQ ID NOS: 2 to 18), which encodes the polypeptide required for the synthesis of the 107891 dehydratase enzyme. In yet another embodiment, the nucleic acid comprises an ORF selected from ORFs 1 to 17 (SEQ ID NOS: 2 to 18), which encodes the polypeptide required for the synthesis of the lanthionine and methyl-lanthionine residues of 107891.

[0022] According to another embodiment, in a nucleic acid of this invention, an ORF selected from ORFs 1 to 17 (SEQ ID NOS: 2 to 18) is provided, which encodes a polypeptide required for the chlorination of the triptophan residue of amino acid 4 of 107891.

[0023] In yet another embodiment, nucleic acid comprising an ORF selected from ORFs 1 to 17 (SEQ ID NOS: 2 to 18) is provided, which encodes a polypeptide required for the hydroxylation of the proline residue of aminoacid 14 of 107891.

[0024] In yet another embodiment, nucleic acid comprising an ORF selected from ORFs 1 to 17 (SEQ ID NOS: 2 to 18) is provided, which encodes a flavoprotein required for the oxidative decarboxylation that yields the S--[(Z)-2-aminovinyl]-(3S)-3-methyl-D-cysteine (AviMeCys) residues present at positions 21 and 24 of 107891.

[0025] According to another embodiment, in the nucleic acid of this invention, an ORF selected from ORFs 1 to 17 (SEQ ID NOS: 2 to 18) is provided which encodes the polypeptide required for the reduction of the flavoprotein.

[0026] According to yet another embodiment, nucleic acids are provided which comprise combinations of ORFs selected from ORFs 1 to 17 (SEQ ID NOS: 2 to 18), encoding polypeptides required for the export of and resistance to 107891.

[0027] In yet another embodiment, nucleic acids are provided which comprise combinations of ORFs selected from ORFs 1 to 17 (SEQ ED NOS: 2 to 18), encoding polypeptides required for regulating the expression of the mlb gene cluster.

[0028] Those skilled in the art understand that the present invention, having provided the nucleotide sequences encoding polypeptides of the 107891 biosynthetic pathway, also provides nucleotides encoding fragments derived from such polypeptides. According to the invention, those skilled in the art understand that, since the genetic code is degenerate, the same polypeptides encoded by SEQ ID NOS: 2 to 18 can be encoded by natural or artificial variants of ORFs 1 to 17, i.e. by nucleotide sequences other than the genomic nucleotide sequences specified by ORFs 1 to 17 but which encode the same polypeptides.

[0029] Furthermore, it is also understood that naturally occurring or artificially manufactured variants can occur of the polypeptides encoded by SEQ ID NOS: 2 to 18, said variants having the same function(s) as the above mentioned original polypeptides but containing addition, deletion or substitution of amino acid not essential for folding or catalytic function, or conservative substitution of essential amino acids.

[0030] Those skilled in the art understand also that, having provided the nucleotide sequence of the entire cluster required for 107891 biosynthesis, the present invention also provides nucleotide sequences required for the expression of the genes present in said cluster. Such regulatory sequences include but are not limited to promoter and enhancer sequences, antisense sequences, transcription terminator and antiterminator sequences. These sequences are useful for regulating the expression of the genes present in the mlb gene cluster. Cells carrying said nucleotide sequences, alone or fused to other nucleotide sequences, fall also within the scope of the present invention.

[0031] In one aspect, the present invention provides isolated nucleic acids comprising nucleotide sequences encoding the ORF6 polypeptide (SEQ ID NO: 7), or naturally occurring variants or derivatives of said polypeptide, encoding the prepropeptide of 107891.

[0032] In another aspect, the present invention provides nucleic acids comprising nucleotide sequences encoding the ORF7 polypeptide (SEQ ID NO: 8), or naturally occurring variants or derivatives of said polypeptide, useful for the dehydratation of serine and threonine residues in the lantibiotic precursor.

[0033] In yet another aspect, the present invention provides a nucleic acid comprising nucleotide sequences encoding the ORF8 polypeptide (SEQ ID NO: 9), or naturally occurring variants or derivatives of said polypeptide, useful for lanthionine and methyl-lanthionine formation in the lantibiotic precursor.

[0034] In another aspect, the present invention provides nucleic acids comprising nucleotide sequences encoding the ORF9 polypeptide (SEQ ID NO: 10), or naturally occurring variants or derivatives of said polypeptide, useful for AviMeCys formation in the antibiotic precursor. In another aspect, the present invention provides nucleic acids comprising nucleotide sequences encoding the ORF15 polypeptide (SEQ ID NO: 16), or naturally occurring variants or derivatives of said polypeptide, useful for the chlorinating the tryptophan residue in the lantibiotic precursor.

[0035] In yet another aspect, the present invention provides nucleic acids comprising nucleotide sequences encoding the ORF2 polypeptide (SEQ ID NO: 3), or naturally occurring variants or derivatives of said polypeptide, useful for hydroxylating the proline residue in the lantibiotic precursor.

[0036] In another aspect, the present invention provides nucleic acids comprising nucleotide sequences encoding the polypeptides specified by ORFs 1, 10 to 11, 13 to 14 and 17 (SEQ ID NOS: 2, 11 to 12, 14 to 15, and 18), or naturally or artificially occurring variants or derivatives of said polypeptides, useful for export out of the cells of 107891 or a 107891 precursor.

[0037] In another aspect, the present invention provides nucleic acids comprising nucleotide sequences encoding the ORFs 3 and 5 polypeptides (SEQ ID NO: 4 and 6), or naturally or artificially occurring variants or derivatives of said polypeptides, useful for regulating lantibiotic production.

[0038] In one embodiment, the present invention provides a lantibiotic-producing strain carrying extra copies of the nucleotide sequences specifying at least one ORF selected from any of ORFs 1 through 17 (SEQ ID NOS: 2 to 18).

[0039] In one preferred embodiment, such lantibiotic-producing strain is any strain belonging to the order Actinomycetales.

[0040] In yet another preferred embodiment, such lantibiotic producing strain is a member of the genus Microbispora.

[0041] In one preferred embodiment, the present invention provides a Microbispora strain containing one or more variations in the nucleotide sequence specified in SEQ ID NO: 1, such variation resulting in an increased or decreased expression of one or more of ORFs 1 through 17 (SEQ ID NOS: 2 to 18).

[0042] In one preferred embodiment, the present invention provides nucleic acids comprising a nucleotide sequence specified by SEQ ID NO: 1, or a portion thereof, carried on one or more vectors, useful for the production of 107891, one or more of its precursors or a derivative thereof by another cell.

[0043] In one preferred embodiment, said nucleotide sequence or portion thereof is carried on a single vector. Suitable vector are any cosmid, fosmid, BAC, PAC, ESAC vector capable of carrying the entire mlb cluster as defined herein. Suitable vectors are well known to those skilled in the art and described in the literature (Kieser et al., 2000, and references described therein).

[0044] In one aspect, the present invention provides a method for increasing the production of 107891, said method comprising the following steps:

[0045] transforming with a recombinant DNA vector a microorganism that produces 107891 or homologues thereof or precursors of 107891 or homologues thereof by means of a biosynthetic pathway, said vector comprising a DNA sequence, chosen from any of ORFs 1 through 17 (SEQ ID NO: 2 through 18), that codes for an activity that is rate limiting in said pathway;

[0046] culturing said microorganism transformed with said vector under conditions suitable for cell growth, expressing said gene and producing said antibiotic or antibiotic precursor.

[0047] Suitable host cell are Actinomycetales, to the families Streptosporangiaceae, Micromonosporaceae, Pseudonocardiaceae and Streptomycetaceae, to the genera Microbispora, Actinoplanes, Planomonospora, Streptomyces and the like.

[0048] In another aspect, the present invention provides a method for producing derivatives of 107891 and homologues thereof, said method comprising the following steps:

[0049] cloning in a suitable vector a segment chosen from the nucleotide sequence defined by SEQ ID NO:1, said segment containing at least a portion of one of ORFs 1 through 17 (SEQ ID NO: 2 through 18), said ORF encoding a polypeptide that catalyzes a biosynthetic step that one wishes to bypass;

[0050] inactivating said ORF by removing or replacing one or more codons that specify for amino acids that are essential for the activity of said polypeptide;

[0051] transforming with said recombinant DNA vector a microorganism that produces 107891 or homologues thereof or 107891 precursor thereof by means of a biosynthetic pathway;

[0052] screening the resulting transformants for those where said DNA sequence has been replaced by the mutated copy;

[0053] culturing mutant cells under conditions suitable for cell growth, expressing of said pathway and producing of said pathway analogue.

[0054] In yet another aspect, the present invention provides a method for producing novel lantibiotics, said method comprising the following steps:

[0055] transforming with a recombinant DNA vector a microorganism that produces a lantibiotic or homologues thereof or precursor thereof by means of a biosynthetic pathway, said vector comprising one or more ORFs, chosen among ORFs 1 through 17 (SEQ ID NOS: 2 through 18), that codes for enzyme(s) capable of modifying said lantibiotic or lantibiotic precursor;

[0056] culturing said microorganism transformed with said vector under conditions suitable for cell growth, expression of said gene and production of said lantibiotic or homologues thereof or lantibiotic precursor thereof.

[0057] In yet another aspect, the present invention provides a method for producing novel lantibiotics, said method comprising the following steps:

[0058] transforming with a recombinant DNA vector a microorganism, said vector comprising one or more ORFs, chosen among ORFs 1 through 17 (SEQ ID NOS: 2 through 18), that codes for enzyme(s) capable of modifying a lantibiotic or lantibiotic precursor;

[0059] culturing said microorganism transformed with said vector under conditions suitable for cell growth, expressing of said gene, in the presence of said lantibiotic or lantibiotic precursor.

[0060] In yet another aspect, the present invention provides a method for producing novel lantibiotics, said method comprising the following steps:

[0061] transforming with a recombinant DNA vector a microorganism, said vector comprising one or more ORFs, chosen among ORFs 1 through 17 (SEQ ID NOS: 2 through 18), that codes for one or more polypeptides that modify a lantibiotic or lantibiotic precursor;

[0062] preparing a cell extract or cell fraction of said microorganism under conditions suitable for the presence of active polypeptide(s), said cell extract or cell fraction containing at least said polypeptide(s);

[0063] adding a lantibiotic or lantibiotic precursor to said cell extract or cell fraction, and incubating said mixture under conditions where said polypeptide(s) can modify said lantibiotic or lantibiotic precursor.

[0064] A further aspect of this invention includes an isolated polypeptide involved in the biosynthetic pathway of 107891 selected from

[0065] an ORF polypeptide encoded by any one of mlb ORFs 1 to 17 (SEQ ID NOS: 2 through 18) and

[0066] a polypeptide which is at least, over its full length, 65%, preferably 95% or more, identical in amino acid sequence to a polypeptide encoded by any one of mlb ORFs 1 to 17 (SEQ ID NOS: 2 through 18), preferably by any one of the mlb ORFs 1 to 3, 5 to 11, 13 to 17 (SEQ NOS: 2 to 4, 6 to 12, 14 to 18).

[0067] A preferred group of polypeptides comprises any ORF polypeptide encoded by any of the mlb ORFs 1 to 3, 5 to 11, 13 to 17 (SEQ ID NOS: 2 to 4, 6 to 12, 14 to 18), or any polypeptide which is at least, over its full length, 65%, preferably 86%, more preferably 90%, most preferably 95% or more, identical in amino acid sequence to a polypeptide encoded by any of said mlb ORFs.

DEFINITIONS

[0068] The term "isolated nucleic acid" herein refers to a DNA molecule, either as genomic DNA or a complementary DNA (cDNA), which can be single or double stranded, of natural or synthetic origin. This term refers also to an RNA molecule, of natural or synthetic origin.

[0069] The term "nucleotide sequence" herein refers to full length or partial length sequences of ORFs and intergenic regions as disclosed herein.

[0070] The term "nucleotide sequence" herein is also referred to and/or comprises any one of the nucleotide sequence of the invention as show in the sequence listing. Any one of the nucleotide sequences of the sequence listing is

[0071] A) a coding sequence,

[0072] B) an RNA molecule derived from transcription of (A),

[0073] C) a coding sequence which uses the degeneracy of the genetic code to encode an identical polypeptide,

[0074] D) an intergenic region, containing promoters, enhancers, terminator and antiterminator sequences.

[0075] The terms "gene cluster", "cluster" and "biosynthesis cluster" herein designate a contiguous segment of a microorganism's genome that contains all the genes required for the synthesis of a secondary metabolite.

[0076] The term "mlb" herein refers to a genetic element responsible for 107891 biosynthesis in Microbispora sp. PTA-5024. The term is an acronym of Microbispora LantiBiotic.

[0077] The term "ORF" herein refers to a genomic nucleotide sequence that encodes one polypeptide. In the context of the present invention, the term ORF is synonymous with "gene".

[0078] The term "ORF polypeptide" herein refers to a polypeptide encoded by an ORF. The term "mlb ORF" herein refers to an ORE comprised within the mlb gene cluster.

[0079] The term "secondary metabolite" herein refers to a bioactive substance produced by a microorganism through the expression of a set of genes specified by a gene cluster.

[0080] The term "vector" herein is defined to include, inter alia, any plasmid, cosmid, phage, which can transform prokaryotic host by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication)

[0081] The term "production host", "host cell" herein is a microorganism where the formation of a secondary metabolite is directed by a gene cluster derived from a donor organism.

[0082] The term "homologue" herein refers to a polypeptide, encoded by a biosynthetic gene cluster, which shares at least 65% sequence identity over its entire length with any of the polypeptides encoded by the mlb cluster.

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG. 1 shows isolated DNA segments derived from the chromosome of Microbispora sp. PTA-5024. The thick line denotes the segment described in SEQ ID NO: 1. The cosmids carrying said isolated DNA segments are designated 1G6 and 6H6.

[0084] FIG. 2 shows genetic organization of the mlb cluster. Each ORF is represented by an arrow, and numbered as in Table 1 (FIG. 4). The orientation is the same as in FIG. 1. Numbers on the scale bars indicate sequence coordinates (in kb).

[0085] FIG. 3 shows structure of the components of the 107891 complex.

[0086] FIG. 4 shows the main features of the ORFs.

A. THE MLB GENES ISOLATED FROM MICROBISPORA

[0087] 107891 is a complex of closely related peptide antibiotics produced by Microbispora sp. PTA-5024. The present invention provides nucleic acid sequences and characterization of the mlb gene cluster for 107891 biosynthesis. The physical organization of the mlb gene cluster, together with flanking DNA sequences, is reported in FIG. 1, which illustrates the physical map of a 20-kb genomic segment from the genome of Microbispora sp. PTA-5024, together with two cosmids defining such segment. The genetic organization of the DNA segment governing 107891 biosynthesis is shown in FIG. 2 and its nucleotide sequence is reported as SEQ ID NO: 1.

[0088] The precise boundary of the cluster can be established from the functions of its gene products. Therefore, on the left end (FIG. 1), the mlb cluster is delimited by mlb ORF1, encoding the ABC transporter (SEQ ID No: 2), involved in the export of 107891. On the right side, the mlb cluster is delimited by mlb ORF17, a membrane ion antiporter (SEQ ID No: 18). The mlb cluster spans approximately 20,000 base pairs and contains 17 ORFs, designated mlb ORF1 through mlb ORF17. The contiguous nucleotide sequence of SEQ ID NO: 1 (20000 base pairs) encodes the 17 deduced proteins listed in SEQ ID NOS: 2 to 18.

[0089] ORF1 (SEQ ID NO: 2) represents 300 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 67 to 969.

[0090] ORF2 (SEQ ID NO: 3) represents 414 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 966 to 2210.

[0091] ORF3 (SEQ ID NO: 4) represents 260 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 2941 to 3723.

[0092] ORF4 (SEQ ID NO: 5) represents 221 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 3948 to 4614.

[0093] ORF5 (SEQ ID NO: 6) represents 220 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 5283 to 4621 on the complementary strand.

[0094] ORF6 (SEQ ID NO: 7) represents 57 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 5414 to 5587.

[0095] ORF7 (SEQ ID NO: 8) represents 1115 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 5706 to 9053.

[0096] ORF8 (SEQ ID NO: 9) represents 475 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 9080 to 10507.

[0097] ORF9 (SEQ ID NO: 10) represents 215 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 10537 to 11184.

[0098] ORF10 (SEQ ID NO: 11) represents 316 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 11181 to 12131.

[0099] ORF11 (SEQ ID NO: 12) represents 242 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 12253 to 12981.

[0100] ORF12 (SEQ ID NO: 13) represents 211 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 13357 to 13992.

[0101] ORF13 (SEQ ID NO: 14) represents 249 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 14795 to 15544.

[0102] ORF14 (SEQ ID NO: 15) represents 236 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 15546 to 16256.

[0103] ORF15 (SEQ ID NO: 16) represents 541 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 16370 to 17995.

[0104] ORF16 (SEQ ID NO: 17) represents 178 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 17992 to 18528.

[0105] ORF17 (SEQ ID NO: 18) represents 430 amino acids deduced from translating SEQ ID NO: 1 from nucleotides 18525 to 19817.

The Genomic Organization and Primary Sequence of the Mlb Cluster Places 107891 in Type-A Lantibiotics.

[0106] Comparison between mlb and other lantibiotic gene clusters reveals important differences. In fact, the mlb cluster is characterized by the presence of several ORFs that do not find homologs in other lantibiotic clusters. These include mlb ORFs 1 through 6, 12, 15 through 18 (SEQ ID NOS: 2 through 7, 13, 16 through 19). In conclusion, the organization of the mlb cluster as described herein is substantially different from those of other clusters involved in the synthesis of other lantibiotic. It therefore represents the first example of a cluster with such a genomic organization.

B. ROLES OF THE MLB GENES

[0107] The present invention discloses the DNA sequence responsible for the synthesis of the prepropeptide precursor of 107891. The 107891 prepropeptide consists of a leader peptide, 33-aa long, and of a 24-aa propeptide. The nucleic acid sequences referred to herein are those encoding the 107891 prepropeptide or fragments thereof. The 57-aa 107891 prepropeptide represents a novel element that shows only, over its entire length, 41% identity with UniProt accession number P21838, the prepropeptide of the lantibiotic gallidermin from Staphylococcus gallinarum.

[0108] Other genes present in the mlb cluster represent novel genetic elements useful for increasing production of 107891 or for synthesizing novel metabolites. Among these, mlb ORFs 7 to 8 (SEQ ID NO: 8 through 9) encode the proteins involved in post-translational modification of the translation product of mlb ORF6, to introduce the lanthionine residues in mature 107891. In particular, the mlb ORF7 polypeptide is responsible for dehydration of the Ser and Thr residues in the prepropeptide portion of 107891 to generate dehydroalanine and dehybutyrine residues, respectively. The mlb ORF8 polypeptide catalyzes the nucleophilic attack of cysteine residues within the prepropeptide onto the dehydro amino acid residues. These genes can be cloned and expressed in a heterologous host to yield active enzymes capable of introducing lanthionine residue to other prepropeptides.

[0109] Yet other preferred nucleic acid molecules of the present invention include mlb ORF9 (SEQ ID NO: 10) that encodes a protein involved in the oxidative decarboxylation yielding the S--[(Z)-2-aminovinyl]-(3S)-3-methyl-D-cysteine residue (Formula I).

[0110] Yet other preferred nucleic acid molecules of the present invention include mlb ORF16 (SEQ ID NO: 17) that encodes a tryptophan halogenase, responsible for the addition of a chlorine atom to amino acid 4 of 107891. mlb ORF16 represents a novel and unique genetic element, previously not reported in other lantibiotic clusters. Indeed, chlorination is a rather unique feature of 107891 among known lantibiotics. This gene can be cloned and expressed in a heterologous host to yield an active enzyme capable of chlorinating tryptophan residues of lantibiotic molecules. Alternatively, mlb ORF16 can be inactivated in the producing strain, resulting in the formation of 107891 derivatives devoid of the chlorine attached to amino acid 4.

[0111] Yet other preferred nucleic acid molecules of the present invention include mlb ORF2 (SEQ ID NO: 3 that encodes a cytochrome P450 hydroxylase responsible for post-translational modification of the 107891 prepropeptide, by the addition of one or two oxygen(s) to the proline residue at position 14. mlb ORF2 represents a novel and unique genetic element, that has never been reported in other lantibiotic clusters. Indeed, the 107891 proline hydroxylation profile is rather unique among lantibiotics. This gene can be cloned and expressed in a heterologous host to yield an active enzyme capable of oxidizing proline residues present in a lantibiotic molecule. Alternatively, mlb ORF2 can be inactivated in the producing strain, resulting in the formation of 107891 derivatives devoid of oxygen atoms at amino acid 14.

[0112] The mlb cluster also includes a number of regulatory genes, responsible for activating, directly or indirectly, the expression of biosynthesis and resistance genes during 107891 production. These genes include mlb ORFs 3 and 5 (SEQ ID NOS: 4 and 6): mlb ORF3 (SEQ ID NO: 4) represents a separate quorum-sensing peptide, responsible, in part, for the regulation of 107891 production; and mlb ORF5 (SEQ ID NO: 6) is highly related to Sigma-70, an extracytoplasmic function family of RNA polymerase sigma factors that act as positive transcriptional regulator. mlb ORFs 3 and 5 represent novel genetic elements, absent from other lantibiotic clusters. The two genes, mlb ORF 3 and 5, can be cloned and expressed, either individually or in any combination of them, in another lantibiotic producer strains to increase the yield of product formed.

[0113] Host strains include but are not limited to strains belonging to the order Actinomycetales, to the families Streptosporangiaceae, Micromonosporaceae, Pseudonocardiaceae and Streptomycetaceae, to the genera Microbispora, Actinoplanes, Planomorzospora, Streptomyces and the like. Alternatively, these genes can be overexpressed, individually or in any combination of them, in the 107891 producing strain to increase the yield of 107891.

[0114] The mlb cluster also includes a number of genes responsible for exporting lantibiotic intermediates or finished products out of the cytoplasm and for conferring resistance to the producer cell. These genes include mlb ORFs 1, 10 to 11, 13 to 14 and 17 (SEQ ID NOS: 2, 11 to 12, 14 to 15 and 18). mlb ORFs 1, 10 to 11, 13 to 14 encode transporters of the ABC class, responsible for the ATP-dependent excretion of 107891 or its intermediates. mlb ORF17 encodes an Na/K ion-antiporter, responsible for exporting 107891 or its intermediates against a proton gradient. These genes can be cloned and expressed, either individually or in any combination of them, in another (antibiotic producer strain to increase the yield of product formed. Host strains include but are not limited to strains belonging to the order Actinomycetales, to the families Streptosporangiaceae, Micromonosporaceae, Pseudonocardiaceae and Streptomycetaceae, to the genera Microbispora, Actinoplanes, Planomonospora, Streptomyces and the like.

[0115] Alternatively, these genes can be overexpressed, individually or in any combination of them, in the 107891 producing strain to increase the yield of 107891.

C. USES OF THE MLB CLUSTER

[0116] The present invention provides also nucleic acids for the expression of the entire 107891 molecule, any of its precursors or a derivative thereof. Such nucleic acids include isolated gene cluster(s) comprising ORFS encoding polypeptides sufficient to direct the assembly of 107891. In one example, the entire mlb cluster (SEQ ID NO: 1) can be introduced into a suitable vector and used to transform a desired production host. In another aspect, the mlb cluster is cloned as two separate segments into two distinct vectors, which can be compatible in the desired production host. In yet another aspect, the mlb cluster can be subdivided into three segments, each cloned into a separate, compatible vector. Examples of the use of one-, two- or three-vector systems have been described in the literature.

[0117] Once the mlb cluster has been suitably cloned into one or more vectors, it can be introduced into a number of suitable production hosts, where production of lantibiotics might occur with greater efficiency than in the native host. Preferred host cells are those of species or strains that can efficiently express actinomycetes genes. Such host include but are not limited to Actinomycetales, Streptosporangiaceae, Micromonosporaceae, Pseudonocardiaceae and Streptomycetaceae, Microbispora, Actinoplanes, Planomonospora and Streptomyces and the like. Alternatively, a second copy of the mlb cluster, cloned into one or more suitable vectors, can be introduced the 107891 producing strain, where the second copy of mlb genes will increase the yield of 107891.

[0118] The transfer of the producing capability to a well characterized host can substantially improve several portions of the process of lead optimization and development: the titer of the natural product in the producing strain can be more effectively increased; the purification of the natural product can be carried out in a known background of possible interfering activities; the composition of the complex can be more effectively controlled; altered derivatives of the natural product can be more effectively produced through manipulation of the fermentation conditions or by pathway engineering.

[0119] Alternatively, the biosynthetic gene cluster can be modified, inserted into a host cell and used to synthesize or chemically modify a wide variety of metabolites: for example the open reading frames can be re-ordered, modified and combined with other lantibiotic biosynthesis gene cluster.

[0120] Using the information provided herein, cloning and expression of 107891 nucleic acids can be accomplished using routine and well known methods.

[0121] In another possible use, selected ORFs from the mlb gene cluster are isolated and inactivated by the use of routine molecular biology techniques. The mutated ORF, cloned in a suitable vector containing DNA segments that flank said ORF in the Microbispora sp. PTA-5024 chromosome, is introduced into said Microbispora strain, where two double cross-over events of homologous recombination result in the inactivation of said ORF in the producer strain. This procedure is useful for the production of precursors or derivatives of 107891 in an efficient manner.

[0122] In another possible use, selected ORFs from the mlb gene cluster are isolated and placed under the control of a desirable promoter. The engineered ORF, cloned in a suitable vector, is then introduced into Microbispora sp. PTA-5024, either by replacing the original ORF as described above, or as an additional copy of said ORF. This procedure is useful for increasing or decreasing the expression level of ORFs that are critical for production of the 107891 molecule, precursors or derivatives thereof.

Experimental Section

[0123] The following examples serve to illustrate the principles and methodologies through which the 107891 gene cluster is identified and the principles and methodologies through which all the mlb genes are identified and analyzed. These examples serve to illustrate the principles and methodologies of the present invention, but are not meant to limit its scope.

General Methods

[0124] Unless otherwise indicated, bacterial strains and cloning vectors can all be obtained from public collections or commercial sources. Standard procedures are used for molecular biology. Microbispora was grown in HT agar and in V6 medium (20 g/l glucose, 5 g/l yeast extract, 3 g/l casein hydrolysate, 5 g/l meat extract, 5 g/l peptone, 1.5 g/l NaCl, 0.5% glycerol). Lantibiotics are isolated following published procedures. Sequence analyses are performed using standard programs. Database searches are performed with Blast or Fasta programs at public sites.

Example 1

Isolation of 107891 Biosynthesis Genes

[0125] A genomic library is made with DNA from Microbispora sp. PTA-5024 in the conjugative cosmid vector Supercos 3. This was constructed by insertion of the aacIV-oriT-intΦC31 cassette from pSET152 into Supercos 1 (Stratagene, La Jolla, Calif. 92037) as follows: the aacIV-oriT-intΦ31 cassette is obtained by PCR as a NruI fragment, 3.8 kb long, from the vector pSET152 and inserted into the same site of supercos1. Total DNA from Microbispora sp. PTA-5024 is partially digested with Sau3AI in order to optimize fragment sizes in the 40 kb range. The partially digested DNA is treated with alkaline phosphatase and ligated to Supercos3 previously digested with BamHI. The ligation mixture is packaged in vitro and used to transfect E. coli XL1Blue cells. The resulting cosmid library is screened by hybridization with the oligonucleotide probe 5'-GTS ACS WSS TGG WSS YTS WSS ACS GGS CCS TGC ACS WSS CCS GGS GGS WSS AAC WSS WSS TCC WSS TG-3' (SEQ ID NO: 19). The oligonucleotide is designed from the amino acid sequence deduced from the structure of 107891. Two cosmids positive to this probe are isolated and physically mapped with restriction enzymes. From such experiments, the cosmids reported in FIG. 1 are identified. The segment thus identified from the genome of Microbispora sp. PTA-5024 contains the mlb gene cluster responsible for the synthesis of the antibiotic 107891.

[0126] The above example serves to illustrate the principle and methodologies through which the mlb cluster can be isolated. It will occur to those skilled in the art that the mlb cluster can be cloned in a variety of vectors. However, those skilled in the art understand that, given the 20-kb size of the mlb cluster, preferred vectors are those capable of carrying large inserts, such as lambda, cosmid and BAC vectors. Those skilled in the art understand that other probes can be used to identify the mlb cluster from such a library. From the sequence reported in SEQ ID NO: 1, any fragment can be PCR-amplified from Microbispora sp. PTA-5024 DNA and used to screen a library made with such DNA. One or more clones from said library can be identified that include any segment covered by SEQ ID NO: 1. Furthermore, it is also possible to identify the mlb cluster through the use of heterologous probes, such as those derived from other mlb cluster, using the information provided in Table 1. Alternatively, other gene clusters directing the synthesis of secondary metabolites contain genes sufficiently related to the mlb genes as to allow heterologous hybridizations. All these variations fall within the scope of the present invention.

Example 2

Sequence Analysis of 107891 Gene Cluster

[0127] The mlb cluster, identified as described under Example 1, is sequenced by the shotgun approach. The sequence of the mlb cluster is provided herein as SEQ ID NO: 1. The resulting DNA sequence is analyzed to identify likely coding sequences, which are compared against other Ian clusters or searched against GenBank. The exact start codon for each ORF is established by multiple alignment of related sequences or by searching for an upstream ribosomal binding site. In total, 17 ORFs, denominated nub ORF1 through ORF17, are identified. The results of these analyses are summarized in Table 1, and provided herein in the sequence listing as SEQ ID No: 2 through SEQ ID No: 18. Details are given below.

2A. Synthesis of the 107891 Prepropeptide

[0128] mlb ORFs 6 is responsible for the synthesis of prepropeptide. The prepropeptide contains a 49-aa leader sequence and a 24-aa propeptide (SEQ ID NOS: 7) which is post-translationally modified to produce the mature lantibiotic. Two common features of lantibiotic leader peptides are preserved in 107891: the conserved sequence of Type-A lantibiotics (e.g. the F-D/N-L-D/E motif) and the proline residue at position-2. The C-terminal part of the prepropeptide (SEQ ID NOS: 7) is in agreement with the published 107891 primary structure and its proposed propeptide sequence.

2B. Post-Translationally Modification of the 107891 Propeptide

[0129] Four proteins, encoded by mlb ORFs 7 through 9 (SEQ ID NOS: 8 through 10) and mlb ORF 17 (SEQ ID NOS: 18) are involved in the post-translational modification of the 107891 prepropeptide. Homologs of these gene products are found in many lantibiotic clusters. On the basis of the sequence identities with the dehydratases and cyclases found in other lantibiotic clusters, and their roles, the following predictions can be made. The mlb ORF7 polypeptide is responsible for dehydration of the serine residues at positions 3, 5, 13, 18 and 21 and of the theonine residue at position 2 and 8 of the 107891 propeptide, to generate the corresponding dehydrated residues. The mlb ORF8 polypeptide catalyzes the regio- and stereospecific conjugate addition of the cysteine residues present in the 107891 propeptide to four dehydroalanine and one dehydrobutyrine residues to generate the corresponding five thioethers. Specifically, the mlb ORF8 polypeptide is involved in the formation of the 3-7, 13-20, 18-23 and 21-24 lanthionines and of the 8-11 methylanthionine.

[0130] On the basis of the sequence identities observed with the decarboxylases encoded by the epidermin and mersacidin clusters, mlb ORF9 encodes the enzyme responsible for the decarboxylation of the 21-24 lanthionine moiety. On the basis of the sequence identities observed with the flavin reductase encoded by other antibiotic clusters, mlb ORF16 encoded a flavoprotein reductase. Considering the roles predicted for oxidative decarboxylation during epidermin and mersacidin formation, the mlb ORFs 9 and 16 polypeptides catalyze the formation of the S--[(Z)-2-aminovinyl]-D-cysteine residue present in 107891 (Formula I).

2C. Formation of §-Hydroxyproline and Tryptophan Chlorination

[0131] Two proteins, encoded by mlb ORFs 2 and 15 (SEQ ID NOS: 3 and 16) are involved in the addition of one or two β-hydroxyl groups to the proline residue at position 14 and in the chlorination of the tryptophan residue at position 4 of the 107891 propeptide. The mlb ORF2 polypeptide show significant identity to P450 monooxygenases (Table 1) and is involved in hydroxylation of the proline residue. No homologs of mlb ORF2 have been found in other lantibiotic clusters, thereby this gene represents a unique example of a P450 monoxygenase involved in hydroxylation of a lantibiotic molecule. In addition, on the basis of the level of identities with other halogenases, the mlb ORF15 polypeptide is involved tryptophan chlorination and represents a unique example of a halogenase involved in modification of a lantibiotic molecule.

2D. Export and Resistance

[0132] Five proteins, encoded by ORFs 1, 10, 11, 13, 14 and 17 (SEQ ID NOS: 2, 11, 12, 14, 15 and 18) are involved in exporting 107891 or its precursor outside the cytoplasm and in conferring resistance to the producing strain. Their predicted roles are as follows.

[0133] Homologs of ORF1 (SEQ ID NO: 2) are not found in other lantibiotic clusters. This gene encodes additional ABC-type transporters (Table 1), and is therefore involved in conferring resistance to 107891 in the producing strain Microbispora sp. PTA. Homologs of ORFs 10, 13 to 14 and 17 (SEQ ID NOS: 11, 14 to 15 and 18) are present in other lantibiotic clusters (Table 1). They encode ABC-type and ion-dependent transmembrane transporters, respectively. They are thus involved in export and/or compartimentalization of 107891 or its precursors. mlb ORF17 encodes an Na/K ion-antiporter, responsible for exporting 107891 or its intermediates against a proton gradient.

2E. Regulation

[0134] Two proteins, encoded by ORFs 3 and 5 (SEQ ID NOS: 4 and 6), are involved in regulating the expression of one or more of the mlb genes. The mlb ORF5 polypeptide (SEQ ID NO: 6) represents a novel genetic element, homologs of which are not found in the other lantibiotic clusters. This protein belongs to the extracytoplasmic function family of sigma factors that act as positive transcriptional regulators. The ORF3 polypeptide (SEQ ID NO: 4) belongs to the family of LuxR-type transcriptional regulators. ORFs 3 (SEQ ID NOS: 4) is therefore likely to be required for the expression of one or more of the mlb genes.

2F. Additional Functions

[0135] Two additional ORFs are present in the mlb cluster: ORF4 (SEQ ID NO: 5) and ORF12 (SEQ ID NO: 13). Both ORFs are related to proteins of unknown function present in Salinispora tropica and Streptomyces ambofaciens, respectively (Table 1). However, their precise role in 107891 biosynthesis cannot be predicted yet.

Example 3

Manipulation of the 107891 Pathway by Gene Replacement

[0136] Using the information provided in Example 2, an in frame deletion in ORF 2 is constructed as follows. Fragment A was obtained through amplification with oligos 5'-AAGCTTGCATCTGCGTGGGCGTCCTGC-3' (SEQ ID NO: 20) and 5'-TCTAGACGGTCCGAAGATCATGGCCGCGG-3' (SEQ ID NO: 21); and fragment B is obtained through amplification with oligos 5'-TCTAGATCCATGTGAACCGGCGGGTGGCCG-3' (SEQ ID NO: 22) and 5'-GAATTCCGGTCGCTCTCCTCGTCCTTTGCC-3' (SEQ ID NO: 23)

[0137] Next, fragment A is digested with EcoRI and XbaI, fragment B with XbaI and HindIII, and both are ligated to pSET152 previously digested with EcoRI and HindIII. After transformation of E. coli DH5α cells, the resulting plasmid, designated pDM1, is recognized by the presence of fragments of 4 kb and 1.5 kb after digestion with EcoRI and HindIII. An aliquot of pDM1 is transferred into E. coli ET12567(pUB307) cells, yielding strain DM1. Then, about 10⁸ CFU of DM1 cells, from an overnight culture in LB, are mixed with about 10⁷ CFU of Microbispora PTA 5024 grown in Rare3 medium for about 80 h. The resulting mixture is spread onto HT plates, which are then incubated at 28° C. for about 20 h. After removing excess E. coli cells with a gentle wash with water, plates are overlaid with 3 ml soft agar containing 200 μg nalidixic acid and 15 μg/ml apramycin. After further incubation at 28° C. for 3-5 weeks, Microbispora ex-conjugants are streaked onto fresh medium containing apramycin. One such ex-conjugant, named strain Mb-DM1, is further processed. Strain Mb-DM1 is then grown for several passages in HT medium without apramycin and appropriate dilutions are plated on HT agar without apramycin. Individual colonies are then analyzed by PCR, using oligos 5'-CGCGCTGCTCGGGGCCAAC-3' (SEQ ID NO: 24) and 5'-AGGAAACGGCCAGCCCGTGG-3' (SEQ ID NO: 25). Colonies containing the deleted allele of ORF2 are recognized by the presence of a 1.5 kb band. One such colony, designated Mb-DM2, is grown in HT medium and the formation of dehydroxyl-107891 is confirmed by comparison with an authentic standard.

[0138] The above example serves to illustrate the principle and methodologies through which an ORE chosen among any of those specified by SEQ ID NOS: 2 to 18 can be replaced by a mutated copy in the 107891 producing strain Microbispora sp. PTA 5024. It will occur to those skilled in the art that ORF2 (SEQ ID NO: 3) is just an example of the methodologies for creating in frame deletions in the cluster specified by SEQ ID NO: 1.

[0139] Those skilled in the art understand also that in frame-deletions are just one method for generating mutations, and that other methods including but not limited to frame-shift mutations, insertions and site-directed mutations can also be used to generate null mutants in any of the ORFs specified by SEQ ID NOS: 2 to 18

[0140] Those skilled in the art also understand that, having established a method for generating mutations in any of the ORFs specified by SEQ ID NOS: 1, these same methodologies can be applied for altering the expression levels of these same ORFs. Examples for how this can be achieved include but are not limited to integration of multiple copies of said ORFs into any place in the Microbispora sp. PTA 5024 genome, alteration in the promoters controlling the expression of said ORFS, removal of antisense RNAs or transcription terminators interfering with their expression.

[0141] Finally, variations in the vectors used for introducing the mutated alleles into Microbispora sp. PTA5024, in the conditions for conjugation and cultivation of the donor and recipient strain, in the method for selecting and screening ex-conjugants and their derivatives, all fall within the scope of the present invention.

Example 4

In Vitro Halogenation of a Lantibiotic

[0142] Using the information provided in Example 2, mlb ORF15 (SEQ ID NO: 16) is overexpressed in E. coli as follows. A 1.6 kb fragment, obtained by amplification with oligos 5'-TTTTTCATATGGGTGGGAGTGATCGGCGGCG-3' (SEQ ID NO: 26) and 5'-TTTTTGTCGACCTACTGCTGGCCGCGGTCCGGACT-3' (SEQ ID NO: 27), is digested with NcoI and SalI and ligated to pET22b, previously digested with NcoI and XhoI. After transformation of E. coli DH5a cells, the resulting plasmid pHAL, recognizable for the presence of fragments of 5.5 kb and 1.6 kb after digestion with NdeI and XhoI, is introduced into E. coli BL21(DE3) cells. Cultures of E. coli BL21(DE3) cells harbouring pHAL are grown at 20° C. in LB to an OD600 of 0.6. Then, IPTG is added to 1 mM and cells grown for further 6 h. Cells are harvested, ruptured by sonication and the His-tagged ORF15 polypeptide recovered from a Ni-agarose column. In vitro halogenation of substrates, such as lacticin 481 or 97518 is carried out and formation of the chlorine-derivative of the lantibiotic is established by MS analysis.

[0143] The above example serves to illustrate the principle and methodologies through which an ORF chosen among any of those specified by SEQ ID NOS: 2 to 18 can be overexpressed in a convenient host, the resulting enzyme overproduced and used for transforming a lantibiotic natural product into a different compound. It will occur to those skilled in the art that ORF15 (SEQ ID NO: 16) is just used as an example of the methodologies for overproducing any polypeptide encoded by SEQ ID NO: 1. Those skilled in the art understand also that other methods for overproducing proteins, including but not limited to the use of different affinity tags, the use of different vectors, of different host strains, and of methods of induction, can also be used to overproduce the polypeptides specified by ORFs 1 to 17 (SEQ ID NOS: 2 to 18).

[0144] It will occur to those skilled in the art that other lantibiotic substrates containing or not triptophan residue, can be used for adding chlorine atoms by ORF15 (SEQ ID NO: 16). It will also occur to those skilled in the art that other ORF polipeptides specified by mlb cluster (SEQ ID NO: 1) can be used for the post-translational modification of other lantibiotic prepropeptides. ORF polipeptides that can be used for this purpose include but are not limited to ORF2 (SEQ ID NO 3). Specifically, ORFs 7 and 8 (SEQ ID NO 8 and 9) can be used to dehydrate and introduce thioether bridges in other lantibiotic prepropeptides; ORFs 9 and 16 (SEQ ID NO 10 and 17) can be used to decarboxylate other lantibiotic containing a C-terminal Cys residue; and ORF 2 (SEQ ID NO 3) can be used to hydroxylate other proline containing lantibiotics.

Example 5

Expression of the Mlb Cluster in a Heterologous Host

[0145] Using the information provided in Examples 1 and 2, cosmid 1G6 (FIG. 1) containing the entire mlb cluster (SEQ ID NO: 1) is introduced into Streptomyces albus by conjugation as described by Kieser et al., 2000. Apramycin resistant exconjugants are grown under appropriate conditions and 107891 is purified as described.

[0146] Those skilled in the art understand also that S. albus is just one producer strain, and that other strains can also be used for introducing the entire mlb cluster (SEQ ID NO: 1) and for the production of 107891. Preferred host cells are those of species or strains that can efficiently express actinomycete genes. Such host include but are not limited to Actinomycetales, Streptosporangiaceae, Micromonosporaceae, Pseudonocardiaceae and Streptomycetaceae, Microbispora, Actinoplanes, Planomonospora and Streptomyces and the like. Those skilled in the art understand that, provided that suitable promoters are placed in front of mlb operons, production hosts need not to be limited to those cells that can efficiently expressed actinomycete genes. Suitable production hosts of this latter category can be found among those that are easy to manipulate genetically or among those that naturally produce other lantibiotics. Examples of such production hosts include but not limited to Escherichia coli and related species, Bacillus spp., Streptococcus spp, Lactobacillus spp., Staphylococcus spp., and the like.

[0147] Those skilled in the art understand also that using the methodologies described in this example, the mlb cluster can be introduced as a second copy into the original 107891 producer strain Microbispora sp. PTA 5024 where the second copy of the mlb genes will increase the yield of 107891.

[0148] Those skilled in the art understand also that different vectors, different methodologies for introducing the mlb cluster, different methods of selecting recombinant clones carrying the mlb cluster, different methods and conditions for growing said recombinant clones and different methods for detecting 107891 production, can be effective for expression of the mlb cluster in a heterologous host.

Example 6

Generation of a Library of 107891 Variants

[0149] Using the information provided in Examples 2, 3 and 5, mlb ORF 6 (SEQ ID NO: 7) is modified to produce variants of 107891 as follows. First, a vector carrying an in frame deletion in ORF 6 (SEQ ID NO: 7) is constructed according to the methodologies of Example 3 utilizing OLIGOS 5'-GCAGCCAGGCTCGCACCGGC-3' and CGCCCGTAACGAGCGA (SEQ ID NO 28 and 29) for fragment A and OLIGOS 5'-GCAGCTTCTGCTGCTGA-3' and 5'-TCCCGGCCAGCCACTT-3' (SEQ ID NO 30 and 31) for fragment B to amplify ORF 6. The resulting construct is used to replace ORF 6 in. the S. albus strain obtained as described on Example 5 according to the methodologies of Example 3 and generating SA-D6 strain. Next, the prepeptide portion of mlb ORF6 (SEQ ID NO: 7), obtained by amplification with oligos 5'-CCGGAAAGGAGCGAGCATATG-3' (SEQ ID NO: 32) and 5'-CAGATCTGCCAATACAGT-3' (SEQ ID NO: 33), is digested with NdeI and BglII and ligated to pIJ18600 (Kieser et al., 2000), previously digested with NdeI and BglII generating plasmid pPRE1. In a parallel experiment aligos A 5'-C GGT GTC GAG GAG ATC ACC GCC GGG CCG GCG NNN NNN AGC NNN NNN NNN TGC ACC NNN NNN TGC NNN AGC NNN NNN NNN NNN AGC NNN TGC AGC NNN TGC TGC TGA AGA TCT-3' and oligo B 5'-T TCA GCA GCA NNN GCT GCA NNN GCT NNN NNN NNN NNN GCT NNN GCA NNN NNN GGT GCA NNN NNN NNN GCT NNN NNN CGC CGG CCC GGC GGT GAT CTC CTC GAC ACC GAT CGA-3'

[0150] (SEQ ID 34 and 35) are denatured and annealed to generate a mixture of DNA segments (SEQ ID NO: 36) which encodes polypeptides with all possible changes in the amino acid sequence of the propeptide region of the ORF6 polipeptide expect for those involved in thioether formation (specifically amino acid 3, 7, 8, 11, 13, 18, 20, 21, 23 and 24). This mixture of DNA segments is ligated to plasmid pPRE1, previously digested with BSA OI and BglII, to generate a library of plasmids carrying all possible variant forms (expect for the (methyl)lanthionine bridges) of the ORF 6 segment encoding the propeptide fused to the ORF 6 segment encoding the leader peptide. This plasmid library is introduced into SA-D6 strain and the resulting exconjugants, grown in the presence of μg/ml thiostrepton, are screened for the production of 107891 variants by biological assays or HPLC analysis. Interesting variants are further characterized by sequencing the propeptide portion of the ORF 6 variant and by structure elucidation.

[0151] Those skilled in the art understand that the leader peptides portion of the ORF 6 polipeptide can also be modified insofar as the enzymes involved in post-translational modifications (SEQ ID NO: 8 and 9) can recognize said different leader peptides. Variations in the leader peptide portion of the ORF 6 polipeptide fall within the scope of this invention.

[0152] It will occur to those skilled in the art that other method can be used for constructing a library of ORF 6 variants, including but not limited to the use of different oligos, vectors, induction system and host strains. Furthermore those skill in the art understand that methyllanthionine residues can be replaced by lanthionine residues, and viceversa, to generate additional ORF 6 variants. Those skill in the art understand also that site directed mutagenesis can be used to generate selected variants of ORF 6 resulting in the production of specified derivatives of 107891.

Sequence CWU 1

1

36120000DNAMicrobispora sp. PTA-5024 1actcgtcgtg ctggcgtgca cgctggtgct gctggccgtg gctcggatct ggacggagcg 60ttaccggtga acaccagcat cagcatcacc gacctgacga agcgttaccg gcgcggcggc 120gagaggccgg ccctgaacgg ggtgagcctg accgtggacg gcgggatgac cgcgctgctc 180ggggccaacg gcgccggcaa gacgaccctc atgcgcatct gcgtgggcgt cctgcggccg 240gacggaggcc gcgtggtggt gggcggccac gacctcggta cggcggccgg ccggagggcg 300gtcaagcgcg tcctgggcta cctgccgcag gagctgtcca tgtacgacga cctgaccggg 360cgggagttcc tcgactacat cgccctgctg aagggcgtcg acgacaagcg tgtccgccgc 420gaccagatcg agcagatgct cgagctgacc ggcctgtccg agcatgcggg ccggagactg 480ggcggttact ccggcgggat gaagcggcgg ctgggaatcg cccaggcgct actcgccgag 540ccggagctga tcgtcgtgga cgagcccacg gccgggctgg acccctccga acggatgcgg 600ttccgctcct tgctggccgg cctcggcggg gcgcggcgta ccgtggtgct gtccacccac 660atcctcgacg acgccgcgca gacctgcccc aacaccatcg tgctgcacca ggggcgggtc 720gcctaccagg gcagcaccgc cgggctcgcc gcggtcgccg agggccgcac ctacctgctg 780ccgcccggag cgcaggcgcc gcccgaggcc gtggtcgtga acgcggcggc ggaggtggag 840gggacgcgct atcgcgtgat cagcgcgcga ccgccgatcg gcggcaccct catgacgccg 900acgctcgaag acggatacgc ggcactgtta cagctcggcg aaccctcccc cacgggtccg 960cggccatgat cttcggaccg gacttccacc gggaccccta tccggtctac cggcggctga 1020gggacgaggc gccgtgccac cacgagccgg ccctcgggct ctacgccctg tccaggtacg 1080aggacgtgct ggccgcgctc aggcagccgg cggtgttctc ctcggccgcc cgcgctgtgg 1140cgagttcggc ggcgggcgcg gggccgtacc gcggagcgga taccgcgtcg ccggagcggg 1200agaccgccgc ggaggggccg gcgcggtcgc tgctcttcct ggacccgccg gagcaccagg 1260tgctgcggca ggcggtgtcg cgggggttca cgccccaggc ggtgctacgg cttgagccgg 1320ccgtacggga catcgccgcc gggctggccg accggatcgc cgaccgcggc ggcggcgagt 1380tcgtcaccga gttcgccgcc ccgctggcga tcgctgtgat cctgcgcctg ctcggcgtcc 1440ccgaagccga ccgggcacgc gtgagcgagt tgctgtccgc ctccgccccc tccggcgccg 1500aggccgaact gcgctcctac tggctcggcc tgagcgccct gctgcgtggc cgcgaggacg 1560cgggcaaggg cgacggcgag gaccggggcg tggtggcgga actcgtccgg cccgatgccg 1620gcctgaggga cgccgacgcg tcggcgggcc cggcctgccg cgcacccctg accgacgagc 1680aggtcgccgc cttctgcgcg ctcgtcggcc aggccggcac cgagtcggtg gcgatggcgt 1740tgtcgaacgc tctggtgctg ttcggccgcc accacgacca gtggcgcacg ctgtgcgcga 1800ggcccgacgc gatccccgcc gccttcgagg aggtcctgag atattgggcg ccgacccagc 1860accagggccg taccctcacc gccgatgtgc gcctgcacgg ccggctgctc ccagccggag 1920cacacgtgct gctgctgacc ggatcggcgg gcagggacga gcgcgcctac cccgacccgg 1980acgtcttcga catcggcagg ttccaccccg accggcgacc gagcacagcg ctcgggttcg 2040gtctcggcgc gcacttctgc ctgggggcgg cgctcgcgcg gctgcaggcc agggtcgccc 2100ttcgagaact gacgcgtcgc ttcccccgtt accggacgga cgaggagcgg accgtgcgat 2160cggaggtcat gaacgggttc ggccacagcc gggtcccgtt ctccatgtga accggcgggt 2220ggccggacgc tacgtacagg gcatgacgaa cacgaccaga gcccgcctgt ccggcgccgg 2280tctcctcgcc gcggccctgc tgctggccgg ttgcacgggc ggcggcagag ccgatccggc 2340gcacaggtcc cccgtgccgt tgccgagtcc cacgagcaac aagcaggaca tcagcgaggc 2400gaacctcgcc tatctgtggc cgctcacggt cgaccacggc acgatcgagt gcctgccctc 2460cgacaacgcc gtcttcgtgg cgcccgacgg cacgacctac gctctcaacg accgcgctga 2520gaaggcgggg caccccccga tcacaccgat tcgcgccaag ggcagcggtg gcggatacat 2580cagcctcggc gccctgctca gcaccaccct caatctctgc ggaaagggct gagaccagat 2640ccgggaccac aggagacggg ccgtccggtg aggcggcggt ccgaaaactt acccgagtgt 2700gggacggaaa atccggctcc tgcgtgaacc ttcgtgcgcc atctcgctac gtacacctcc 2760gaaagatcga aactgccgga ggtaacaggg acaggtgcac gggggagata cgcgatgccg 2820atggtgcgcg agtgcggtgc ggcacaaccg gccggaacgg aggcgatgtg cgcggcacgg 2880acagggcctg tgacgggggg acgaccagcg acaccgggag ggacatcggg cacaccgggg 2940gtgcccgacc ggcaaaggac gaggagagcg accgaggggt ccgccgacgc ccggggccgc 3000ggcgaggtcg tcgtcgcatg cgccagcctg cgcgacacga acgcgccgtt ggcggagtgg 3060ctgggagacc tggacgtcgg ctgcacgttc tgcggcgacg tctattcggc cgcggagacg 3120tatgacccgc ggctggcctt gctgcccgtc ctcaccgagg cccaggcgca gcgcctgggc 3180aggctgatgg agcggtgccc gggcacgtcg gtgctcggca tcgtcatgga cgtgaccggc 3240caccatactc atcgggccat ccagaacggg gcgagctggg tgctcaacac gctgctcccg 3300gccgcgtgct gccgcaacct gctgcgcatg gtcattcagg cggtggtgct cgggcccacg 3360gtccccgagc cgctggtcgc cgagccggcg gttccggagg gcgcggagcc gcccacgcgg 3420ccgggcgacc cccgtgcgga accgccggcg gaggcgcgga aggtcgccga cgcgcaggag 3480gaggaactgc tcacgctgct gtgcgggccg gagtccatcg ccgagatcgc cagacgcttc 3540tactgttccg aacggtcgat gtaccgtcag ctacgcgatc tgtaccgaag ttacggagtc 3600accggacgcc gggagctccg ccgggagatc gctcttcgga gcgtcacgcg ccaccaggag 3660acgctgtcgg cccaccttct cgccccgcct cggcccgtcc gccggggcgg tttgtcgtgc 3720tgagggctgg cacaccgccg ggctgtggca caccacgacg ccgggttgcg gctggcagct 3780ttggcagagc cggcagcgcg ccccggacgc cgggcgcggc gtcccgcgta gcgggccccc 3840cgaacccgcc ccccgctccc gtcagccgtc cgacccggac agccgcggcc ggcgggccag 3900ccagacggcc gcggccagca gtgccagagc gcacgagagc atccaggggt gcgtgccgga 3960caccacggcg ttcgttccgg tccgcgtacc catccgagca gccagaccac gactcccgcc 4020atcgttccga aggcgggtga ccgccagatc gcgaagacgg cgcccgcgcc cgccgccagg 4080agggcgcggc cgagccagcc ggcgacgacg gcgagcatcc ccgtagtcgc accggcggac 4140acgaggacgc ccgaggcgac gaccgagagc gcgaggtcga ttcccaggac gagcgcgagt 4200ctggtggcga acgccgtggc gggagagacc ggcatcgccg acagcagctc caggcgtgga 4260tcggatcggt gtgagcacgc cgtgaccgag ccgagcagca gcaccaccgt caccaccgag 4320ctgaacaact gcacgaccgc tcccggggtc ggcgccgtgc gcgcgagcaa caccgccgcg 4380accaggccca gagcggtcag cgggccgagc gaccgcggta tcagccgtgc ctgcgcccgc 4440acgagaccgg tggtcagccg ccacgacgcg cgaagcgtgg ggccggtcac gggcgccttc 4500cggtgtccgg cgatgtccgg caggcggccg aggagctcgt cgtacgccgg gaccgtgagc 4560ggcccgatca gttcctcgtc ggcttcgcgg accgcggccg tcagggcgtc ctgatcctcg 4620tcatctatca ccagtccgcc cccctttcgg atttgtcgtt tttgtcagtg cctccgtcag 4680caaccggcgg gccatgtgca tccggctctt gaccgtgccg gttggaattc caagtattac 4740ggccacctgt ggatagggga gatcctcggc gagcacgagc acgagcacct cgcgcaggtg 4800ctcgggcagc tccgccaccg cggccaccag gtcccgccgc tcggcgcggg cgagcacctg 4860ctcgtcgacc gccggctcca gatcgggcac gtccgcggcc ttctccagat ccacgaggac 4920cggttcggcc cgccgtagcc ggttgtgcgc ctgacgccgt gccaccccca gcagccaggc 4980tcgcaccggc gcctcgcccc ggaaactgcc ggccgagcgc cacaccgcca gccaggactc 5040ctgaaggatc tcctccgcca cctcgcgctg ggaggtgagc cggcggatga gccgcagcat 5100cccggcggcg tggcgttcgt acagcatacg gagcgccacc tcctcgccgt cggccacgcg 5160tctcatgagg acggcatcct cggacacctc cgcggagagc tccggttcac gcaactgctg 5220cacacgttcg tatgtagcgt cggctgccgt accggttcgg ggggatctgc ggcgtagcgg 5280cacgagtgcg ctccgtcctc gtttctgtca ttcctgccat tcattgagcc gttattgaca 5340ctagtagtcc gaaatgttcg actcaatgcg attccggaat cttgtccgaa cgaacaccgg 5400aaaggagcga gcaatgcccg ctgacatcct ggagacccgg acttccgaga ccgaggacct 5460gctcgacctc gacctctcga tcggtgtcga ggagatcacc gccgggccgg cggtgacgag 5520ctggtcgctg tgcacccccg ggtgcaccag ccccggcggc ggcagcaact gcagcttctg 5580ctgctgacat aaccgcagac gacaggggct gtagccagcc cgggccggag cgcgtcccgc 5640cccggtccgg gccaccggcc ggaacacgag ccggcgcggg cagggcagaa aggaccaggt 5700ggaaaatgac agattcgcca tttcgtgcgt gggatgtctt tatggtccgg gcaccggttg 5760gttatgcata tcctactcca ctgccgaact ccgaattcga ttctccggca tcctcacctg 5820gccttgacga agcggagttc ccgcctgacg cgcccgttct gtccgatgtc tccggacaca 5880gagccggctc gtccgaggcg tccgcacgca cgtccgggcc gccgccggcc gacgatcatc 5940tctcgctgct gcgggcggcg tgcgaagacg ggccgctgat ggaggccgtg gagctggcct 6000cacccagcct ggccggtctg ctcgccaggg tcgcgcgcgg cgacacgggc gggctcaagg 6060acaagcggct gcgccgggcc gccctcgcgc tcctgcgcta cgacatccgg atgcgaaccc 6120ggccgactcc gttcggcctg ttcgccggcg tcagcggcgg ccggttcgac acgtccgcga 6180agtggctggc cgggacgggt catcgcacca ggacgcgcgc cgacatggag tggctgctgt 6240cggcggtgca ccggctcgaa cgggatcgtg tgctgctcgc cggcgtcacg gtgcaggcgc 6300accagaccct gaccgtgcgc ggcgaccgga tcgtccttga ctgtccttcc gctctcggca 6360aacccctcaa cggatccacc cgttcgaccg tctccgcgcg gcgctcgccg gtggtcgccg 6420agatccttgg cgccgcccgg cgccccgtcc tcgcgggaag gctcgcgcag agcgtggcgc 6480agcggttcga gctgccggtc gaccgggtga cgggcctcct cgcggacatg gccgcccagg 6540aactgttgat caccgcgctg cggccgccct tggacggggg tgacccgctc cagcacgtgc 6600tcgacgtcgt ggcgacggcg gaggcgaggg cgggttcgcc cgccgaggcg atgagctccg 6660attcggccgc cctggtggcg gcgctgcgcg aggtggacgc gcgctgccac gcctacgatc 6720ggaccgccgt cgggcagggg cgccgggagc tggccgagct gatccaggcc acgcggcggg 6780tccacccgca cgacactccg ctgcacgtcg accttcggat cgacctggag gtgcgactcc 6840ccgagatcgt gcgcacggag atcgagcggg cggccgaggc gctgtggcgg ttgtccccgc 6900cgcggcgtgg catgcgggcc ctgcgccgtt accacgaggc gttcctggag aggtacggcg 6960cggaccgtgc ggtgccgctg ctcgaactcc tcgacgacac gcgcggactc ggcccgcccg 7020ccggctacaa gtggccgccg agcgagacac cggcgggccc gcaagaggag cctcgccgca 7080gtgcggcgct cgcccggctg gtcgccacgg cggcccggca cggggagcgt gagatcgtca 7140tcgacgagga gacgatcgcc gaactcgtct acgacgaggc ggctccggcc gatctgccga 7200actccttgga gctgggggtg cacgtggtgg ccccgtcgct ggacgagctg tccgccggaa 7260ccttccgagt cgtcctggcg cccgggccgg gctcgcacca cgcgggcgcg acgctcggca 7320ggttcaccgg tctgctgccc gatgtggacg ccgagtcggc ggccaggcag gccggccggc 7380ccctgcacat ccaggacgcg gtcgccgccg atgtcgcctt cattccgcgg tccggccggg 7440cggccaacct cgcgcacacc ccctcgtact ccggccggcg catcagcgtg gggctcccgg 7500acagcggacg cgcgcaggaa ctccccctcg acgagctggg ggtcgcggcc aacctggaac 7560gtctctgcct cgtccacctg ccgacaggcc gtgaggtggt ccccgcactg ccgaacatgg 7620tcagcgcctt cgcgcaggcc ccgaaccccg ctcgcctgtt gttcgagctg ggcctggagg 7680ggcagcggct ctgggaaccg tgggactggg gggcgctgag cgagatgcca ttcctgcctg 7740gtgtgcgtta cggccgtacg cttctggccg ctccgatctg gcggatggat cagctccgcg 7800ggccggccgc cgactccggt ccggcggcgg actgggacgc ggcgctcgac cggtggcggg 7860cggagtggaa cgtcccccgg cgggtgctcg ccgtcagcat ggaccagcgc ctgctcctcg 7920acctcggcga cgcctggcac cgcgtactgc tccgcgacga actgcgcagg acacccgagc 7980tgatcgcgca gcaggtggcc ggcgacgagg agggctggct ggaccgcggt gacgggggct 8040ttcccgggca cctcgccgag atcgtggttc cgctggaacg ccgtgaccgc cacgccgccc 8100ggcctccgca catcagggcc accgtcggcg gccgcgagcc gaccggagcg gggggaccgt 8160ggctctacct caggctgcgg gtcccgcgcc ggaaccagga cgacttcctg cgcgaccagg 8220tgcccgtcct ggtacgggcg gggatcgggc acggcgccga ccgctggttc ttcatccgct 8280acagcgacac cgccggacag cacctgcggg tcaggttccg cggcgagcgg gagaaactgt 8340gggccgggct gctccccgag atcggcgcgc ggctcgtcga gtggcagcgg caggggctgc 8400tggcgggcca cgagctcggc cagtacgacc ccgagtacga gcgatacggc ggcgacgcgc 8460tggccgagtt caccgaggcc gccttccagc acgacagcgc cgcggccatc tcgctcctgc 8520gcctcaccag gcgcgccggg ttccgctaca cgctcgacga ggtgacggcg atctcggccg 8580cggcgctggc gcacgccttc ggtcctcccg ccccggtcgt cgagccggtg ccgctggtgg 8640gcggcctcca gtgggctccg gatctgttcg acggcgaccc ggccgccgcg tggatgagca 8700ccaccggagc ccgcagggaa ctaccgcccg actaccggcg tgagccggcc cggtggcaga 8760agctgatcga ccccaccgga ggctggcggg tgctgcgggc ggatgaggac ggctgtcagg 8820tgctggcagc cctggaatcc agggatgagg ccgtccggcg gttcgggact gccttccgcg 8880aggcgtccag acccacggac tccccgtcga cgcagctcag actggtcggc agcctgcttc 8940acatgacctg caaccggctg atcggcggat ccgcggaacg agagcgaagc gtgctcgggc 9000tcgcccgggg cgccgtccag gacaacctga accgcaggag gcaccgggca tgaccacagt 9060cggtccgaca tcgtgcgggg tgacccctga ccggcacccg gcccggttcc tgcgcggcag 9120cgcggcccgc cgggccgcgc gcctggtgcg gctggtcgcc gaacggctcg ccgacccgga 9180cgaggtcgcc gggatcgcgg cccgccccgg caactccgtg ccggcgaacg ggctgtcgat 9240gtggagcccg gccacgctgt cgcacggatt tccgggcatc gcggtcttct acgcggagct 9300ggggcgggtc gaccccgcct ggtcggcgtt ggcacatcgc cacctcaggg cgggggccgc 9360ggctgtggag acggcgcctt cgggcggact gttcgccggt ccggcctctc tcctggccgc 9420cgcgcagagc tgcgcaggcc cggcggggca ctaccggggc ctgcgacgca cgctgaccgc 9480atggctcgcc gctgatcacg ccggacggct cgccgccgcg cgtgaccggc ccggccccgg 9540tgtcgcctgg accgactacg acgtcgttca cggactgtcc gggtccaccc gtctgctgct 9600ggacgccgcg cgcgatcccg acgacgagac ggcggcaaag gcctccggcg cggtgaccga 9660caccctgcgg cacctggtgc ggctcaccga gccgatcaca gtggacggac acgaggttcc 9720cggctggtgg gtgccgtccc atctgcagcc ggtcgaacag gaccggcgcg actacccccg 9780gggagacctg aacctgggat tggcccatgg agcggcgggg ccgctgtcgg tgctggccac 9840ggccaccctt cacggggtgg aggtcccggg ccagcgtgag gccgtcgcgc ggctggccga 9900atggctgctg ggctggacga tgaccgacga cacgggcgcc tactggccct gccgggtcag 9960ctgggacgag cagatcgccg ccgtccgccc ggacacctcc ttcacccgta cggcctggtg 10020ctacggggcc cccggagtct gcgcggcact ccaccgggcc ggactggcgc tcggcgtgac 10080ggagtggcgc gaagtcgcgg tcaccgcgct gctggacggg ctgcgccgcg accggtccgc 10140ctggcgggtg gacggctcga ccgtctgtca cgggtacgcg ggcctgctgc aggtgctgtc 10200gcgggtcggc gccgagtccg gcgacccgcg gctgctggac ggctgcctgg acgtcgcgcg 10260catggtcctc ggcgaggccg acgaatcggc tccgttcgtc ttcccccatc tcgtccccga 10320ttcgcccgac gggtggcgca acgccacggg atacctgccg ctggacggcg ccggcttgct 10380ggaaggggcg gccggagtcg cctgtgcgct gctctcggtc atcccgccgt cgtcgctcgg 10440tggaacggac ccggccccgg agcgtgcgga cctcccgccc tgggacaggt gcctggccct 10500gtgctgacgc ccccgaccca tcaggagagc cagaccatga cagcgcacag cgacgcgggc 10560ggcgttccgc ggcccccgga gcggctgctg ctcggcgtga gcggctcggt cgccgcgctg 10620aacctgcccg cctacgtcta cgcgttccgg gccgcgggcg tcgcccggct ggccgtcgtc 10680ctgaccccgg cggccgaggg tttcctgccc gcgggcgcgc tgcgcccgat cgtcgacgcc 10740gtccacacgg agcacgacca gggaaagggg cacgtggcgc tgtcccgatg ggcgcagcac 10800ctgctcgtcc tgcccgcgac ggcgaacctg ctcggttgcg cggcgtcggg gctggcgccg 10860aacttcctgg cgacagtgct gctggcggcc gactgcccga tcacgttcgt gcccgcgatg 10920aacccggtga tgtggcgcaa gcccgccgta cggcggaacg tcgccaccct ccgcgcggac 10980ggtcaccgtg tggtggaccc gctgcctggc gcggtgtacg aggcggcctc gcggtcgatc 11040gtcgacggac tgaccatgcc tcggccggag gcgctggtcc ggttgctggg cggcggggac 11100gacggcagcc cgtccggtca ggacggtccg gtcggcaggg cggagcacgc ggagcacgcg 11160gaggccgcgg aggccttggc atgacggtcc cggcgttcga gctcagcgac ctgacggtcc 11220gctacggccc ggtcacggcg gtcgacggcg tctcggccgg ttcggcgccc ggcctcgtca 11280ccgccctgct cggacccaac ggtgcgggca agtccagcct gctccgggtg ctgtcgacgg 11340tcgcgccgcc ctcatcgggc acggcgaggg tgttcggcca cgacacgcgc gcggagccgc 11400tggccgcacg taggcggatc gggctggtct tccaggaacg cgcgctcgac accgacctgt 11460ccgcggagca gaacctgcgc tttcacgcgc ggctgttcgg ggtggggcgg gcccgggccg 11520cggaggacat cctcgtgctg ctggaacgct tcgggctggc cggccgcggc cgcgaccggg 11580tcgagaccct ctccggcggg ctggcccgcc ggctggagat cgcgcgggcg ctgttgcacc 11640ggcccggcct gctgatcctg gacgagccga ccaacggcct cgacccggag gcgcgccaga 11700ccgtatggga cgacctcatc cggctgcgct ccgaactcgg cgtcacggtg ctgtactcca 11760cgcactacat ggacgaggcc gagctggccg accagatcat catcctcagc gagggccgcg 11820tcgcggggtt cggctcgccc ggccggctga agagcgagct gcgatcgtcg cgcatcgtgc 11880tcgtcaccca cgacgacgac acggtgctcg cccggctcgc ggaggccggc ttcgacgctg 11940tgatcgactc ggacggcgtc gccgtgcgct gccgcgaacc ggagagccgg atggcggagg 12000tcatacgggc ggcggggccg ctcgtccgcg cggcgtccgt gcaccacccc tcgatgaacg 12060acgtgttcct ggcccacacg gccgcgaacc gcgacaggga ggcggccgat ggcactgtca 12120gctgtccgtg acaccggtgg cacggcgggg agtactcccg gcggccccgc cggaggcggg 12180tcggccggcg cgggcgaggc gaggaggctc gtctctgcgg ccctgcggtc cggggtccgc 12240ggaacgctcg tcgtggcgca ccgcgacgtg ctgcggcagg tgcggcaccc gggggtcgtg 12300gtcgcccagg ccgcgcagat cgtctttttc gtcctcgtct acgccgtggg cttccggtcg 12360atgatcggat ccgtcggcgg cgtgtcgttc ggcgcctacg tctatccggg gatcatcgcc 12420atccaggtcg tcatgctggg cgtcggcacg ggcctgacgt acgcgatgga ccgcgagttc 12480ggcgtgctgc gcgagatgca ggtcgcgccc gtgccacgca tgtgcctgcc cctcggaaag 12540atccttgcga gctgcgtgct gctcaccgcg caggccatgc tcatgctcct gcccgccccg 12600ctgctcgggc tgcccctgac gcccgcgcga tacgcggcgg gcgccgcggt ctacctggcc 12660acggcggcgg ccttcagctt gatcggcctg ctgctcgcgg tgagcgtccg ccggatcgag 12720acgctgcagg cgaccgtcca gctcgcgatg tacccgctgc tgttcctgtc gggttcggtg 12780ttcaaacccg acgccgtgcc cggctggctg gcggccctca tgcggctcaa cccgatgacg 12840tacgcggtcg atctggcgcg gcacgtgctg ctgccgtcgg ccccgggcgt gtcgtacctg 12900cccgtctggc gggacctcct ggtgatcgcc gcgctggtgg cggccgcgtc ggcggcgctg 12960cggctgcggg tggggaggtg accggccggt gaccaccgcg acgacgacag ggccgaccgg 13020ccgcgagacg ctgcttgaag gcgcggtgtc ctggctcgcg gcacgactgc gctggttcga 13080ccccgagcag tgggcgaggc atctgcagcc gagaggcttc gcgccgagcg cgctgctgga 13140actccttatc atctgccgca acctgaactc ggtctacggc cctccgcgcc cgccggaccc 13200ggaggacggc tcccgcgggt ccggcgtacg ggcgacgggt gcggccggcc cggccgtcga 13260actgagcggg cgtgcgctgg acctggccga ggaggtcgtg ggacggcccg atttcggcgc 13320ggccctctac cgcggcgacg ccgccttccc caccacgtgt ggctggtcgc gcttctcgcg 13380gagggcggcc ggtccgtcga gccgtggcct gccgtcgccc agcggatcat cgacgcgggc 13440tgcgccgagc cgatccggcc gggacggccg acggcggccc ggctggaggc ccgctacgtc 13500ttcgacctcg ccggcctccg ccacgggctg cccgccatgg gcgaactggc cggtcgtacg 13560gctctgggcc ccggtgcgga cccgctgcac ctcaccgacg cggacctcta tgtgatcacg 13620cacatgctct tctacctcac cgacttcggg cggcgcccgt tctccgcgga cgaggccgaa 13680agccggcggg tccgcgggct cgtcgaggtg ctcctcggcc gccagctcgc ggtcggcgac 13740ctcgatctgg cggccgagct gctcgcctgc gcgggcctca ccggcgccga cgaccggctg 13800tccggctgcg cgtggaaccg gttgtcggcc gcgcgccgtc cggacggctc ggtgcccagc 13860ccgctgttcc ggcaagccgc gctggaccgg ctgagcgggg agaaggccga ggcgtacgcg 13920ttcggaacct gctaccacac caccctcgcc atggtgctcg cggcgacgct gacggacggc 13980gccgatggct gacccgttgc tgaccggggc cctcgcacgc gtcgtgccgg cgctggagaa 14040gctggacggc gagcgcgccg cggcggtgct cctgctgtgc cgggcggctc tcgacggccc 14100atcgcggccc aggccgggcg gccgccggcc tgaagcgccg ccggttcccg gatcggtgga 14160cccggctttc cgacctgctc cacgacgctc ccggaaccgc gcagggcctc gccgggcaga 14220tcgccgagac gaccgctttc ggggccgtcc ggcccgccgc cgccgtccgg gaggtctggg 14280agctcgccgt gcccgcgctg ctggccgccg ccgcgcgcag ggccgacctg atgaccctgg 14340ctccgctcgt ccgggccggc gtcttcctcc aggcggtctc gtccgccggc gacgtgcccg 14400agcgggacga ccggcccgct cctgcgggcc tgaccacgcg cgccgcgtgg cgcctggcgg 14460cgtggcagcg tccggacggc gggttcgggg cgccggccgc gaccgccgac tgtggctggg 14520cgctcgcgga ggccgcggtt cccgggctga ccgcctcgca gcgcgagttc gccgtgcccc 14580tcccggacgg gaagcctccg gcacggccgc gaaccgccca ctccccaccc gctacgtacg 14640gcgggaagga gtgatgcaga tgaacgacca tgacgcggcc ggcgtcccgt ccggcagccc 14700ggcccaccac cgaccggcgg accccgccgc caccctggcc gcggagacca cagggggccc 14760aggcgggacc gccggcccga ccgactcccc gtcgatggcg gccctgatat ccacggagtt 14820gctcagactg cgttccggct tcgtcggctg gtacatcctg ctgtcgccga tcgtgatcgc 14880cattccgctc tacctggggt cgatcttctc cccggagggc cggtccggcc gcctctggga 14940gaccttcagc aacgtgacgc tggagttctg gggcgtactg attccgatga cggccggtct 15000gatcgcggcc ctcgcggtcc gtgccgacac

cgaaccctgg cgtttcctgt tctcgtacgc 15060gatcccacgc tggcgctact tcaccgcgaa ggtcgccgcg ctggccgtcg cccagttgct 15120gtcggcgacc atcctggtcg tgatgctggc cgggggagcg ctgctgaccg ggcagctctc 15180caacgccgcc tcgatgatcc tcaaagtcgc ctacctgccg tgggcggcgg ggttggcggc 15240gaccgcgctc gccgtcctcg tctgcaccgt ctggggactg ggccccggca tcgcccttgg 15300tgtggccggg atgatggcag gcgcgctcat ctccgacaag tcgttctggt acgccatccc 15360gcccgcctgg ccgatgcggg tcatcctccc gctggcggac atccgcccca acggcctggc 15420gctcgacgcg agcagcccgc tccacgacac ctcggtcatc ccgctggccg tcgcgctctc 15480ggccgcggcg acgatcgtga tcctgctgat cggcggccgg cacatggcgc ggaaggaggt 15540ctgacatggc cgctctcgag atcagggacc tgcacaagca ctacgacgac ttccacgcgc 15600tcgacggcgc gaacctgacc gtgccggacg gctccctcta cggcctgctc ggcccgaacg 15660gcgcgggcaa gaccaccctg atgaaggcgg tcaccgggct gcggcatccg acctcgggcc 15720acatcagcct tttcgggcgg ccctacgaac ggcggctgct gacccaggtc ggcgcgctgc 15780tcgagtcgcc gggcctgtgg acccagctcg acgcggtgtc ccacctgcgc atccacgccc 15840ggctgcgcgg cgtgcccgag acacggatcg gcgaggtgct gagcctgatg aacctcaccg 15900aggtcagcac ccgcaaggtg gcgaagtact ccctcggcat gcggtggcgt ctcggcatcg 15960ccatcgccct gctcggccgg ccccggctgg tcgtgctcga cgagccgatg aacggactcg 16020acccggtcgg catcagggac atgcgggcga cgctgcgcgc gctgaccgcc gccgggacca 16080ccgtcatggt ctccagtcac cagctggcgg agatcgcgca catctgcgat cacgtcggcg 16140tgctggtggc cgggcggacc gcgtacgagg gcccgctgcc cggcctcgcc gtggacggcg 16200acctcgaaca gggcttcttc cgcctgctcg agaaggcggg ttccgcagtg agatgacctg 16260acccacgaac ggaagggcga tcgggaacat ggcgaggtcc gaagagagca acaccctggc 16320caggctgttc gacgtgctcg gcgacgacgc cgcggcggcg agggaatggg tgaccgagcc 16380gcaccggctc atcgcctcga acgagaggct cggcaccgcc ccggaagcgc cggccgacga 16440cgaccccggg gcgatccgca cggtgggagt gatcggcggc ggcaccgccg gttacctcac 16500cgcgctggca ctcaaggcca agcgcccctg gttggatgtg gctctcgtcg agtccgccga 16560catccccatc atcggcgtcg gcgaggcgac ggtctcgtac atggtgatgt tcctgcatca 16620ctacctcggc atcgacccgg cggagttcta ccagcacgta cgccccacgt ggaagctcgg 16680catcaggttc gagtggggca gccggccgga gggattcgtc gcgccgttcg actgggggac 16740cggctctgtg ggactggtcg gctcgctgcg cgagacgggg aacgtcaacg aggccaccct 16800gcaggccatg ctgatgaccg aggaccgcgt ccccgtctac cggggggagg gcgggcacgt 16860ctccttgatg aagtacctgc ccttcgccta ccacatggac aacgcgcgcc tcgtccgcta 16920tctcaccgag ctggcggctc ggcgcggcgt gcgccatgtg gacgcgacgg tcgccgaggt 16980acggctcgac ggcccggacc acgtcggcgg cctgatcacc accgacggcc ggcggctgca 17040ctacgacttc tacgtcgact gcacgggctt ccgctccctg ctgctggaga aggcgctggg 17100catcccgttc gagagctacg cgagcagcct gttcaccgac gccgccgtca ccggcactct 17160cgcgcacggc ggccacctca agccgtacac gacggcgacc acgatgaacg cggggtggtg 17220ctggacgatc ccgacgcccg agagcgacca tctcggctac gtgttctcgt cggcggcgat 17280cgatcccgac gacgcggcgg cggagatggc gcgccgattc ccgggggtca ccagggaggc 17340gctcgtccgg ttccggtccg gacggcaccg ggaggcctgg cgcggcaacg tcatggcggt 17400cggcaactcc tacgcgttcg tggagccgct ggagtccagc ggtctgctga tgatcgccac 17460ggcggtgcag atcctggtga gtctgcttcc ctcctcccgc cgggacccgc tgcccagcga 17520cgccgccaac caggccctgg cacaccgctg ggacgcgatc cgctggttcc tctcgatcca 17580ctaccggttc aacggacggt tggacacccc gttctggaag gaggccaggg ccgagaccga 17640catctccggg atcgagccgc tcctgcggct gttcgcggcc ggagcgccgc tgaccggccg 17700cgactccttc acccgatacc tcgcggacgg ggcggcgccg ctgttctacg gtcttgaggg 17760cgtcgacacg ctgctgctcg ggcaggaggt gccggcccgg ctgctgccac cccgcgagcc 17820cccggagcag tggagggcac gggcggcggc ggccagaagc ctggcctcgc gggggctgcg 17880gcagagcgag gcgctggacg cctacgccgc cgacccgtgc ctcaacgccg aactgctcag 17940cgactccgac agctgggcgg gcgagcgggt cgccgtccgg gccggcctga gatgacgacc 18000ggcgccacgg tcgcccatgt cgtcgaaccg gacgggttcc gggcggtcat ggccaccctt 18060ccggcagccg tggcgatcgt caccgccgcg gccgcggacg ggaggccctg ggggatgacc 18120tgcagttcgg tctgcagtgt gaccctgacg ccgccgaccc tgctggtctg cctgcggacg 18180gcgagcccca ctctcgcggc cgtcgtctcc ggccgggcgt tctcggtgaa cctgctgtgc 18240gcccggtcgt accccgtcgc ggagctgttc gcctcggcgg cggccgaccg cttcgaccgg 18300gtccgctggc ggcggcccac cggtacgggc ggcccccacc tggccgacga cgcccgtgcc 18360gtcctcgact gccggctctc ggagtccgcc gaggtgggag accacatggt ggtcttcggc 18420gaagtgaggg cgatccgcag gctgtccgac gagccgccgc tgatgtacgg ctaccgccgc 18480tacgccccct ggcccgccga ccggggaccg ggagcggtgg gggggtgaac gccgagcagc 18540tcaccggtgt ggtcatcgcc gatctcgggg tgatcgtcgt cgtgtcggcg ctcttcgggg 18600cactggcacg gcgatgcggc cacccgaccg tcatcggcca gatcgtcgcc gggatcgcgc 18660tgggaccgac cctgctgggc cggctgcccg gcgacccggc cgggtggctg ttccccgccc 18720aggtccggcc gtcgctgtcg gtcctgtccc agatcgccgt cgtgatcttc atgttcgcgg 18780tgggttacga ggtcgacctg cggcttctgc gccggggtgg ccgcagcgcg ctctgcgtgg 18840cgtcgctgtc gctggcggtg cccatgacgc tcggcgcggc ggtcgccgtg ctgttccgcg 18900aggttttcac ggtcggctcc cctggggggc cgggaggccc gacgttcgtg ctgttcatgg 18960ccgtggcgat ctcgatcacc gccctgccgg tgctggcggc gatcgtacgg gagcggggcc 19020tcgcgggaac cgcggcggga accgtggcca cggcggccgc cgggctgatg gacgtggccg 19080catggaccac actggccgcg gttctggccg agaccggcga tgccgatgag ccgacggtgt 19140cacacgtgcc ctggatgctg gctcttccgg ccctcacggc gttcgcggtg gccatgttcc 19200tggtcgtgcg tccccttctc gggtggttga ccaggaggcc cggagccatg tgggggcggc 19260tgccggcggc gttcgcactg gcgctcggca gcgcctgggg caccgccgca cttggcctgc 19320acccggtgtt cggcggtctg ctggccgggc tcgtcatgcc gcgccgcgac ggcgcccccg 19380agccggaggt gctgcggccg atggagcaga ccgccgagtt gctcctgccg ctgttcttcg 19440tgatgaccgg gctgtcggcc gacatatcgg cgatcgaacc gggtgggctg atcctgctgg 19500cggtgctcct ggtcgccgcc atcgggggca agctcgtgcc cgcctacgcg gcctcccggc 19560tgaccggtct cgactccggt gagtcggccg tggtcgccgt gctggtgaac acccggggcc 19620tcaccgagct gatcgtgctc gatgtggggc tgtcggcgca cgtcatcgat gagcggctgt 19680tcaccgtcct ggtcgtcatg gccctgatca ccacggccat gaccgcgcca ctgctgaccg 19740cgctgagacg gcgcgaagag cggagacgcg gtcgtcaggc ggccccgctg tcgagggcga 19800cggcctggcg gatgtagtcg cgcagggcgt cccggtcgag ggcgcccagg ttcttgatct 19860tgacgtgcct cgtcgtcttg cccacgccct ccagcaggcc gtgcccgtcg tcgaactccg 19920ccccccgggc gaaggcgaag gtgacgtgcg ttttgctgtg gctgatgatg gcgaggatct 19980tgtcgccctt ccaggcgggc 200002300PRTMicrobispora sp. PTA-5024 2Val Asn Thr Ser Ile Ser Ile Thr Asp Leu Thr Lys Arg Tyr Arg Arg 1 5 10 15 Gly Gly Glu Arg Pro Ala Leu Asn Gly Val Ser Leu Thr Val Asp Gly 20 25 30 Gly Met Thr Ala Leu Leu Gly Ala Asn Gly Ala Gly Lys Thr Thr Leu 35 40 45 Met Arg Ile Cys Val Gly Val Leu Arg Pro Asp Gly Gly Arg Val Val 50 55 60 Val Gly Gly His Asp Leu Gly Thr Ala Ala Gly Arg Arg Ala Val Lys 65 70 75 80 Arg Val Leu Gly Tyr Leu Pro Gln Glu Leu Ser Met Tyr Asp Asp Leu 85 90 95 Thr Gly Arg Glu Phe Leu Asp Tyr Ile Ala Leu Leu Lys Gly Val Asp 100 105 110 Asp Lys Arg Val Arg Arg Asp Gln Ile Glu Gln Met Leu Glu Leu Thr 115 120 125 Gly Leu Ser Glu His Ala Gly Arg Arg Leu Gly Gly Tyr Ser Gly Gly 130 135 140 Met Lys Arg Arg Leu Gly Ile Ala Gln Ala Leu Leu Ala Glu Pro Glu 145 150 155 160 Leu Ile Val Val Asp Glu Pro Thr Ala Gly Leu Asp Pro Ser Glu Arg 165 170 175 Met Arg Phe Arg Ser Leu Leu Ala Gly Leu Gly Gly Ala Arg Arg Thr 180 185 190 Val Val Leu Ser Thr His Ile Leu Asp Asp Ala Ala Gln Thr Cys Pro 195 200 205 Asn Thr Ile Val Leu His Gln Gly Arg Val Ala Tyr Gln Gly Ser Thr 210 215 220 Ala Gly Leu Ala Ala Val Ala Glu Gly Arg Thr Tyr Leu Leu Pro Pro 225 230 235 240 Gly Ala Gln Ala Pro Pro Glu Ala Val Val Val Asn Ala Ala Ala Glu 245 250 255 Val Glu Gly Thr Arg Tyr Arg Val Ile Ser Ala Arg Pro Pro Ile Gly 260 265 270 Gly Thr Leu Met Thr Pro Thr Leu Glu Asp Gly Tyr Ala Ala Leu Leu 275 280 285 Gln Leu Gly Glu Pro Ser Pro Thr Gly Pro Arg Pro 290 295 300 3414PRTMicrobispora sp. PTA-5024 3Met Ile Phe Gly Pro Asp Phe His Arg Asp Pro Tyr Pro Val Tyr Arg 1 5 10 15 Arg Leu Arg Asp Glu Ala Pro Cys His His Glu Pro Ala Leu Gly Leu 20 25 30 Tyr Ala Leu Ser Arg Tyr Glu Asp Val Leu Ala Ala Leu Arg Gln Pro 35 40 45 Ala Val Phe Ser Ser Ala Ala Arg Ala Val Ala Ser Ser Ala Ala Gly 50 55 60 Ala Gly Pro Tyr Arg Gly Ala Asp Thr Ala Ser Pro Glu Arg Glu Thr 65 70 75 80 Ala Ala Glu Gly Pro Ala Arg Ser Leu Leu Phe Leu Asp Pro Pro Glu 85 90 95 His Gln Val Leu Arg Gln Ala Val Ser Arg Gly Phe Thr Pro Gln Ala 100 105 110 Val Leu Arg Leu Glu Pro Ala Val Arg Asp Ile Ala Ala Gly Leu Ala 115 120 125 Asp Arg Ile Ala Asp Arg Gly Gly Gly Glu Phe Val Thr Glu Phe Ala 130 135 140 Ala Pro Leu Ala Ile Ala Val Ile Leu Arg Leu Leu Gly Val Pro Glu 145 150 155 160 Ala Asp Arg Ala Arg Val Ser Glu Leu Leu Ser Ala Ser Ala Pro Ser 165 170 175 Gly Ala Glu Ala Glu Leu Arg Ser Tyr Trp Leu Gly Leu Ser Ala Leu 180 185 190 Leu Arg Gly Arg Glu Asp Ala Gly Lys Gly Asp Gly Glu Asp Arg Gly 195 200 205 Val Val Ala Glu Leu Val Arg Pro Asp Ala Gly Leu Arg Asp Ala Asp 210 215 220 Ala Ser Ala Gly Pro Ala Cys Arg Ala Pro Leu Thr Asp Glu Gln Val 225 230 235 240 Ala Ala Phe Cys Ala Leu Val Gly Gln Ala Gly Thr Glu Ser Val Ala 245 250 255 Met Ala Leu Ser Asn Ala Leu Val Leu Phe Gly Arg His His Asp Gln 260 265 270 Trp Arg Thr Leu Cys Ala Arg Pro Asp Ala Ile Pro Ala Ala Phe Glu 275 280 285 Glu Val Leu Arg Tyr Trp Ala Pro Thr Gln His Gln Gly Arg Thr Leu 290 295 300 Thr Ala Asp Val Arg Leu His Gly Arg Leu Leu Pro Ala Gly Ala His 305 310 315 320 Val Leu Leu Leu Thr Gly Ser Ala Gly Arg Asp Glu Arg Ala Tyr Pro 325 330 335 Asp Pro Asp Val Phe Asp Ile Gly Arg Phe His Pro Asp Arg Arg Pro 340 345 350 Ser Thr Ala Leu Gly Phe Gly Leu Gly Ala His Phe Cys Leu Gly Ala 355 360 365 Ala Leu Ala Arg Leu Gln Ala Arg Val Ala Leu Arg Glu Leu Thr Arg 370 375 380 Arg Phe Pro Arg Tyr Arg Thr Asp Glu Glu Arg Thr Val Arg Ser Glu 385 390 395 400 Val Met Asn Gly Phe Gly His Ser Arg Val Pro Phe Ser Met 405 410 4260PRTMicrobispora sp. PTA-5024 4Val Pro Asp Arg Gln Arg Thr Arg Arg Ala Thr Glu Gly Ser Ala Asp 1 5 10 15 Ala Arg Gly Arg Gly Glu Val Val Val Ala Cys Ala Ser Leu Arg Asp 20 25 30 Thr Asn Ala Pro Leu Ala Glu Trp Leu Gly Asp Leu Asp Val Gly Cys 35 40 45 Thr Phe Cys Gly Asp Val Tyr Ser Ala Ala Glu Thr Tyr Asp Pro Arg 50 55 60 Leu Ala Leu Leu Pro Val Leu Thr Glu Ala Gln Ala Gln Arg Leu Gly 65 70 75 80 Arg Leu Met Glu Arg Cys Pro Gly Thr Ser Val Leu Gly Ile Val Met 85 90 95 Asp Val Thr Gly His His Thr His Arg Ala Ile Gln Asn Gly Ala Ser 100 105 110 Trp Val Leu Asn Thr Leu Leu Pro Ala Ala Cys Cys Arg Asn Leu Leu 115 120 125 Arg Met Val Ile Gln Ala Val Val Leu Gly Pro Thr Val Pro Glu Pro 130 135 140 Leu Val Ala Glu Pro Ala Val Pro Glu Gly Ala Glu Pro Pro Thr Arg 145 150 155 160 Pro Gly Asp Pro Arg Ala Glu Pro Pro Ala Glu Ala Arg Lys Val Ala 165 170 175 Asp Ala Gln Glu Glu Glu Leu Leu Thr Leu Leu Cys Gly Pro Glu Ser 180 185 190 Ile Ala Glu Ile Ala Arg Arg Phe Tyr Cys Ser Glu Arg Ser Met Tyr 195 200 205 Arg Gln Leu Arg Asp Leu Tyr Arg Ser Tyr Gly Val Thr Gly Arg Arg 210 215 220 Glu Leu Arg Arg Glu Ile Ala Leu Arg Ser Val Thr Arg His Gln Glu 225 230 235 240 Thr Leu Ser Ala His Leu Leu Ala Pro Pro Arg Pro Val Arg Arg Gly 245 250 255 Gly Leu Ser Cys 260 5221PRTMicrobispora sp. PTA-5024 5Val Arg Ala Gly His His Gly Val Arg Ser Gly Pro Arg Thr His Pro 1 5 10 15 Ser Ser Gln Thr Thr Thr Pro Ala Ile Val Pro Lys Ala Gly Asp Arg 20 25 30 Gln Ile Ala Lys Thr Ala Pro Ala Pro Ala Ala Arg Arg Ala Arg Pro 35 40 45 Ser Gln Pro Ala Thr Thr Ala Ser Ile Pro Val Val Ala Pro Ala Asp 50 55 60 Thr Arg Thr Pro Glu Ala Thr Thr Glu Ser Ala Arg Ser Ile Pro Arg 65 70 75 80 Thr Ser Ala Ser Leu Val Ala Asn Ala Val Ala Gly Glu Thr Gly Ile 85 90 95 Ala Asp Ser Ser Ser Arg Arg Gly Ser Asp Arg Cys Glu His Ala Val 100 105 110 Thr Glu Pro Ser Ser Ser Thr Thr Val Thr Thr Glu Leu Asn Asn Cys 115 120 125 Thr Thr Ala Pro Gly Val Gly Ala Val Arg Ala Ser Asn Thr Ala Ala 130 135 140 Thr Arg Pro Arg Ala Val Ser Gly Pro Ser Asp Arg Gly Ile Ser Arg 145 150 155 160 Ala Cys Ala Arg Thr Arg Pro Val Val Ser Arg His Asp Ala Arg Ser 165 170 175 Val Gly Pro Val Thr Gly Ala Phe Arg Cys Pro Ala Met Ser Gly Arg 180 185 190 Arg Pro Arg Ser Ser Ser Tyr Ala Gly Thr Val Ser Gly Pro Ile Ser 195 200 205 Ser Ser Ser Ala Ser Arg Thr Ala Ala Val Arg Ala Ser 210 215 220 6220PRTMicrobispora sp. PTA-5024 6Val Pro Leu Arg Arg Arg Ser Pro Arg Thr Gly Thr Ala Ala Asp Ala 1 5 10 15 Thr Tyr Glu Arg Val Gln Gln Leu Arg Glu Pro Glu Leu Ser Ala Glu 20 25 30 Val Ser Glu Asp Ala Val Leu Met Arg Arg Val Ala Asp Gly Glu Glu 35 40 45 Val Ala Leu Arg Met Leu Tyr Glu Arg His Ala Ala Gly Met Leu Arg 50 55 60 Leu Ile Arg Arg Leu Thr Ser Gln Arg Glu Val Ala Glu Glu Ile Leu 65 70 75 80 Gln Glu Ser Trp Leu Ala Val Trp Arg Ser Ala Gly Ser Phe Arg Gly 85 90 95 Glu Ala Pro Val Arg Ala Trp Leu Leu Gly Val Ala Arg Arg Gln Ala 100 105 110 His Asn Arg Leu Arg Arg Ala Glu Pro Val Leu Val Asp Leu Glu Lys 115 120 125 Ala Ala Asp Val Pro Asp Leu Glu Pro Ala Val Asp Glu Gln Val Leu 130 135 140 Ala Arg Ala Glu Arg Arg Asp Leu Val Ala Ala Val Ala Glu Leu Pro 145 150 155 160 Glu His Leu Arg Glu Val Leu Val Leu Val Leu Ala Glu Asp Leu Pro 165 170 175 Tyr Pro Gln Val Ala Val Ile Leu Gly Ile Pro Thr Gly Thr Val Lys 180 185 190 Ser Arg Met His Met Ala Arg Arg Leu Leu Thr Glu Ala Leu Thr Lys 195 200 205 Thr Thr Asn Pro Lys Gly Gly Arg Thr Gly Asp Arg 210 215 220 757PRTMicrobispora sp. PTA-5024 7Met Pro Ala Asp Ile Leu Glu Thr Arg Thr Ser Glu Thr Glu Asp Leu 1 5 10 15 Leu Asp Leu Asp Leu Ser Ile Gly Val Glu Glu Ile Thr Ala Gly Pro 20 25 30 Ala Val Thr Ser Trp Ser Leu Cys Thr Pro Gly Cys Thr Ser Pro Gly 35 40 45 Gly Gly Ser Asn Cys Ser Phe Cys Cys 50 55 81115PRTMicrobispora sp. PTA-5024 8Met Thr Asp Ser Pro Phe Arg Ala Trp Asp Val Phe Met Val Arg Ala 1 5 10 15 Pro Val Gly Tyr Ala Tyr Pro Thr Pro Leu Pro Asn Ser Glu Phe Asp 20 25 30 Ser Pro Ala Ser Ser Pro Gly

Leu Asp Glu Ala Glu Phe Pro Pro Asp 35 40 45 Ala Pro Val Leu Ser Asp Val Ser Gly His Arg Ala Gly Ser Ser Glu 50 55 60 Ala Ser Ala Arg Thr Ser Gly Pro Pro Pro Ala Asp Asp His Leu Ser 65 70 75 80 Leu Leu Arg Ala Ala Cys Glu Asp Gly Pro Leu Met Glu Ala Val Glu 85 90 95 Leu Ala Ser Pro Ser Leu Ala Gly Leu Leu Ala Arg Val Ala Arg Gly 100 105 110 Asp Thr Gly Gly Leu Lys Asp Lys Arg Leu Arg Arg Ala Ala Leu Ala 115 120 125 Leu Leu Arg Tyr Asp Ile Arg Met Arg Thr Arg Pro Thr Pro Phe Gly 130 135 140 Leu Phe Ala Gly Val Ser Gly Gly Arg Phe Asp Thr Ser Ala Lys Trp 145 150 155 160 Leu Ala Gly Thr Gly His Arg Thr Arg Thr Arg Ala Asp Met Glu Trp 165 170 175 Leu Leu Ser Ala Val His Arg Leu Glu Arg Asp Arg Val Leu Leu Ala 180 185 190 Gly Val Thr Val Gln Ala His Gln Thr Leu Thr Val Arg Gly Asp Arg 195 200 205 Ile Val Leu Asp Cys Pro Ser Ala Leu Gly Lys Pro Leu Asn Gly Ser 210 215 220 Thr Arg Ser Thr Val Ser Ala Arg Arg Ser Pro Val Val Ala Glu Ile 225 230 235 240 Leu Gly Ala Ala Arg Arg Pro Val Leu Ala Gly Arg Leu Ala Gln Ser 245 250 255 Val Ala Gln Arg Phe Glu Leu Pro Val Asp Arg Val Thr Gly Leu Leu 260 265 270 Ala Asp Met Ala Ala Gln Glu Leu Leu Ile Thr Ala Leu Arg Pro Pro 275 280 285 Leu Asp Gly Gly Asp Pro Leu Gln His Val Leu Asp Val Val Ala Thr 290 295 300 Ala Glu Ala Arg Ala Gly Ser Pro Ala Glu Ala Met Ser Ser Asp Ser 305 310 315 320 Ala Ala Leu Val Ala Ala Leu Arg Glu Val Asp Ala Arg Cys His Ala 325 330 335 Tyr Asp Arg Thr Ala Val Gly Gln Gly Arg Arg Glu Leu Ala Glu Leu 340 345 350 Ile Gln Ala Thr Arg Arg Val His Pro His Asp Thr Pro Leu His Val 355 360 365 Asp Leu Arg Ile Asp Leu Glu Val Arg Leu Pro Glu Ile Val Arg Thr 370 375 380 Glu Ile Glu Arg Ala Ala Glu Ala Leu Trp Arg Leu Ser Pro Pro Arg 385 390 395 400 Arg Gly Met Arg Ala Leu Arg Arg Tyr His Glu Ala Phe Leu Glu Arg 405 410 415 Tyr Gly Ala Asp Arg Ala Val Pro Leu Leu Glu Leu Leu Asp Asp Thr 420 425 430 Arg Gly Leu Gly Pro Pro Ala Gly Tyr Lys Trp Pro Pro Ser Glu Thr 435 440 445 Pro Ala Gly Pro Gln Glu Glu Pro Arg Arg Ser Ala Ala Leu Ala Arg 450 455 460 Leu Val Ala Thr Ala Ala Arg His Gly Glu Arg Glu Ile Val Ile Asp 465 470 475 480 Glu Glu Thr Ile Ala Glu Leu Val Tyr Asp Glu Ala Ala Pro Ala Asp 485 490 495 Leu Pro Asn Ser Leu Glu Leu Gly Val His Val Val Ala Pro Ser Leu 500 505 510 Asp Glu Leu Ser Ala Gly Thr Phe Arg Val Val Leu Ala Pro Gly Pro 515 520 525 Gly Ser His His Ala Gly Ala Thr Leu Gly Arg Phe Thr Gly Leu Leu 530 535 540 Pro Asp Val Asp Ala Glu Ser Ala Ala Arg Gln Ala Gly Arg Pro Leu 545 550 555 560 His Ile Gln Asp Ala Val Ala Ala Asp Val Ala Phe Ile Pro Arg Ser 565 570 575 Gly Arg Ala Ala Asn Leu Ala His Thr Pro Ser Tyr Ser Gly Arg Arg 580 585 590 Ile Ser Val Gly Leu Pro Asp Ser Gly Arg Ala Gln Glu Leu Pro Leu 595 600 605 Asp Glu Leu Gly Val Ala Ala Asn Leu Glu Arg Leu Cys Leu Val His 610 615 620 Leu Pro Thr Gly Arg Glu Val Val Pro Ala Leu Pro Asn Met Val Ser 625 630 635 640 Ala Phe Ala Gln Ala Pro Asn Pro Ala Arg Leu Leu Phe Glu Leu Gly 645 650 655 Leu Glu Gly Gln Arg Leu Trp Glu Pro Trp Asp Trp Gly Ala Leu Ser 660 665 670 Glu Met Pro Phe Leu Pro Gly Val Arg Tyr Gly Arg Thr Leu Leu Ala 675 680 685 Ala Pro Ile Trp Arg Met Asp Gln Leu Arg Gly Pro Ala Ala Asp Ser 690 695 700 Gly Pro Ala Ala Asp Trp Asp Ala Ala Leu Asp Arg Trp Arg Ala Glu 705 710 715 720 Trp Asn Val Pro Arg Arg Val Leu Ala Val Ser Met Asp Gln Arg Leu 725 730 735 Leu Leu Asp Leu Gly Asp Ala Trp His Arg Val Leu Leu Arg Asp Glu 740 745 750 Leu Arg Arg Thr Pro Glu Leu Ile Ala Gln Gln Val Ala Gly Asp Glu 755 760 765 Glu Gly Trp Leu Asp Arg Gly Asp Gly Gly Phe Pro Gly His Leu Ala 770 775 780 Glu Ile Val Val Pro Leu Glu Arg Arg Asp Arg His Ala Ala Arg Pro 785 790 795 800 Pro His Ile Arg Ala Thr Val Gly Gly Arg Glu Pro Thr Gly Ala Gly 805 810 815 Gly Pro Trp Leu Tyr Leu Arg Leu Arg Val Pro Arg Arg Asn Gln Asp 820 825 830 Asp Phe Leu Arg Asp Gln Val Pro Val Leu Val Arg Ala Gly Ile Gly 835 840 845 His Gly Ala Asp Arg Trp Phe Phe Ile Arg Tyr Ser Asp Thr Ala Gly 850 855 860 Gln His Leu Arg Val Arg Phe Arg Gly Glu Arg Glu Lys Leu Trp Ala 865 870 875 880 Gly Leu Leu Pro Glu Ile Gly Ala Arg Leu Val Glu Trp Gln Arg Gln 885 890 895 Gly Leu Leu Ala Gly His Glu Leu Gly Gln Tyr Asp Pro Glu Tyr Glu 900 905 910 Arg Tyr Gly Gly Asp Ala Leu Ala Glu Phe Thr Glu Ala Ala Phe Gln 915 920 925 His Asp Ser Ala Ala Ala Ile Ser Leu Leu Arg Leu Thr Arg Arg Ala 930 935 940 Gly Phe Arg Tyr Thr Leu Asp Glu Val Thr Ala Ile Ser Ala Ala Ala 945 950 955 960 Leu Ala His Ala Phe Gly Pro Pro Ala Pro Val Val Glu Pro Val Pro 965 970 975 Leu Val Gly Gly Leu Gln Trp Ala Pro Asp Leu Phe Asp Gly Asp Pro 980 985 990 Ala Ala Ala Trp Met Ser Thr Thr Gly Ala Arg Arg Glu Leu Pro Pro 995 1000 1005 Asp Tyr Arg Arg Glu Pro Ala Arg Trp Gln Lys Leu Ile Asp Pro 1010 1015 1020 Thr Gly Gly Trp Arg Val Leu Arg Ala Asp Glu Asp Gly Cys Gln 1025 1030 1035 Val Leu Ala Ala Leu Glu Ser Arg Asp Glu Ala Val Arg Arg Phe 1040 1045 1050 Gly Thr Ala Phe Arg Glu Ala Ser Arg Pro Thr Asp Ser Pro Ser 1055 1060 1065 Thr Gln Leu Arg Leu Val Gly Ser Leu Leu His Met Thr Cys Asn 1070 1075 1080 Arg Leu Ile Gly Gly Ser Ala Glu Arg Glu Arg Ser Val Leu Gly 1085 1090 1095 Leu Ala Arg Gly Ala Val Gln Asp Asn Leu Asn Arg Arg Arg His 1100 1105 1110 Arg Ala 1115 9475PRTMicrobispora sp. PTA-5024 9Val Thr Pro Asp Arg His Pro Ala Arg Phe Leu Arg Gly Ser Ala Ala 1 5 10 15 Arg Arg Ala Ala Arg Leu Val Arg Leu Val Ala Glu Arg Leu Ala Asp 20 25 30 Pro Asp Glu Val Ala Gly Ile Ala Ala Arg Pro Gly Asn Ser Val Pro 35 40 45 Ala Asn Gly Leu Ser Met Trp Ser Pro Ala Thr Leu Ser His Gly Phe 50 55 60 Pro Gly Ile Ala Val Phe Tyr Ala Glu Leu Gly Arg Val Asp Pro Ala 65 70 75 80 Trp Ser Ala Leu Ala His Arg His Leu Arg Ala Gly Ala Ala Ala Val 85 90 95 Glu Thr Ala Pro Ser Gly Gly Leu Phe Ala Gly Pro Ala Ser Leu Leu 100 105 110 Ala Ala Ala Gln Ser Cys Ala Gly Pro Ala Gly His Tyr Arg Gly Leu 115 120 125 Arg Arg Thr Leu Thr Ala Trp Leu Ala Ala Asp His Ala Gly Arg Leu 130 135 140 Ala Ala Ala Arg Asp Arg Pro Gly Pro Gly Val Ala Trp Thr Asp Tyr 145 150 155 160 Asp Val Val His Gly Leu Ser Gly Ser Thr Arg Leu Leu Leu Asp Ala 165 170 175 Ala Arg Asp Pro Asp Asp Glu Thr Ala Ala Lys Ala Ser Gly Ala Val 180 185 190 Thr Asp Thr Leu Arg His Leu Val Arg Leu Thr Glu Pro Ile Thr Val 195 200 205 Asp Gly His Glu Val Pro Gly Trp Trp Val Pro Ser His Leu Gln Pro 210 215 220 Val Glu Gln Asp Arg Arg Asp Tyr Pro Arg Gly Asp Leu Asn Leu Gly 225 230 235 240 Leu Ala His Gly Ala Ala Gly Pro Leu Ser Val Leu Ala Thr Ala Thr 245 250 255 Leu His Gly Val Glu Val Pro Gly Gln Arg Glu Ala Val Ala Arg Leu 260 265 270 Ala Glu Trp Leu Leu Gly Trp Thr Met Thr Asp Asp Thr Gly Ala Tyr 275 280 285 Trp Pro Cys Arg Val Ser Trp Asp Glu Gln Ile Ala Ala Val Arg Pro 290 295 300 Asp Thr Ser Phe Thr Arg Thr Ala Trp Cys Tyr Gly Ala Pro Gly Val 305 310 315 320 Cys Ala Ala Leu His Arg Ala Gly Leu Ala Leu Gly Val Thr Glu Trp 325 330 335 Arg Glu Val Ala Val Thr Ala Leu Leu Asp Gly Leu Arg Arg Asp Arg 340 345 350 Ser Ala Trp Arg Val Asp Gly Ser Thr Val Cys His Gly Tyr Ala Gly 355 360 365 Leu Leu Gln Val Leu Ser Arg Val Gly Ala Glu Ser Gly Asp Pro Arg 370 375 380 Leu Leu Asp Gly Cys Leu Asp Val Ala Arg Met Val Leu Gly Glu Ala 385 390 395 400 Asp Glu Ser Ala Pro Phe Val Phe Pro His Leu Val Pro Asp Ser Pro 405 410 415 Asp Gly Trp Arg Asn Ala Thr Gly Tyr Leu Pro Leu Asp Gly Ala Gly 420 425 430 Leu Leu Glu Gly Ala Ala Gly Val Ala Cys Ala Leu Leu Ser Val Ile 435 440 445 Pro Pro Ser Ser Leu Gly Gly Thr Asp Pro Ala Pro Glu Arg Ala Asp 450 455 460 Leu Pro Pro Trp Asp Arg Cys Leu Ala Leu Cys 465 470 475 10215PRTMicrobispora sp. PTA-5024 10Met Thr Ala His Ser Asp Ala Gly Gly Val Pro Arg Pro Pro Glu Arg 1 5 10 15 Leu Leu Leu Gly Val Ser Gly Ser Val Ala Ala Leu Asn Leu Pro Ala 20 25 30 Tyr Val Tyr Ala Phe Arg Ala Ala Gly Val Ala Arg Leu Ala Val Val 35 40 45 Leu Thr Pro Ala Ala Glu Gly Phe Leu Pro Ala Gly Ala Leu Arg Pro 50 55 60 Ile Val Asp Ala Val His Thr Glu His Asp Gln Gly Lys Gly His Val 65 70 75 80 Ala Leu Ser Arg Trp Ala Gln His Leu Leu Val Leu Pro Ala Thr Ala 85 90 95 Asn Leu Leu Gly Cys Ala Ala Ser Gly Leu Ala Pro Asn Phe Leu Ala 100 105 110 Thr Val Leu Leu Ala Ala Asp Cys Pro Ile Thr Phe Val Pro Ala Met 115 120 125 Asn Pro Val Met Trp Arg Lys Pro Ala Val Arg Arg Asn Val Ala Thr 130 135 140 Leu Arg Ala Asp Gly His Arg Val Val Asp Pro Leu Pro Gly Ala Val 145 150 155 160 Tyr Glu Ala Ala Ser Arg Ser Ile Val Asp Gly Leu Thr Met Pro Arg 165 170 175 Pro Glu Ala Leu Val Arg Leu Leu Gly Gly Gly Asp Asp Gly Ser Pro 180 185 190 Ser Gly Gln Asp Gly Pro Val Gly Arg Ala Glu His Ala Glu His Ala 195 200 205 Glu Ala Ala Glu Ala Leu Ala 210 215 11316PRTMicrobispora sp. PTA-5024 11Met Thr Val Pro Ala Phe Glu Leu Ser Asp Leu Thr Val Arg Tyr Gly 1 5 10 15 Pro Val Thr Ala Val Asp Gly Val Ser Ala Gly Ser Ala Pro Gly Leu 20 25 30 Val Thr Ala Leu Leu Gly Pro Asn Gly Ala Gly Lys Ser Ser Leu Leu 35 40 45 Arg Val Leu Ser Thr Val Ala Pro Pro Ser Ser Gly Thr Ala Arg Val 50 55 60 Phe Gly His Asp Thr Arg Ala Glu Pro Leu Ala Ala Arg Arg Arg Ile 65 70 75 80 Gly Leu Val Phe Gln Glu Arg Ala Leu Asp Thr Asp Leu Ser Ala Glu 85 90 95 Gln Asn Leu Arg Phe His Ala Arg Leu Phe Gly Val Gly Arg Ala Arg 100 105 110 Ala Ala Glu Asp Ile Leu Val Leu Leu Glu Arg Phe Gly Leu Ala Gly 115 120 125 Arg Gly Arg Asp Arg Val Glu Thr Leu Ser Gly Gly Leu Ala Arg Arg 130 135 140 Leu Glu Ile Ala Arg Ala Leu Leu His Arg Pro Gly Leu Leu Ile Leu 145 150 155 160 Asp Glu Pro Thr Asn Gly Leu Asp Pro Glu Ala Arg Gln Thr Val Trp 165 170 175 Asp Asp Leu Ile Arg Leu Arg Ser Glu Leu Gly Val Thr Val Leu Tyr 180 185 190 Ser Thr His Tyr Met Asp Glu Ala Glu Leu Ala Asp Gln Ile Ile Ile 195 200 205 Leu Ser Glu Gly Arg Val Ala Gly Phe Gly Ser Pro Gly Arg Leu Lys 210 215 220 Ser Glu Leu Arg Ser Ser Arg Ile Val Leu Val Thr His Asp Asp Asp 225 230 235 240 Thr Val Leu Ala Arg Leu Ala Glu Ala Gly Phe Asp Ala Val Ile Asp 245 250 255 Ser Asp Gly Val Ala Val Arg Cys Arg Glu Pro Glu Ser Arg Met Ala 260 265 270 Glu Val Ile Arg Ala Ala Gly Pro Leu Val Arg Ala Ala Ser Val His 275 280 285 His Pro Ser Met Asn Asp Val Phe Leu Ala His Thr Ala Ala Asn Arg 290 295 300 Asp Arg Glu Ala Ala Asp Gly Thr Val Ser Cys Pro 305 310 315 12242PRTMicrobispora sp. PTA-5024 12Val Ala His Arg Asp Val Leu Arg Gln Val Arg His Pro Gly Val Val 1 5 10 15 Val Ala Gln Ala Ala Gln Ile Val Phe Phe Val Leu Val Tyr Ala Val 20 25 30 Gly Phe Arg Ser Met Ile Gly Ser Val Gly Gly Val Ser Phe Gly Ala 35 40 45 Tyr Val Tyr Pro Gly Ile Ile Ala Ile Gln Val Val Met Leu Gly Val 50 55 60 Gly Thr Gly Leu Thr Tyr Ala Met Asp Arg Glu Phe Gly Val Leu Arg 65 70 75 80 Glu Met Gln Val Ala Pro Val Pro Arg Met Cys Leu Pro Leu Gly Lys 85 90 95 Ile Leu Ala Ser Cys Val Leu Leu Thr Ala Gln Ala Met Leu Met Leu 100 105 110 Leu Pro Ala Pro Leu Leu Gly Leu Pro Leu Thr Pro Ala Arg Tyr Ala 115 120 125 Ala Gly Ala Ala Val Tyr Leu Ala Thr Ala Ala Ala Phe Ser Leu Ile 130 135 140 Gly Leu Leu Leu Ala Val Ser Val Arg Arg Ile Glu Thr Leu Gln Ala 145 150 155 160 Thr Val Gln Leu Ala Met Tyr Pro Leu Leu Phe Leu Ser Gly Ser Val 165 170 175 Phe Lys Pro Asp Ala Val Pro Gly Trp Leu Ala Ala Leu Met Arg Leu 180 185 190 Asn Pro Met Thr Tyr Ala Val Asp Leu

Ala Arg His Val Leu Leu Pro 195 200 205 Ser Ala Pro Gly Val Ser Tyr Leu Pro Val Trp Arg Asp Leu Leu Val 210 215 220 Ile Ala Ala Leu Val Ala Ala Ala Ser Ala Ala Leu Arg Leu Arg Val 225 230 235 240 Gly Arg 13211PRTMicrobispora sp. PTA-5024 13Val Trp Leu Val Ala Leu Leu Ala Glu Gly Gly Arg Ser Val Glu Pro 1 5 10 15 Trp Pro Ala Val Ala Gln Arg Ile Ile Asp Ala Gly Cys Ala Glu Pro 20 25 30 Ile Arg Pro Gly Arg Pro Thr Ala Ala Arg Leu Glu Ala Arg Tyr Val 35 40 45 Phe Asp Leu Ala Gly Leu Arg His Gly Leu Pro Ala Met Gly Glu Leu 50 55 60 Ala Gly Arg Thr Ala Leu Gly Pro Gly Ala Asp Pro Leu His Leu Thr 65 70 75 80 Asp Ala Asp Leu Tyr Val Ile Thr His Met Leu Phe Tyr Leu Thr Asp 85 90 95 Phe Gly Arg Arg Pro Phe Ser Ala Asp Glu Ala Glu Ser Arg Arg Val 100 105 110 Arg Gly Leu Val Glu Val Leu Leu Gly Arg Gln Leu Ala Val Gly Asp 115 120 125 Leu Asp Leu Ala Ala Glu Leu Leu Ala Cys Ala Gly Leu Thr Gly Ala 130 135 140 Asp Asp Arg Leu Ser Gly Cys Ala Trp Asn Arg Leu Ser Ala Ala Arg 145 150 155 160 Arg Pro Asp Gly Ser Val Pro Ser Pro Leu Phe Arg Gln Ala Ala Leu 165 170 175 Asp Arg Leu Ser Gly Glu Lys Ala Glu Ala Tyr Ala Phe Gly Thr Cys 180 185 190 Tyr His Thr Thr Leu Ala Met Val Leu Ala Ala Thr Leu Thr Asp Gly 195 200 205 Ala Asp Gly 210 14249PRTMicrobispora sp. PTA-5024 14Met Ala Ala Leu Ile Ser Thr Glu Leu Leu Arg Leu Arg Ser Gly Phe 1 5 10 15 Val Gly Trp Tyr Ile Leu Leu Ser Pro Ile Val Ile Ala Ile Pro Leu 20 25 30 Tyr Leu Gly Ser Ile Phe Ser Pro Glu Gly Arg Ser Gly Arg Leu Trp 35 40 45 Glu Thr Phe Ser Asn Val Thr Leu Glu Phe Trp Gly Val Leu Ile Pro 50 55 60 Met Thr Ala Gly Leu Ile Ala Ala Leu Ala Val Arg Ala Asp Thr Glu 65 70 75 80 Pro Trp Arg Phe Leu Phe Ser Tyr Ala Ile Pro Arg Trp Arg Tyr Phe 85 90 95 Thr Ala Lys Val Ala Ala Leu Ala Val Ala Gln Leu Leu Ser Ala Thr 100 105 110 Ile Leu Val Val Met Leu Ala Gly Gly Ala Leu Leu Thr Gly Gln Leu 115 120 125 Ser Asn Ala Ala Ser Met Ile Leu Lys Val Ala Tyr Leu Pro Trp Ala 130 135 140 Ala Gly Leu Ala Ala Thr Ala Leu Ala Val Leu Val Cys Thr Val Trp 145 150 155 160 Gly Leu Gly Pro Gly Ile Ala Leu Gly Val Ala Gly Met Met Ala Gly 165 170 175 Ala Leu Ile Ser Asp Lys Ser Phe Trp Tyr Ala Ile Pro Pro Ala Trp 180 185 190 Pro Met Arg Val Ile Leu Pro Leu Ala Asp Ile Arg Pro Asn Gly Leu 195 200 205 Ala Leu Asp Ala Ser Ser Pro Leu His Asp Thr Ser Val Ile Pro Leu 210 215 220 Ala Val Ala Leu Ser Ala Ala Ala Thr Ile Val Ile Leu Leu Ile Gly 225 230 235 240 Gly Arg His Met Ala Arg Lys Glu Val 245 15236PRTMicrobispora sp. PTA-5024 15Met Ala Ala Leu Glu Ile Arg Asp Leu His Lys His Tyr Asp Asp Phe 1 5 10 15 His Ala Leu Asp Gly Ala Asn Leu Thr Val Pro Asp Gly Ser Leu Tyr 20 25 30 Gly Leu Leu Gly Pro Asn Gly Ala Gly Lys Thr Thr Leu Met Lys Ala 35 40 45 Val Thr Gly Leu Arg His Pro Thr Ser Gly His Ile Ser Leu Phe Gly 50 55 60 Arg Pro Tyr Glu Arg Arg Leu Leu Thr Gln Val Gly Ala Leu Leu Glu 65 70 75 80 Ser Pro Gly Leu Trp Thr Gln Leu Asp Ala Val Ser His Leu Arg Ile 85 90 95 His Ala Arg Leu Arg Gly Val Pro Glu Thr Arg Ile Gly Glu Val Leu 100 105 110 Ser Leu Met Asn Leu Thr Glu Val Ser Thr Arg Lys Val Ala Lys Tyr 115 120 125 Ser Leu Gly Met Arg Trp Arg Leu Gly Ile Ala Ile Ala Leu Leu Gly 130 135 140 Arg Pro Arg Leu Val Val Leu Asp Glu Pro Met Asn Gly Leu Asp Pro 145 150 155 160 Val Gly Ile Arg Asp Met Arg Ala Thr Leu Arg Ala Leu Thr Ala Ala 165 170 175 Gly Thr Thr Val Met Val Ser Ser His Gln Leu Ala Glu Ile Ala His 180 185 190 Ile Cys Asp His Val Gly Val Leu Val Ala Gly Arg Thr Ala Tyr Glu 195 200 205 Gly Pro Leu Pro Gly Leu Ala Val Asp Gly Asp Leu Glu Gln Gly Phe 210 215 220 Phe Arg Leu Leu Glu Lys Ala Gly Ser Ala Val Arg 225 230 235 16541PRTMicrobispora sp. PTA-5024 16Val Thr Glu Pro His Arg Leu Ile Ala Ser Asn Glu Arg Leu Gly Thr 1 5 10 15 Ala Pro Glu Ala Pro Ala Asp Asp Asp Pro Gly Ala Ile Arg Thr Val 20 25 30 Gly Val Ile Gly Gly Gly Thr Ala Gly Tyr Leu Thr Ala Leu Ala Leu 35 40 45 Lys Ala Lys Arg Pro Trp Leu Asp Val Ala Leu Val Glu Ser Ala Asp 50 55 60 Ile Pro Ile Ile Gly Val Gly Glu Ala Thr Val Ser Tyr Met Val Met 65 70 75 80 Phe Leu His His Tyr Leu Gly Ile Asp Pro Ala Glu Phe Tyr Gln His 85 90 95 Val Arg Pro Thr Trp Lys Leu Gly Ile Arg Phe Glu Trp Gly Ser Arg 100 105 110 Pro Glu Gly Phe Val Ala Pro Phe Asp Trp Gly Thr Gly Ser Val Gly 115 120 125 Leu Val Gly Ser Leu Arg Glu Thr Gly Asn Val Asn Glu Ala Thr Leu 130 135 140 Gln Ala Met Leu Met Thr Glu Asp Arg Val Pro Val Tyr Arg Gly Glu 145 150 155 160 Gly Gly His Val Ser Leu Met Lys Tyr Leu Pro Phe Ala Tyr His Met 165 170 175 Asp Asn Ala Arg Leu Val Arg Tyr Leu Thr Glu Leu Ala Ala Arg Arg 180 185 190 Gly Val Arg His Val Asp Ala Thr Val Ala Glu Val Arg Leu Asp Gly 195 200 205 Pro Asp His Val Gly Gly Leu Ile Thr Thr Asp Gly Arg Arg Leu His 210 215 220 Tyr Asp Phe Tyr Val Asp Cys Thr Gly Phe Arg Ser Leu Leu Leu Glu 225 230 235 240 Lys Ala Leu Gly Ile Pro Phe Glu Ser Tyr Ala Ser Ser Leu Phe Thr 245 250 255 Asp Ala Ala Val Thr Gly Thr Leu Ala His Gly Gly His Leu Lys Pro 260 265 270 Tyr Thr Thr Ala Thr Thr Met Asn Ala Gly Trp Cys Trp Thr Ile Pro 275 280 285 Thr Pro Glu Ser Asp His Leu Gly Tyr Val Phe Ser Ser Ala Ala Ile 290 295 300 Asp Pro Asp Asp Ala Ala Ala Glu Met Ala Arg Arg Phe Pro Gly Val 305 310 315 320 Thr Arg Glu Ala Leu Val Arg Phe Arg Ser Gly Arg His Arg Glu Ala 325 330 335 Trp Arg Gly Asn Val Met Ala Val Gly Asn Ser Tyr Ala Phe Val Glu 340 345 350 Pro Leu Glu Ser Ser Gly Leu Leu Met Ile Ala Thr Ala Val Gln Ile 355 360 365 Leu Val Ser Leu Leu Pro Ser Ser Arg Arg Asp Pro Leu Pro Ser Asp 370 375 380 Ala Ala Asn Gln Ala Leu Ala His Arg Trp Asp Ala Ile Arg Trp Phe 385 390 395 400 Leu Ser Ile His Tyr Arg Phe Asn Gly Arg Leu Asp Thr Pro Phe Trp 405 410 415 Lys Glu Ala Arg Ala Glu Thr Asp Ile Ser Gly Ile Glu Pro Leu Leu 420 425 430 Arg Leu Phe Ala Ala Gly Ala Pro Leu Thr Gly Arg Asp Ser Phe Thr 435 440 445 Arg Tyr Leu Ala Asp Gly Ala Ala Pro Leu Phe Tyr Gly Leu Glu Gly 450 455 460 Val Asp Thr Leu Leu Leu Gly Gln Glu Val Pro Ala Arg Leu Leu Pro 465 470 475 480 Pro Arg Glu Pro Pro Glu Gln Trp Arg Ala Arg Ala Ala Ala Ala Arg 485 490 495 Ser Leu Ala Ser Arg Gly Leu Arg Gln Ser Glu Ala Leu Asp Ala Tyr 500 505 510 Ala Ala Asp Pro Cys Leu Asn Ala Glu Leu Leu Ser Asp Ser Asp Ser 515 520 525 Trp Ala Gly Glu Arg Val Ala Val Arg Ala Gly Leu Arg 530 535 540 17178PRTMicrobispora sp. PTA-5024 17Met Thr Thr Gly Ala Thr Val Ala His Val Val Glu Pro Asp Gly Phe 1 5 10 15 Arg Ala Val Met Ala Thr Leu Pro Ala Ala Val Ala Ile Val Thr Ala 20 25 30 Ala Ala Ala Asp Gly Arg Pro Trp Gly Met Thr Cys Ser Ser Val Cys 35 40 45 Ser Val Thr Leu Thr Pro Pro Thr Leu Leu Val Cys Leu Arg Thr Ala 50 55 60 Ser Pro Thr Leu Ala Ala Val Val Ser Gly Arg Ala Phe Ser Val Asn 65 70 75 80 Leu Leu Cys Ala Arg Ser Tyr Pro Val Ala Glu Leu Phe Ala Ser Ala 85 90 95 Ala Ala Asp Arg Phe Asp Arg Val Arg Trp Arg Arg Pro Thr Gly Thr 100 105 110 Gly Gly Pro His Leu Ala Asp Asp Ala Arg Ala Val Leu Asp Cys Arg 115 120 125 Leu Ser Glu Ser Ala Glu Val Gly Asp His Met Val Val Phe Gly Glu 130 135 140 Val Arg Ala Ile Arg Arg Leu Ser Asp Glu Pro Pro Leu Met Tyr Gly 145 150 155 160 Tyr Arg Arg Tyr Ala Pro Trp Pro Ala Asp Arg Gly Pro Gly Ala Val 165 170 175 Gly Gly 18430PRTMicrobispora sp. PTA-5024 18Val Asn Ala Glu Gln Leu Thr Gly Val Val Ile Ala Asp Leu Gly Val 1 5 10 15 Ile Val Val Val Ser Ala Leu Phe Gly Ala Leu Ala Arg Arg Cys Gly 20 25 30 His Pro Thr Val Ile Gly Gln Ile Val Ala Gly Ile Ala Leu Gly Pro 35 40 45 Thr Leu Leu Gly Arg Leu Pro Gly Asp Pro Ala Gly Trp Leu Phe Pro 50 55 60 Ala Gln Val Arg Pro Ser Leu Ser Val Leu Ser Gln Ile Ala Val Val 65 70 75 80 Ile Phe Met Phe Ala Val Gly Tyr Glu Val Asp Leu Arg Leu Leu Arg 85 90 95 Arg Gly Gly Arg Ser Ala Leu Cys Val Ala Ser Leu Ser Leu Ala Val 100 105 110 Pro Met Thr Leu Gly Ala Ala Val Ala Val Leu Phe Arg Glu Val Phe 115 120 125 Thr Val Gly Ser Pro Gly Gly Pro Gly Gly Pro Thr Phe Val Leu Phe 130 135 140 Met Ala Val Ala Ile Ser Ile Thr Ala Leu Pro Val Leu Ala Ala Ile 145 150 155 160 Val Arg Glu Arg Gly Leu Ala Gly Thr Ala Ala Gly Thr Val Ala Thr 165 170 175 Ala Ala Ala Gly Leu Met Asp Val Ala Ala Trp Thr Thr Leu Ala Ala 180 185 190 Val Leu Ala Glu Thr Gly Asp Ala Asp Glu Pro Thr Val Ser His Val 195 200 205 Pro Trp Met Leu Ala Leu Pro Ala Leu Thr Ala Phe Ala Val Ala Met 210 215 220 Phe Leu Val Val Arg Pro Leu Leu Gly Trp Leu Thr Arg Arg Pro Gly 225 230 235 240 Ala Met Trp Gly Arg Leu Pro Ala Ala Phe Ala Leu Ala Leu Gly Ser 245 250 255 Ala Trp Gly Thr Ala Ala Leu Gly Leu His Pro Val Phe Gly Gly Leu 260 265 270 Leu Ala Gly Leu Val Met Pro Arg Arg Asp Gly Ala Pro Glu Pro Glu 275 280 285 Val Leu Arg Pro Met Glu Gln Thr Ala Glu Leu Leu Leu Pro Leu Phe 290 295 300 Phe Val Met Thr Gly Leu Ser Ala Asp Ile Ser Ala Ile Glu Pro Gly 305 310 315 320 Gly Leu Ile Leu Leu Ala Val Leu Leu Val Ala Ala Ile Gly Gly Lys 325 330 335 Leu Val Pro Ala Tyr Ala Ala Ser Arg Leu Thr Gly Leu Asp Ser Gly 340 345 350 Glu Ser Ala Val Val Ala Val Leu Val Asn Thr Arg Gly Leu Thr Glu 355 360 365 Leu Ile Val Leu Asp Val Gly Leu Ser Ala His Val Ile Asp Glu Arg 370 375 380 Leu Phe Thr Val Leu Val Val Met Ala Leu Ile Thr Thr Ala Met Thr 385 390 395 400 Ala Pro Leu Leu Thr Ala Leu Arg Arg Arg Glu Glu Arg Arg Arg Gly 405 410 415 Arg Gln Ala Ala Pro Leu Ser Arg Ala Thr Ala Trp Arg Met 420 425 430 1968PRTartificial sequencesynthetic primer 19Gly Thr Ser Ala Cys Ser Trp Ser Ser Thr Gly Gly Trp Ser Ser Tyr 1 5 10 15 Thr Ser Trp Ser Ser Ala Cys Ser Gly Gly Ser Cys Cys Ser Thr Gly 20 25 30 Cys Ala Cys Ser Trp Ser Ser Cys Cys Ser Gly Gly Ser Gly Gly Ser 35 40 45 Trp Ser Ser Ala Ala Cys Trp Ser Ser Trp Ser Ser Thr Cys Cys Trp 50 55 60 Ser Ser Thr Gly 65 2027DNAartificial sequencesynthetic primer 20aagcttgcat ctgcgtgggc gtcctgc 272129DNAartificial sequencesynthetic primer 21tctagacggt ccgaagatca tggccgcgg 292230DNAartificial sequencesynthetic primer 22tctagatcca tgtgaaccgg cgggtggccg 302330DNAartificial sequencesynthetic primer 23gaattccggt cgctctcctc gtcctttgcc 302419DNAartificial sequencesynthetic primer 24cgcgctgctc ggggccaac 192520DNAartificial sequencesynthetic primer 25aggaaacggc cagcccgtgg 202631DNAartificial sequencesynthetic primer 26tttttcatat gggtgggagt gatcggcggc g 312735DNAartificial sequencesynthetic primer 27tttttgtcga cctactgctg gccgcggtcc ggact 352820DNAartificial sequencesynthetic primer 28gcagccaggc tcgcaccggc 202916DNAartificial sequencesynthetic primer 29cgcccgtaac gagcga 163017DNAartificial sequencesynthetic primer 30gcagcttctg ctgctga 173116DNAartificial sequencersynthetic primer 31tcccggccag ccactt 163221DNAartificial sequencesynthetic primer 32ccggaaagga gcgagcatat g 213318DNAartificial sequencesynthetic primer 33cagatctgcc aatacagt 1834112DNAartificial sequencesynthetic primer 34cggtgtcgag gagatcaccg ccgggccggc gnnnnnnagc nnnnnnnnnt gcaccnnnnn 60ntgcnnnagc nnnnnnnnnn nnagcnnntg cagcnnntgc tgctgaagat ct 11235112DNAartificial sequencesynthetic primer 35ttcagcagca nnngctgcan nngctnnnnn nnnnnnngct nnngcannnn nnggtgcann 60nnnnnnngct nnnnnncgcc ggcccggcgg tgatctcctc gacaccgatc ga 11236171DNAartificial sequencesynthetic primer 36atgcccgctg acatcctgga gacccggact tccgagaccg aggacctgct cgacctcgac 60ctctcgatcg gtgtcgagga gatcaccgcc gggccggcgn nnnnnagcnn nnnnnnntgc 120accnnnnnnt gcnnnagcnn nnnnnnnnnn agcnnntgca gcnnntgctg c 171

Claims

1-34. (canceled)

35. A recombinant DNA vector which comprises a nucleotide sequence encoding a polypeptide that, over its entire length, is at least 65% identical in amino acid sequence to a polypeptide encoded by SEQ ID NO: 7.

36. A host cell transformed with the vector of claim 35.

37. The transformed host cell of claim 36 which belongs to the order Actinomycetales.

38. The transformed host cell of claim 37 which belongs to the family selected from the group consisting of Streptosporangiaceae, Micromonosporaceae, Pseudonocardiaceae and Streptomycetaceae.

39. The transformed host cell according to claim 38 which belongs to the genera selected from the group consisting of Microbispora, Actinoplanes, Planomonospora, and Streptomyces.

40. The transformed host cell of claim 36 which belongs to the order Bacillales.

41. The transformed host cell of claim 37 which belongs to the family selected from the group consisting of Bacillaceae, Lactobacillaceae, Streptococcaceae and Staphylococcaccae.

42. The transformed host cell of claim 38 which belongs to the genera selected from the group consisting of Bacillus, Lactococcus, Streptococcus, and Staphylococcus.

43. The transformed host cell of claim 36 which belongs to the species Escherichia coli.

44. A method for increasing production of 107891 by a microorganism capable of producing 107891, said method comprising: transforming with a recombinant DNA vector of claim 35 a microorganism that produces 107891, wherein said DNA vector is an expression vector.

45. A recombinant DNA vector which comprises a nucleotide sequence encoding a polypeptide that, over its entire length, is at least 65% identical in amino acid sequence to a polypeptide encoded by SEQ ID NO: 8.

46. A host cell transformed with the vector of claim 45.

47. The transformed host cell of claim 46 which belongs to the order Actinomycetales.

48. The transformed host cell of claim 47 which belongs to the family selected from the group consisting of Streptosporangiaceae, Micromonosporaceae, Pseudonocardiaceae and Streptomycetaceae.

49. The transformed host cell according to claim 48 which belongs to the genera selected from the group consisting of Microbispora, Actinoplanes, Planomonospora, and Streptomyces.

50. The transformed host cell of claim 46 which belongs to the order Bacillales.

51. The transformed host cell of claim 47 which belongs to the family selected from the group consisting of Bacillaceae, Lactobacillaceae, Streptococcaceae and Staphylococcaccae.

52. The transformed host cell of claim 48 which belongs to the genera selected from the group consisting of Bacillus, Lactococcus, Streptococcus, and Staphylococcus.

53. The transformed host cell of claim 46 which belongs to the species Escherichia coli.

54. A method for increasing production of 107891 by a microorganism capable of producing 107891, said method comprising: transforming with a recombinant DNA vector of claim 45 a microorganism that produces 107891, wherein said DNA vector is an expression vector.

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