Patent application title: MATERIALS AND METHODS FOR THE BIOSYNTHESIS OF SEVEN CARBON CHEMICALS IN THE PRESENCE OF METHANOL OXIDATION
Inventors:
IPC8 Class: AC12N1552FI
USPC Class:
528310
Class name: Synthetic resins (class 520, subclass 1) from carboxylic acid or derivative thereof from imide- or lactam-containing compound, or from an amino-nitrogen containing carboxylic acid, or derivative of an amino-nitrogen-containing carboxylic acid
Publication date: 2018-01-25
Patent application number: 20180023088
Abstract:
This disclosure describes methods for regulating the biosynthesis of
pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate,
heptamethylenediamine, 7-aminoheptanol, or 1,7-heptanediol by channeling
increased flux through the biosynthesis pathway to obtain an intermediate
required for growth of the host microorganism.Claims:
1. A method for regulating biosynthesis of a product chosen from pimelic
acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine,
7-aminoheptanol, and 1,7-heptanediol, or salts and derivatives thereof,
using a pathway having a pimeloyl-ACP intermediate, the method comprising
converting methanol to formate via at least one spontaneous enzymatic
reaction, wherein the formate is used in the conversion of
tetrahydrofolate to N.sup.10-formyl-tetrahydrofolate.
2. The method of claim 1, wherein: the methanol is produced during BioH enzyme activity; and/or the method comprises the step of downregulating the activity of FolD.
3. The method of claim 2, wherein: BioH removes the methyl group from pimeloyl-ACP methyl ester during conversion of pimeloyl-ACP methyl ester to pimeloyl-ACP; and/or the step of downregulating the activity of FolD comprises a step of attenuating folD.
4. (canceled)
5. (canceled)
6. The method of claim 3, wherein the method comprises the step of cloning in a formate-tetrahydrofolate ligase (fhs); the method comprises the step of downregulating the activity of PflB and TdcE; the method comprises a step of cloning in an alcohol dehydrogenase (adh); the method comprises a step of cloning in a S-(hydroxymethyl), glutathione dehydrogenase (frmA); and/or the method comprises a step of cloning in a S-formylglutathione hydrolase (frmB).
7. The method of claim 6, wherein: the formate-tetrahydrofolate ligase has at least 70%, at least 80%, or at least 90% sequence identity or homology to an amino acid sequence chosen from SEQ ID NOs: 18-22; the alcohol dehydrogenase has at least 70%, at least 80%, or at least 90% sequence identity or homology to the amino acid sequence set forth in SEQ ID NO: 31; the S-(hydroxymethyl) glutathione dehydrogenase has at least 70%, at least 80%, or at least 90% sequence identity or homology to an amino acid sequence chosen from SEQ ID NOs: 23-26; and/or the S-formylglutathione hydrolase has at least 70%, at least 80%, or at least 90% sequence identity or homology to an amino acid sequence chosen from SEQ ID NOs: 27-30.
8. (canceled)
9. The method of claim 6, wherein: the step of downregulating the activity of PflB and TdcE comprises a step of deleting pflB and tdcE; the alcohol dehydrogenase converts the methanol to formaldehyde; the S-(hydroxymethyl) glutathione dehydrogenase converts S-hydroxymethylglutathione to S-formylgluathione; and/or the S-formylglutathione hydrolase converts S-formylglutathione to formate.
10-18. (canceled)
19. The method of claim 6, wherein: the expression of adh, frmA, and frmB allows conversion of the methanol to formate; and the biosynthesis of the product is increased.
20. (canceled)
21. The method of claim 1, wherein the product is chosen from salts and derivatives thereof of pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, 7-aminoheptanol, and 1,7-heptanediol.
22. The method of claim 1, wherein the method is performed in a recombinant host.
23. The method of claim 22, wherein: the host is subjected to a cultivation strategy under aerobic, anaerobic, micro-aerobic, or mixed oxygen/denitrification cultivation conditions; the host is cultured under conditions of nutrient limitation; the host is retained using a ceramic hollow fiber membrane to maintain a high cell density during fermentation; and/or the host's tolerance to high concentrations of a C7 building block is improved through continuous cultivation in a selective environment.
24. (canceled)
25. (canceled)
26. The method of claim 22, wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks.
27. The method of claim 26, wherein: the biological feedstock is, or derives from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste; or the non-biological feedstock is, or derives from, natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, benzoate, non-volatile residue (NVR) caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/I isophthalic acid mixture waste streams.
28. (canceled)
29. (canceled)
30. The method of claim 29, wherein: the host is a prokaryote chosen from the following genera: Escherichia; Corynebacteria; Cupriavidus; Pseudomonas; Delftia; Bacillus; Lactobacillus; Lactococcus and Rhodococcus; or the host is a eukaryote chosen from the following genera: Aspergillus; Saccharomyces; Pichia; Yarrowia; Issatchenkia; Debaryomyces; Arxula; and Kluyveromyces.
31. The method of claim 33, wherein the host is Escherichia coli.
32. (canceled)
33. The method of claim 30, wherein: the prokaryote is chosen from Escherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans, Bacillus subtillis, Lactobacillus delbrueckii, Lactococcus lactis, and Rhodococcus equi; or the eukaryote is chosen from Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans, and Kluyveromyces lactis.
34. (canceled)
35. The method of claim 22, wherein: the host comprises one or more of the following attenuated enzymes: polyhydroxyalkanoate synthase; an acetyl-CoA thioesterase; an acetyl-CoA specific .beta.-ketothiolase; a phosphotransacetylase forming acetate; an acetate kinase; a lactate dehydrogenase; a menaquinol-fumarate oxidoreductase; a 2-oxoacid decarboxylase producing isobutanol; an alcohol dehydrogenase forming ethanol; a triose phosphate isomerase; a pyruvate decarboxylase; a glucose-6-phosphate isomerase; a transhydrogenase dissipating the NADH or NADPH imbalance; an glutamate dehydrogenase dissipating the NADH or NADPH imbalance; a NADH/NADPH-utilizing glutamate dehydrogenase; a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase accepting C7 building blocks and central precursors as substrates; a glutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase; and/or the host overexpresses an acetyl-CoA synthetase; a 6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a formate dehydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase; a fructose 1.6 diphosphatase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase specific to the NADH or NADPH used to generate a co-factor imbalance; a methanol dehydrogenase; a formaldehyde dehydrogenase; a diamine transporter a dicarboxylate transporter an S-adenosylmethionine synthetase; and/or a multidrug transporter.
36. (canceled)
37. A recombinant host for producing pimeloyl-ACP, wherein: the host comprises at least one exogenous nucleic acid encoding (i) a formate-tetrahydrofolate ligase, (ii) a S-(hydroxymethyl) glutathione dehydrogenase, and an (iii) a S-formylglutathione hydrolase; and the host optionally further comprises at least one exogenous nucleic acid encoding an alcohol dehydrogenase.
38. The recombinant host of claim 37, wherein: the host further comprises a deletion of folD; and/or the host further comprises a deletion of pflB and tdcE.
39. (canceled)
40. (canceled)
41. The recombinant host of claim 37, said host comprising at least one exogenous nucleic acid encoding one or more of a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a glutaconate CoA-transferase, a reversible succinyl-CoA ligase, an acetylating aldehyde dehydrogenase, or a carboxylate reductase, and said host producing pimelic acid or pimelate semialdehyde.
42. The recombinant host of claim 37, said host further comprising: at least one exogenous nucleic acid encoding a .omega.-transaminase, wherein the host produces 7-aminoheptanoate; one or more of a 4-hydroxybutirate dehydrogenase, a 5-hydroxypentanoate dehydrogenase or a 6-hydroxyhexanoate dehydrogenase, said host producing 7-hydroxyheptanoic acid; at least one exogenous nucleic acid encoding a .omega.-transaminase, a deacetylase, an N-acetyltransferase or an alcohol dehydrogenase, said host producing heptamethylenediamine; or at least one exogenous nucleic acid encoding a (i) carboxylate reductase enhanced by a phosphopantetheinyl transferase or (ii) an alcohol dehydrogenase, said host producing 1,7-heptanediol.
43-53. (canceled)
54. A nucleic acid construct or expression vector comprising: a polynucleotide encoding a polypeptide having formate-tetrahydrofolate ligase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having formate-tetrahydrofolate ligase activity is selected from: (a) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 18; (b) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 19; (c) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 20; (d) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 21; and (e) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 22; a polypeptide having alcohol dehydrogenase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having alcohol dehydrogenase is a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 31; a polypeptide having S-(hydroxymethyl) glutathione dehydrogenase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having S-(hydroxymethyl) glutathione dehydrogenase activity is selected from: (a) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 23; (b) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 24; (c) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 25; and (d) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 26; or a polypeptide having S-formylglutathione hydrolase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having S-formylglutathione hydrolase activity is selected from: (a) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 27; (b) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 28; (c) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 29; and (d) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 30.
55-58. (canceled)
59. A bio-derived, bio-based, or fermentation-derived product, wherein said product comprises: (i) a composition comprising at least one bio-derived, bio-based, or fermentation-derived compound produced or biosynthesized according to the methods of claim 1, or any combination thereof, (ii) a bio-derived, bio-based, or fermentation-derived polymer comprising the bio-derived, bio-based, or fermentation-derived composition or compound of (i), or any combination thereof, (iii) a bio-derived, bio-based, or fermentation-derived resin comprising the bio-derived, bio-based, or fermentation-derived compound or bio-derived, bio-based, or fermentation-derived composition of (i) or any combination thereof or the bio-derived, bio-based, or fermentation-derived polymer of (ii) or any combination thereof, (iv) a molded substance obtained by molding the bio-derived, bio-based, or fermentation-derived polymer of (ii) or the bio-derived, bio-based, or fermentation-derived resin of (iii), or any combination thereof, (v) a bio-derived, bio-based, or fermentation-derived formulation comprising the bio-derived, bio-based, or fermentation-derived composition of (i), bio-derived, bio-based, or fermentation-derived compound of (i), bio-derived, bio-based, or fermentation-derived polymer of (ii), bio-derived, bio-based, or fermentation-derived resin of (iii), or bio-derived, bio-based, or fermentation-derived molded substance of (iv), or any combination thereof, or (vi) a bio-derived, bio-based, or fermentation-derived semi-solid or a non-semi-solid stream, comprising the bio-derived, bio-based, or fermentation-derived composition of (i), bio-derived, bio-based, or fermentation-derived compound of (i), bio-derived, bio-based, or fermentation-derived polymer of (ii), bio-derived, bio-based, or fermentation-derived resin of (iii), bio-derived, bio-based, or fermentation-derived formulation of (v), or bio-derived, bio-based, or fermentation-derived molded substance of (iv), or any combination thereof.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S. Provisional Application No. 62/366,549, filed Jul. 25, 2016, which is incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 25, 2017, is named 12444_0685-00000_SL.txt and is 167,292 bytes in size.
TECHNICAL FIELD
[0003] The invention relates to methods for regulating the biosynthesis of one or more seven carbon compounds. This invention relates to materials and methods for biosynthesizing one or more of pimelic acid, 7-aminoheptanoic acid (7-AHA), 7-hydroxyheptanoic acid, heptamethylenediamine, 7-aminoheptanol, and 1,7-heptanediol (hereafter "C7 building blocks") from malonyl-CoA or malonyl-[acp] and optionally acetyl-CoA using polypeptides having the activity of one or more enzymes such as methyltransferases, .beta.-ketoacyl-[acp] synthases, .beta.-ketothiolases, dehydmrogenases, reductases, hydratases, thioesterases, esterases, CoA-transferases, reversible CoA-ligases, and transaminases or using recombinant host cells expressing one or more such enzymes in genetically modified hosts. The invention further relates to coupling a modified tetrahydrofolate metabolic cycle (THF-MC) to the biosynthesis of C7 building blocks by forcing the modified THF-MC to utilize formate derived from methanol oxidation, wherein the methanol is a byproduct of the C7 building block biosynthesis pathway.
BACKGROUND
[0004] Nylons are synthetic polyamides which are sometimes synthesized by the condensation polymerisation of a diamine with a dicarboxylic acid. Similarly, Nylons may be produced by the condensation polymerisation of lactams. A ubiquitous Nylon is Nylon 6,6, which is produced by reaction of hexamethylenediamine (HMD) and adipic acid. Nylon 6 is produced by a ring opening polymerisation of caprolactam (Anton & Baird, Polyamides Fibers, Encyclopedia of Polymer Science and Technology, 2001).
[0005] Nylon 7 and Nylon 7,7 represent novel polyamides with value-added characteristics compared to Nylon 6 and Nylon 6,6. Nylon 7 is produced by polymerisation of 7-aminoheptanoic acid (7-AHA), whereas Nylon 7,7 is produced by condensation polymerisation of pimelic acid and heptamethylenediamine. No economically viable petrochemical routes exist to producing the monomers for Nylon 7 and Nylon 7,7.
[0006] Given no economically cost competitive petrochemical monomer feedstocks, biotechnology offers an alternative approach via biocatalysis. Biocatalysis is the use of biological catalysts, such as enzymes, to perform biochemical transformations of organic compounds.
[0007] Both bioderived feedstocks and petrochemical feedstocks are viable starting materials for the biocatalysis processes.
[0008] However, no wild-type prokaryote or eukaryote naturally overproduces or excretes C7 building blocks to the extracellular environment. Nevertheless, the metabolism of pimelic acid has been reported.
[0009] The dicarboxylic acid, pimelic acid, is converted efficiently as a carbon source by a number of bacteria and yeasts via .beta.-oxidation into central precursors. .beta.-oxidation of coenzyme A (CoA) activated pimelate to CoA-activated 3-oxopimelate facilitates further catabolism via, for example, pathways associated with aromatic substrate degradation. The catabolism of 3-oxopimeloyl-CoA to acetyl-CoA and glutaryl-CoA by several bacteria has been characterized comprehensively (Harwood and Parales, Annual Review of Microbiology, 1996, 50, 553-590).
[0010] The biosynthesis of C7 building blocks by recombinant host microorganisms has been described in U.S. Patent Publication Nos. 2014/0186904 and 2014/0242655.
[0011] The optimality principle states that microorganisms regulate their biochemical networks to support maximum biomass growth. Beyond the need to express heterologous pathways in a host organism, directing carbon flux toward C7 building blocks that serve as carbon sources, rather than to biomass growth constituents, contradicts the optimality principle. For example, transferring the 1-butanol pathway from Clostridium species into other production strains has often fallen short by an order of magnitude compared to the production performance of native producers (Shen et al., Appl. Environ. Microbiol., 2011, 77(9), 2905-2915).
[0012] The synthesis of the seven carbon aliphatic backbone precursor is a key consideration in synthesizing C7 building blocks prior to forming terminal functional groups, such as carboxyl, amine, or hydroxyl groups, on the C7 aliphatic backbone.
SUMMARY
[0013] Accordingly, against this background, it is clear that there is a need for methods for producing pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine, 7-aminoheptanol, 7-hydroxyheptanoic acid, and 1,7-heptanediol (hereafter "C7 building blocks"), wherein the methods are biocatalyst-based. By making the host organism dependent on a byproduct of the biosynthesis of C7 building blocks, the host channels flux through the pathway leading to the C7 building block product, thus increasing yield, titre, and productivity.
[0014] This disclosure is based at least in part on the discovery that it is possible to construct biochemical pathways for producing a seven carbon chain aliphatic backbone precursor, in which one or two functional groups, i.e., carboxyl, amine, or hydroxyl, can be formed, leading to the synthesis of one or more of pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, 7-aminoheptanol, and 1,7-heptanediol (C7 building blocks), or salts or derivatives thereof. Pimelic acid and pimelate, pimelyl and pimeloyl, 7-hydroxyheptanoic acid and 7-hydroxyheptanoate, and 7-aminoheptanoic and 7-aminoheptanoate are used interchangeably herein to refer to the compound in any of its neutral or ionized forms, including any salt forms thereof. It is understood by those skilled in the art that the specific form will depend on pH.
[0015] For compounds containing carboxylic acid groups such as organic monoacids, hydroxyacids, aminoacids, and dicarboxylic acids, these compounds may be formed or converted to their ionic salt form when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. The salt can be isolated as is from the system as the salt or converted to the free acid by reducing the pH to below the pKa through addition of acid or treatment with an acidic ion exchange resin.
[0016] For compounds containing amine groups such as, but not limited to, organic amines, aminoacids, and diamines, these compounds may be formed or converted to their ionic salt form by addition of an acidic proton to the amine to form the ammonium salt, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, or muconic acid. Acceptable inorganic bases are known in the art and include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. The salt can be isolated as is from the system as a salt or converted to the free amine by raising the pH to above the pKb through addition of base or treatment with a basic ion exchange resin.
[0017] For compounds containing both amine groups and carboxylic acid groups such as, but not limited to, aminoacids, these compounds may be formed or converted to their ionic salt form by either 1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like or 2) when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases are known in the art and include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases are known in the art and include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. The salt can be isolated as is from the system or converted to the free acid by reducing the pH to below the pKa through addition of acid or treatment with an acidic ion exchange resin.
[0018] These pathways and metabolic engineering and cultivation strategies described herein rely on fatty acid elongation and synthesis enzymes or homologs accepting methyl-ester shielded dicarboxylic acids as substrates.
[0019] This disclosure is further based on increasing flux through the C7 building block biosynthesis pathway by making methanol, a byproduct of the pathway, a requirement for cell growth. This disclosure is also related to using methanol for cell growth.
[0020] The present disclosure provides methods for biosynthesizing one or more seven carbon compounds (C7 building blocks) and for regulating the biosynthesis of a C7 building block product using a pathway having a pimeloyl-ACP intermediate. In some embodiments, this method comprises converting methanol to formate, wherein the formate is used in the conversion of tetrahydrofolate to N.sup.10-formyl-tetrahydrofolate. In some embodiments, methanol is converted to formate via spontaneous enzymatic reactions. In one embodiment, the methanol is produced during BioH enzyme activity, wherein BioH removes the methyl group from pimelyl-ACP methyl ester during conversion of pimeloyl-ACP methyl ester to pimeloyl-ACP.
[0021] In some embodiments, the product may be a salt or derivative thereof of a C7 building block.
[0022] In some embodiments, the method comprises the step of downregulating activity of bifunctional protein FolD (FolD). In one embodiment, the step of downregulating the activity of FolD comprises a step of deleting folD. In some embodiments, the method comprises the step of cloning in, or knocking in, a formate-tetrahydrofolate ligase. In one embodiment, the formate-tetrahydrofolate ligase has at least 70%, at least 80%, or at least 90% sequence identity or homology to an amino acid chosen from SEQ ID NOs: 18-22.
[0023] In some embodiments, the method comprises the step of downregulating the activity of formate acetyltransferase 1 (PflB) and PFL-like enzyme TdcE (TdcE). In one embodiment, the step of downregulating the activity of PflB and TdcE comprises a step of deleting pflB and tdcE.
[0024] In some embodiments, the method comprises a step of cloning in, or knocking in, an alcohol dehydrogenase. In some embodiments an alcohol dehydrogenase converts the methanol to formaldehyde. In one embodiment, the alcohol dehydrogenase has at least 70%, at least 80%, or at least 90% sequence identity or homology to the amino acid sequence set forth in SEQ ID NO: 31.
[0025] In some embodiments, the method comprises a step of cloning in, or knocking in, a S-(hydroxy methyl) glutathione dehydrogenase (frmA). In some embodiments, a S-(hydroxymethyl) glutathione dehydrogenase converts S-hydroxymethylglutathione to S-formylgluathione. In one embodiment, the S-(hydroxymethyl) glutathione dehydrogenase has at least 70%, at least 80%, or at least 90% sequence identity or homology to an amino acid sequence chosen from SEQ ID NOs: 23-26.
[0026] In some embodiments, the method comprises a step of cloning in, or knocking in, a S-formylglutathione hydrolase (frmB). In some embodiments, a S-formylglutathione hydrolase converts S-formylglutathione to formate. In one embodiment, the S-formylglutathione hydrolase has at least 70%, at least 80%, or at least 90% sequence identity or homology to an amino acid sequence chosen from SEQ ID NOs: 27-30.
[0027] In some embodiments, the expression of adh, frmA, and frmB gene products allows conversion of the methanol to formate.
[0028] In some embodiments, the biosynthesis of the product is increased.
[0029] In some embodiments, the method is performed in a recombinant host.
[0030] In some embodiments, the host is subjected to a cultivation strategy under aerobic, anaerobic, micro-aerobic or mixed oxygen/denitrification cultivation conditions. In one embodiment, the host is cultured under conditions of nutrient limitation.
[0031] In some embodiments, the host is retained using a ceramic hollow fiber membrane to maintain a high cell density during fermentation. In one embodiment, the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks.
[0032] In some embodiment, the biological feedstock is, or derives from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste. In some embodiments, the non-biological feedstock is, or derives from, natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, benzoate, non-volatile residue (NVR) caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.
[0033] In some embodiments, the host is a prokaryote. In one embodiment, the host is from the genus Escherichia such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogenum, or Clostridium kluyveri; from the genus Corynebacteria such as Corynebacterium glutamicum; from the genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans; from the genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida, or Pseudomonas oleavorans; from the genus Delftia such as Delftia acidovorans, from the genus Bacillus such as Bacillus subtillis; from the genus Lactobacillus such as Lactobacillus delbrueckii; from the genus Lactococcus such as Lactococcus lactis; or from the genus Rhodococcus such as Rhodococcus equi. For example, the host may be Escherichia coli.
[0034] In some embodiments, the host is a eukaryote. In one embodiment, the host is from the genus Aspergillus such as Aspergillus niger; from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris; from the genus Yarrowia such as Yarrowia lipolytica, from the genus Issatchenkia such as Issathenkia orientalis, from the genus Debaryomyces such as Debaryomyces hansenii, from the genus Arxula such as Arxula adenoinivorans, or from the genus Kluyveromyces such as Kluyveromyces lactis.
[0035] In some embodiments, the host's tolerance to high concentrations of a C7 building block is improved through continuous cultivation in a selective environment.
[0036] In some embodiments, the host comprises one or more of the following attenuated enzymes: polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, an acetyl-CoA specific .beta.-ketothiolase, a phosphotransacetylase forming acetate, an acetate kinase, a lactate dehydrogenase, a menaquinol-fumarate oxidoreductase, a 2-oxoacid decarboxylase producing isobutanol, an alcohol dehydrogenase forming ethanol, a triose phosphate isomerase, a pyruvate decarboxylase, a glucose-6-phosphate isomerase, a transhydrogenase dissipating the NADH or NADPH imbalance, an glutamate dehydrogenase dissipating the NADH or NADPH imbalance, a NADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase accepting C7 building blocks and central precursors as substrates, a glutaryl-CoA dehydrogenase, or a pimeloyl-CoA synthetase.
[0037] In some embodiments, the host expresses or overexpresses one or more of the following enzymes: an acetyl-CoA synthetase; a 6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a formate dehydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase; a fructose 1, 6 diphosphatase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase specific to the NADH or NADPH used to generate a co-factor imbalance; a methanol dehydrogenase; a formaldehyde dehydrogenase; a diamine transporter; a dicarboxylate transporter; an S-adenosylmethionine synthetase; and/or a multidrug transporter.
[0038] The present disclosure also provides a recombinant host for producing pimeloyl-ACP, wherein the host comprises at least one exogenous nucleic acid encoding (i) a formate-tetrahydrofolate ligase, (ii) a S-(hydroxymethyl) glutathione dehydrogenase, and an (iii) a S-formylglutathione hydrolase, or the host has (i) a formate-tetrahydrofolate ligase, (ii) a S-(hydroxymethyl) glutathione dehydrogenase, and an (iii) a S-formylglutathione hydrolase knocked in.
[0039] In some embodiments, the host comprises deletion of folD. In some embodiments, the host comprises a deletion of pflB and tdcE.
[0040] In some embodiments, the host comprises at least one exogenous nucleic acid encoding an alcohol dehydrogenase. In one embodiment, the host has a knock in of alcohol dehydrogenase. In some embodiments, the host comprises at least one exogenous nucleic acid encoding one or more of a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a glutaconate CoA-transferase, a reversible succinyl-CoA ligase, an acetylating aldehyde dehydrogenase, or a carboxylate reductase, said host producing pimelic acid or pimelate semialdehyde.
[0041] In one embodiment, the host comprises at least one exogenous nucleic acid encoding .omega.-transaminase, wherein the host produces 7-aminoheptanoate.
[0042] In some embodiments, the host further comprises at least one exogenous nucleic acid encoding a .omega.-transaminase, a deacetylase, an N-acetyltransferase, or an alcohol dehydrogenase, said host producing heptamethylenediamine.
[0043] In some embodiments, the host comprises at least one exogenous nucleic acid encoding a (i) carboxylate reductase enhanced by a phosphopantetheinyl transferase or (ii) an alcohol dehydrogenase, said host producing 1,7-heptanediol.
[0044] This disclosure also relates to non-naturally occurring organisms comprising at least one exogenous nucleic acid encoding at least one polypeptide having the activity of at least one enzyme depicted in any one of FIGS. 1 to 11.
[0045] The present disclosure also provides methods of using methanol for cell growth of a recombinant host.
[0046] In some embodiments, the method comprises the step of deleting folD.
[0047] In some embodiments, the method comprises the step of cloning in, or knocking in, a formate-tetrahydrofolate ligase (fhs).
[0048] In some embodiments, the method comprises the step of deleting pflB and tdcE.
[0049] In some embodiments, the method comprises the step of cloning in an alcohol dehydrogenase (adh), a S-(hydroxymethyl) glutathione dehydrogenase (frmA), and a S-formylglutathione hydrolase.
[0050] In some embodiments, the recombinant host converts methanol to formate. In some embodiments, formate is required for the synthesis of purines and initiator tRNA.
[0051] This disclosure also relates to nucleic acid constructs or expression vectors comprising a polynucleotide encoding a polypeptide, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide.
[0052] In some embodiments, the polypeptide has formate-tetrahydrofolate ligase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having formate-tetrahydrofolate ligase activity is selected from a polypeptide having at least 70% sequence identity or homology to the polypeptide of any one of SEQ ID NOs: 18-22.
[0053] In some embodiments, the polypeptide has alcohol dehydrogenase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having alcohol dehydrogenase activity has at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 31.
[0054] In some embodiments, the polypeptide has S-(hydroxymethyl) glutathione dehydrogenase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having S-(hydroxymethyl) glutathione dehydrogenase activity has at least 70% sequence identity or homology to the polypeptide of any one of SEQ ID NOs: 23-26.
[0055] In some embodiments the polypeptide has S-formylglutathione hydrolase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having S-formylglutathione hydrolase activity has at least 70% sequence identity or homology to the polypeptide of any one of SEQ ID NOs: 27-30.
[0056] The present disclosure relates to the following additional embodiments:
[0057] 1. A method for regulating biosynthesis of a product chosen from pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, 7-aminoheptanol, and 1,7-heptanediol, or salts and derivatives thereof, using a pathway having a pimeloyl-ACP intermediate, the method comprising converting methanol to formate via at least one spontaneous enzymatic reaction, wherein the formate is used in the conversion of tetrahydrofolate to N.sup.10-formyl-tetrahydrofolate.
[0058] 2. The method of embodiment 1, wherein the methanol is produced via BioH enzyme activity.
[0059] 3. The method of embodiment 2, wherein BioH removes the methyl group from pimeloyl-ACP methyl ester during conversion of pimeloyl-ACP methyl ester to pimeloyl-ACP.
[0060] 4. The method of any one of embodiments 1 to 3, wherein the method comprises the step of downregulating the activity of FolD.
[0061] 5. The method of embodiment 4, wherein the step of downregulating the activity of FolD comprises a step of attenuating folD.
[0062] 6. The method of embodiment 4, wherein the method comprises the step of cloning in a formate-tetrahydrofolate ligase (fhs).
[0063] 7. The method of embodiment 6, wherein the formate-tetrahydrofolate ligase has at least 70%, at least 80%, or at least 90% sequence identity or homology to an amino acid sequence chosen from SEQ ID NOs: 18-22.
[0064] 8. The method of any one of embodiments 4 to 7, wherein the method comprises the step of downregulating the activity of PflB and TdcE.
[0065] 9. The method of embodiment 8, wherein the step of downregulating the activity of PflB and TdcE comprises a step of deleting pflB and tdcE.
[0066] 10. The method of any one of embodiments 1 to 9, wherein the method comprises a step of cloning in an alcohol dehydrogenase (adh).
[0067] 11. The method of embodiment 10, wherein the alcohol dehydrogenase converts the methanol to formaldehyde.
[0068] 12. The method of embodiment 11, wherein the alcohol dehydrogenase has at least 70%, at least 80%, or at least 90% sequence identity or homology to the amino acid sequence set forth in SEQ ID NO: 31.
[0069] 13. The method of any one of embodiments 1 to 12, wherein the method comprises a step of cloning in a S-(hydroxymethyl) glutathione dehydrogenase (frmA).
[0070] 14. The method of embodiment 13, wherein the S-(hydroxymethyl) glutathione dehydrogenase converts S-hydroxymethylglutathione to S-formylgluathione.
[0071] 15. The method of embodiment 14, wherein the S-(hydroxymethyl) glutathione dehydrogenase has at least 70%, at least 80%, or at least 90% sequence identity or homology to an amino acid sequence chosen from SEQ ID NOs: 23-26.
[0072] 16. The method of any one of embodiments 1 to 15, wherein the method comprises a step of cloning in a S-formylglutathione hydrolase (frmB).
[0073] 17. The method of embodiment 16, wherein the S-formylglutathione hydrolase converts S-formylglutathione to formate.
[0074] 18. The method of embodiment 17, wherein the S-formylglutathione hydrolase has at least 70%, at least 80%, or at least 90% sequence identity or homology to an amino acid sequence chosen from SEQ ID NOs: 27-30.
[0075] 19. The method of any one of embodiments 16 to 18, wherein the expression of adh, frmA, and frmB allows conversion of methanol to formate.
[0076] 20. The method of any of the preceding embodiments, wherein the biosynthesis of the product is increased.
[0077] 21. The method of any of the preceding embodiments, wherein the product is chosen from salts and derivatives thereof of pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, 7-aminoheptanol, and 1,7-heptanediol.
[0078] 22. The method of any of the preceding embodiments, wherein the method is performed in a recombinant host.
[0079] 23. The method of embodiment 22, wherein the host is subjected to a cultivation strategy under aerobic, anaerobic, micro-aerobic, or mixed oxygen/denitrification cultivation conditions.
[0080] 24. The method of embodiment 22 or 23, wherein the host is cultured under conditions of nutrient limitation.
[0081] 25. The method according to any one of embodiments 22 to 24, wherein the host is retained using a ceramic hollow fiber membrane to maintain a high cell density during fermentation.
[0082] 26. The method of any one of embodiments 22 to 25, wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks.
[0083] 27. The method of embodiment 26, wherein the biological feedstock is, or derives from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste.
[0084] 28. The method of embodiment 26, wherein the non-biological feedstock is, or derives from, natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, benzoate, non-volatile residue (NVR) caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.
[0085] 29. The method of any one of embodiments 22 to 28, wherein the host is a prokaryote.
[0086] 30. The method of embodiment 29, wherein the prokaryote is from the genus Escherichia such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogenum, or Clostridium kluyveri; from the genus Corynebacteria such as Corynebacterium glutamicum from the genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans; from the genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida, or Pseudomonas oleavorans; from the genus Delftia such as Delftia acidovorans, from the genus Bacillus such as Bacillus subtillis; from the genus Lactobacillus such as Lactobacillus delbrueckii; from the genus Lactococcus such as Lactococcus lactis; or from the genus Rhodococcus such as Rhodococcus equi.
[0087] 31. The method of embodiment 30, wherein the host is Escherichia coli.
[0088] 32. The method of any one of embodiments 22 to 28, wherein the host is a eukaryote.
[0089] 33. The method of embodiment 32, wherein the eukaryote is from the genus Aspergillus such as Aspergillus niger; from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris; from the genus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkia such as Issathenkia orientalis; from the genus Debaryomyces such as Debaryomyces hansenii; from the genus Arxula such as Arxula adenoinivorans; or from the genus Kluyveromyces such as Kluyveromyces lactis.
[0090] 34. The method of embodiment 22, wherein the host's tolerance to high concentrations of a C7 building block is improved through continuous cultivation in a selective environment.
[0091] 35. The method of any one of embodiments 22 to 34, wherein the host comprises one or more of the following attenuated enzymes: polyhydroxyalkanoate synthase; an acetyl-CoA thioesterase; an acetyl-CoA specific .beta.-ketothiolase; a phosphotransacetylase forming acetate; an acetate kinase; a lactate dehydrogenase; a menaquinol-fumarate oxidoreductase; a 2-oxoacid decarboxylase producing isobutanol; an alcohol dehydrogenase forming ethanol; a triose phosphate isomerase; a pyruvate decarboxylase; a glucose-6-phosphate isomerase; a transhydrogenase dissipating the NADH or NADPH imbalance; an glutamate dehydrogenase dissipating the NADH or NADPH imbalance; a NADH/NADPH-utilizing glutamate dehydrogenase; a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase accepting C7 building blocks and central precursors as substrates; a glutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase.
[0092] 36. The method of any one of embodiments 22 to 34, wherein the host overexpresses one or more genes encoding: an acetyl-CoA synthetase; a 6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a formate dehydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosposphate dehydrogenase; a fructose 1,6 diphosphatase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase specific to the NADH or NADPH used to generate a co-factor imbalance; a methanol dehydrogenase; a formaldehyde dehydrogenase; a diamine transporter; a dicarboxylate transporter; an S-adenosylmethionine synthetase; and/or a multidrug transporter.
[0093] 37. A recombinant host for producing pimeloyl-ACP, wherein the host comprises at least one exogenous nucleic acid encoding (i) a formate-tetrahydrofolate ligase, (ii) a S-(hydroxymethyl) glutathione dehydrogenase, and an (iii) a S-formylglutathione hydrolase.
[0094] 38. The recombinant host of embodiment 37, wherein the host comprises a deletion of folI).
[0095] 39. The recombinant host of embodiment 37 or 38, wherein the host comprises a deletion of pflB and tdcE.
[0096] 40. The recombination host of any one of embodiments 37 to 39, wherein the host further comprises at least one exogenous nucleic acid encoding an alcohol dehydrogenase.
[0097] 41. The recombinant host of any one of embodiments 37 to 40, said host comprising at least one exogenous nucleic acid encoding one or more of a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a glutaconate CoA-transferase, a reversible succinyl-CoA ligase, an acetylating aldehyde dehydrogenase, or a carboxylate reductase, said host producing pimelic acid or pimelate semialdehyde.
[0098] 42. The recombinant host of any one of embodiments 37 to 41, said host comprising at least one exogenous nucleic acid encoding a .omega.-transaminase, wherein the host produces 7-aminoheptanoate.
[0099] 43. The recombinant host of any one of embodiments 37 to 42, said host further comprising one or more of a 4-hydroxybutyrate dehydrogenase, a S-hydroxypentanoate dehydrogenase, or a 6-hydroxyhexanoate dehydrogenase, said host producing 7-hydroxyheptanoic acid.
[0100] 44. The recombinant host of any one of embodiments 37 to 43, wherein the host further comprises at least one exogenous nucleic acid encoding a co-transaminase, a deacetylase, an N-acetyltransferase, or an alcohol dehydrogenase, said host producing heptamethylenediamine.
[0101] 45. The recombinant host of any one of embodiments 37 to 44, wherein the host further comprises at least one exogenous nucleic acid encoding a (i) carboxylate reductase enhanced by a phosphopantetheinyl transferase or (ii) an alcohol dehydrogenase, said host producing 1,7-heptanediol.
[0102] 46. A method for using methanol as a requirement for growth of a recombinant host.
[0103] 47. The method of embodiment 46, wherein the method comprises the step of deleting folD.
[0104] 48. The method of embodiment 46 or 47, wherein the method comprises the step of cloning in a formate-tetrahydrofolate ligase (fhs).
[0105] 49. The method of any one of embodiments 46 to 48, wherein the method comprises the step of deleting pflB and tdcE.
[0106] 50. The method of any one of embodiments 46 to 49, wherein the method comprises the step of cloning in an alcohol dehydrogenase (adh), a S-(hydroxymethyl) glutathione dehydrogenase (frmA), and a S-formylglutathione hydrolase.
[0107] 51. The method of any one of embodiments 46 to 50, wherein the recombinant host converts methanol to formate.
[0108] 52. The method of any one of embodiments 46 to 51, wherein formate is required for the synthesis of purines and initiator tRNA.
[0109] 53. A non-naturally occurring organism comprising at least one exogenous nucleic acid encoding at least one polypeptide having the activity of at least one enzyme depicted in any one of FIGS. 1 to 11.
[0110] 54. A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having formate-tetrahydrofolate ligase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having formate-tetrahydrofolate ligase activity is selected from: (a) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 18; (b) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 19; (c) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 20, (d) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 21; and (e) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 22.
[0111] 55. A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having alcohol dehydrogenase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having alcohol dehydrogenase is a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 31.
[0112] 56. A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having S-(hydroxymethyl) glutathione dehydrogenase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having S-(hydroxymethyl) glutathione dehydrogenase activity is selected from: (a) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 23; (b) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 24; (c) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 25; and (d) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 26.
[0113] 57. A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having S-formylglutathione hydrolase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having S-formylglutathione hydrolase activity is selected from: (a) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 27; (b) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 28; (c) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 29; and (d) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 30.
[0114] 58. A composition comprising the nucleic acid construct or expression vector of any one of embodiments 54 to 57.
[0115] 59. A bio-derived, bio-based, or fermentation-derived product, wherein said product comprises:
[0116] (i) a composition comprising at least one bio-derived, bio-based, or fermentation-derived compound produced or biosynthesized according to the methods of any one of embodiments 1 to 36 or any one of FIGS. 1-7, or any combination thereof,
[0117] (ii) a bio-derived, bio-based, or fermentation-derived polymer comprising the bio-derived, bio-based, or fermentation-derived composition or compound of (i), or any combination thereof,
[0118] (iii) a bio-derived, bio-based, or fermentation-derived resin comprising the bio-derived, bio-based, or fermentation-derived compound or bio-derived, bio-based, or fermentation-derived composition of (i) or any combination thereof or the bio-derived, bio-based, or fermentation-derived polymer of (ii) or any combination thereof,
[0119] (iv) a molded substance obtained by molding the bio-derived, bio-based, or fermentation-derived polymer of (ii) or the bio-derived, bio-based, or fermentation-derived resin of (iii), or any combination thereof,
[0120] (v) a bio-derived, bio-based, or fermentation-derived formulation comprising the bio-derived, bio-based, or fermentation-derived composition of (i), bio-derived, bio-based, or fermentation-derived compound of (i), bio-derived, bio-based, or fermentation-derived polymer of (ii), bio-derived, bio-based, or fermentation-derived resin of (iii), or bio-derived, bio-based, or fermentation-derived molded substance of (iv), or any combination thereof, or
[0121] (vi) a bio-derived, bio-based, or fermentation-derived semi-solid or a non-semi-solid stream, comprising the bio-derived, bio-based, or fermentation-derived composition of (i), bio-derived, bio-based, or fermentation-derived compound of (i), bio-derived, bio-based, or fermentation-derived polymer of (ii), bio-derived, bio-based, or fermentation-derived resin of (iii), bio-derived, bio-based, or fermentation-derived formulation of (v), or bio-derived, bio-based, or fermentation-derived molded substance of (iv), or any combination thereof.
[0122] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and are intended to provide further, non-limiting explanation of the disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0123] FIG. 1A is a schematic of an exemplary biochemical pathway leading to pimeloyl-ACP using NADPH-dependent enzymes and malonyl-ACP as a central precursor.
[0124] FIG. 1B is a schematic of an exemplary biochemical pathway leading to pimeloyl-CoA using NADPH-dependent enzymes and acetyl-CoA and malonyl-CoA as central precursors.
[0125] FIG. 1C is a schematic of an exemplary biochemical pathway leading to pimeloyl-CoA using NADH-dependent enzymes and acetyl-CoA and malonyl-CoA as centralprecursors.
[0126] FIG. 2 is a schematic of exemplary biochemical pathways leading to pimelate using pimeloyl-ACP, pimeloyl-CoA, or pimelate semialdehyde as central precursors.
[0127] FIG. 3 is a schematic of exemplary biochemical pathways leading to 7-aminoheptanoate using pimeloyl-CoA, pimelate, or pimelate semialdehyde as central precursors.
[0128] FIG. 4 is a schematic of exemplary biochemical pathways leading to heptamethylenediamine using 7-aminoheptanoate, 7-hydroxyheptanoate, or pimelate semialdehyde as central precursors.
[0129] FIG. 5 is a schematic of exemplary biochemical pathways leading to 7-hydroxyheptanoate using pimelate, pimeloyl-CoA, or pimelate semialdehyde as central precursors.
[0130] FIG. 6 is a schematic of an exemplary biochemical pathway leading to 1,7-heptanediol using 7-hydroxyheptanoate as a central precursor.
[0131] FIG. 7 shows a simplified schematic of an exemplary biochemical pathway for producing 7-AHA (7-AHA pathway) from the central carbon intermediate acetyl-CoA via the biotin metabolism pathway branching out from classic fatty acid metabolism.
[0132] FIG. 8A shows a schematic of an example cloning strategy for producing a recombinant host strain that can assimilate methanol produced during BioH enzyme activity into essential purines and initiator tRNAs. The left column provides the strains including the appropriate modifications (e.g., knockout genes and exogenous nucleic acids) for each strain. The right column provides the growth conditions for each strain. The remainder of the cloning strategy is represented in FIG. 8B.
[0133] FIG. 8B shows the continuation of the cloning strategy represented in FIG. 8A.
[0134] FIG. 9 shows a schematic of an additional cloning strategy for producing a recombinant host strain that can assimilate methanol produced during BioH enzyme activity into essential purines and initiator tRNAs.
[0135] FIG. 10A shows a schematic of the wild type (WT) tretrahydrofolate metabolic cycle (THF-MC). FolD converts 5,10-methylenetetrahydrofolate (5,10-CH2-THF) to N.sup.10-formyl tetrahydrofolate (N.sup.10-fTHF). N.sup.10-fTHF is a precursor in the pathway of purine nucleotide biosynthesis and formylation of the initiator tRNA.
[0136] FIG. 10B shows a schematic of a modified THF-MC where folD is eliminated and a formate-tetrahydrofolate ligase (fhs) is inserted. In this modified cycle foil) function is replaced by fhs.
[0137] FIG. 11 shows a schematic of a modified THF-MC where folD) function is replaced by a formate-tetrahydrofolate ligase (fhs) and formate acetyltransferase (pflB) and PFL-like enzyme TdcE (tdcE) are deleted, eliminating the source of formate. Introduction of a S-(hydroxymethyl) glutathione dehydrogenase (frmA) and a S-formylglutathione hydrolase (frmB) enables conversion of formaldehyde into formate.
DETAILED DESCRIPTION
[0138] The following detailed description and examples illustrate certain embodiments of the present disclosure. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure that are encompassed by its scope. Accordingly, the description of certain embodiments should not be deemed as limiting.
[0139] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[0140] This disclosure provides enzymes, non-natural pathways, cultivation strategies, feedstocks, host microorganisms and attenuations to the host's biochemical network, which generates a seven carbon chain aliphatic backbone from central precursors in which two terminal functional groups may be formed leading to the synthesis of pimelic acid, 7-aminoheptanoic acid (7-AHA), heptamethylenediamine or 1,7-heptanediol (referred to as "C7 building blocks" herein). As used herein, the term "central precursor" is used to denote any precursor or metabolite leading to the synthesis of a final product, such as, for example, a c C7 building block. The term "central metabolite" is used herein to denote a metabolite that is produced in all microorganisms to support growth.
[0141] The reactions of the pathways described herein can be performed in one or more cell (e.g., host cell) strains (a) naturally expressing one or more relevant enzymes, (b) genetically engineered to express one or more relevant enzymes, or (c) naturally expressing one or more relevant enzymes and genetically engineered to express one or more relevant enzymes. Alternatively, relevant enzymes can be extracted from any of the above types of host cells and used in a purified or semi-purified form. Extracted enzymes can optionally be immobilized to a solid substrate such as the floors and/or walls of appropriate reaction vessels. Moreover, such extracts include lysates (e.g., cell lysates) that can be used as sources of relevant enzymes. In the methods provided by the disclosure, all the steps can be performed in cells (e.g., host cells), all the steps can be performed using extracted enzymes, or some of the steps can be performed in cells and others can be performed using extracted enzymes.
[0142] Host microorganisms described herein can include endogenous pathways that can be manipulated such that one or more C7 building blocks can be produced. Host microorganisms can also include endogenous pathways that can be manipulated to regulate the biosynthesis of one or more C7 building blocks. In an endogenous pathway, the host microorganism naturally expresses all of the enzymes catalyzing the reactions within the pathway. A host microorganism containing an engineered pathway does not naturally express all of the enzymes catalyzing the reactions within the pathway but has been engineered such that all of the enzymes within the pathway are expressed in the host.
[0143] As used herein, a "bio-based product" is a product in which both the feedstock (e.g., sugars from sugar cane, corn, wood; biomass; waste streams from agricultural processes) and the conversion process to the product are biologically based (e.g., fermentation/enzymatic transformation involving a biological host/organism/enzyme). As used herein, a "bio-derived product" is a product in which one of the feedstocks (e.g., sugars from sugar cane, corn, wood; biomass; waste streams from agricultural processes) or the conversion process to the product is biologically based (e.g., fermentation/enzymatic transformation involving a biological host/organism/enzyme).
[0144] As used herein, a "fermentation-derived product" is a product produced by fermentation involving a biological host or organism.
[0145] The term "C7 building block" is used to denote a seven (7) carbon chain aliphatic backbone.
[0146] The term "exogenous" as used herein with reference to a nucleic acid (or a protein) and a host refers to a nucleic acid that does not occur in (and cannot be obtained from) a cell of that particular type as it is found in nature or a protein encoded by such a nucleic acid. Thus, a non-naturally-occurring nucleic acid is considered to be exogenous to a host once in the host. It is important to note that non-naturally-occurring nucleic acids can contain nucleic acid subsequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature. For example, a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a host cell once introduced into the host, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature. Thus, any vector, autonomously replicating plasmid, or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a whole does not exist in nature is considered to be non-naturally-occurring nucleic acid. It follows that genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally-occurring nucleic acid since they exist as separate molecules not found in nature. It also follows that any nucleic acid containing a promoter sequence and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally-occurring nucleic acid. A nucleic acid that is naturally-occurring can be exogenous to a particular host microorganism. For example, an entire chromosome isolated from a cell of yeast x is an exogenous nucleic acid with respect to a cell of yeast y once that chromosome is introduced into a cell of yeast y.
[0147] In contrast, the term "endogenous" as used herein with reference to a nucleic acid (e.g., a gene) or a protein and a host refers to a nucleic acid or protein that does occur in (and can be obtained from) that particular host as it is found in nature. Moreover, a cell "endogenously expressing" a nucleic acid (or protein) expresses that nucleic acid (or protein) as does a host of the same particular type as it is found in nature. Moreover, a host "endogenously producing" or that "endogenously produces" a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound as does a host of the same particular type as it is found in nature.
[0148] For example, depending on the host and the compounds produced by the host, one or more of the following enzymes may be expressed in the host in addition to a malonyl-ACP O-methyltransferase and a pimeloyl-ACP methyl ester methylesterase: a .beta.-ketoacyl-ACP synthase; a .beta.-ketothiolase; a 3-oxoacyl-ACP reductase; acetoacetyl-CoA reductase; a 3-hydroxyacyl-CoA dehydrogenase; a 3-hydroxybutyryl-CoA dehydrogenase; an enoyl-CoA hydratase; 3-hydroxyacyl-ACP dehydratase; an enoyl-ACP reductase; a trans-2-enoyl-CoA reductase; a thioesterase; a reversible CoA ligase; a CoA-transferase; an acetylating aldehyde dehydrogenase; a 6-oxohexanoate dehydrogenase; a 7-oxoheptanoate dehydrogenase; an aldehyde dehydrogenase; a carboxylate reductase; a .omega.-transaminase; a N-acetyltransferase; an alcohol dehydrogenase; a deacetylase a 6-hydroxyhexanoate dehydrogenase; a 5-hydroxypentanoate dehydrogenase; a 4-hydroxybutyrate dehydrogenase; a formate-tetrahydrofolate ligase; a S-(hydroxymethyl) glutathione dehydrogenase; or a S-formylglutathione hydrolase. In recombinant hosts expressing a carboxylate reductase, a phosphopantetheinyl transferase also can be expressed as it enhances activity of the carboxylate reductase.
[0149] For example, a recombinant host can include at least one exogenous nucleic acid encoding at least one polypeptide having the activity of: (i) a malonyl-ACP O-methyltransferase, (ii) a .beta.-ketoacyl-ACP synthase or a .beta.-ketothiolase, (iii) a 3-oxoacyl-ACP reductase, acetoacetyl-CoA reductase, a 3-hydroxyacyl-CoA dehydrogenase, or a 3-hydroxybutyryl-CoA dehydrogenase, (iv) an enoyl-CoA hydratase or 3-hydroxyacyl-ACP dehydratase, (v) an enoyl-ACP reductase or a trans-2-enoyl-CoA reductase and produce pimeloyl-ACP or pimeloyl-CoA.
[0150] For example, a recombinant host, or non-naturally occurring organism, can include at least one exogenous nucleic acid encoding at least one polypeptide having the activity of at least one enzyme depicted in any of FIGS. 1 to 11. For example, the organism can include at least one exogenous nucleic acid encoding at least one polypeptide having the activity of an alcohol dehydrogenase, a formate-tetrahydrofolate ligase, a S-(hydroxymethyl) glutathione dehydrogenase, or a S-formylglutathione hydrolase. See, e.g., FIGS. 8-11.
[0151] Such recombinant hosts producing pimeloyl-ACP or pimeloyl-CoA further can include at least one exogenous nucleic acid encoding at least one polypeptide having the activity of a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a glutaconate CoA-transferase, a reversible succinyl-CoA ligase, an acetylating aldehyde dehydrogenase, or a carboxylate reductase and produce pimelic acid or pimelate semialdehyde. For example, a recombinant host producing pimeloyl-ACP or pimeloyl-CoA further can include a thioesterase, a reversible Co-ligase (e.g., a reversible succinyl-CoA ligase), or a CoA transferase (e.g., a glutaconate CoA-transferase) and produce pimelic acid. For example, a recombinant host producing pimeloyl-CoA further can include an acetylating aldehyde dehydrogenase and produce pimelate semialdehyde. For example, a recombinant host producing pimelate further can include a carboxylate reductase and produce pimelate semialdehyde.
[0152] A recombinant host producing pimelate semialdehyde further can include at least one exogenous nucleic acid encoding at least one polypeptide having the activity of a w-transaminase and produce 7-aminoheptanoate. In some embodiments, a recombinant host producing pimeloyl-CoA includes a carboxylate reductase and a .omega.-transaminase to produce 7-aminoheptanoate.
[0153] A recombinant host producing pimelate or pimelate semialdehyde further can include at least one exogenous nucleic acid encoding at least one polypeptide having the activity of a 6-hydroxyhexanoate dehydrogenase, a S-hydroxypentanoate dehydrogenase, or a 4-hydroxybutyrate dehydrogenase, and produce 7-hydroxyheptanoic acid. In some embodiments, a recombinant host producing pimeloyl-CoA includes an acetylating aldehyde dehydrogenase and a 6-hydroxyhexanoate dehydrogenase, a S-hydroxypentanoate dehydrogenase, or a 4-hydroxybutyrate dehydrogenase to produce 7-hydroxyheptanoate. In some embodiments, a recombinant host producing pimelate includes a carboxylate reductase and a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, or a 4-hydroxybutyrate dehydrogenase to produce 7-hydroxyheptanoate.
[0154] A recombinant host producing 7-aminoheptanoate, 7-hydroxyheptanoate, or pimelate semialdehyde further can include at least one exogenous nucleic acid encoding at least one polypeptide having the activity of a .omega.-transaminase, a deacetylase, a N-acetyltransferase, or an alcohol dehydrogenase, and produce heptamethylenediamine. For example, a recombinant host producing 7-hydroxyheptanoate can include a carboxylate reductase with an optional phosphopantetheine transferase enhancer, a .omega.-transaminase, and an alcohol dehydrogenase.
[0155] Within an engineered pathway, the enzymes can be from a single source, i.e., from one species, or can be from multiple sources, i.e., different species. Nucleic acids encoding the enzymes described herein have been identified from various organisms and are readily available in publicly available databases such as GenBank or EMBL.
[0156] In some embodiments, the host microorganism's endogenous biochemical network is attenuated or augmented to (1) ensure the intracellular availability of acetyl-CoA and malonyl-CoA, (2) create an NAD.sup.+ imbalance that may only be balanced via the formation of a C7 building block, (3) prevent degradation of central metabolites and/or central precursors leading to and including C7 building blocks, (4) ensure efficient efflux from the cell, and/or (5) channel increased flux through the pathway leading to the C7 building block product(s).
Channeling Increased Flux Through C7 Building Block Biosynthesis Pathways
[0157] An exemplary pathway (7-AHA pathway) for producing 7-AHA, a C7 building block, is depicted in FIG. 7. The 7-AHA pathway produces 7-AHA from the central carbon metabolism intermediate acetyl-CoA via the biotin synthesis pathway. The 7-AHA pathway branches out from the biotin pathway at the common intermediate pimelyl-ACP. BioH removes the methyl group of pimelyl-ACP methyl ester, producing pimelyl-ACP. The byproduct of this reaction is methanol, which is excreted by the host microorganism.
[0158] Metabolism can often be seen to be resistant to stimulating biosynthesis by expressing or over-expressing pathway enzymes. Control of the flux soon passes elsewhere, and metabolite concentrations rise. A classic metabolic engineering strategy to mitigate this risk is to exploit the natural homeostatic mechanisms of the cell: pull the products out, rather than push precursors toward them. The metabolic network will tend to replace the material that is removed. Feedback loops in the metabolism transfer control from the `supply` steps near the beginning to the `demand` reactions after the feedback loop.
[0159] A promising strategy to achieve this `flux pulling` mechanism is to couple the growth of the cell to the formation of a growth critical intermediate produced as part of the biosynthetic pathway. In most cases, this ties growth to yield, titre, and productivity, thus regulating the biosynthesis of the product. This can be achieved by linking the byproduct of methanol, through formate, to the protein synthesis machinery through the tetrahydrofolate metabolic cycle (THF-MC), or a modified THF-MC, and hence growth of the microorganism host. A schematic of the wild-type THF-MC is shown in FIG. 10A.
[0160] Work by Sah et al. (Sah et al., J. Bacteriol., 2015, 197(4), 717-726) investigated the physiological and functional importance of FolD and formyltetrahydrofolate synthetase (Fhs) in the THF-MC. As noted by Sah et al., the enzymes that catalyse interconversions of the above pathway intermediates are highly conserved across the three domains of life (Smith et al., J. Bacteriol., 200, 50, 43-53; Bult et al., Science, 1996, 273, 1058-1073; Slesarev et al., Proc. Natl. Acad. Sci. USA, 2002, 99, 4644-4649; Buchenau et al., Arch. Microbiol., 2004, 182, 313-325; Maeder et al., J. Bacteriol., 2006, 188, 7922-7931). While serine hydroxymethyltransferase (GlyA) catalyses the reversible reaction of conversion of serine and THF to glycine and 5,10-methylene-tetrahydrofolate (5,10-CH2-THF), FolD carries out the conversion of 5,10-methenyltetrahydrofolate (5,10-CH1-THF) to N.sup.10-formyltetrahydrofolate (N.sup.10-fTHF) (see FIG. 10A). N.sup.10-fTHF is important for the de novo pathway of purine nucleotide biosynthesis and formylation of the initiator tRNA (tRNAfMet) to initiate protein synthesis in eubacteria and eukaryotic organelles (Pino et al., Mol. Microbiol., 2010, 76, 706-718). Another enzyme, Fhs, can also synthesize N.sup.10-fTHF by utilizing THF, formate, and ATP. The dual scheme of N.sup.10-fTHF synthesis is conserved in eukaryotes and some archaea (Maeder et al., J. Bacteriol., 2006, 188, 7922-7931).
[0161] Many eukaryotic organisms possess FolD (Pino et al., Mol. Microbiol., 2010, 76, 706-718; Paukert et al., J. Biol. Chem., 1976, 251, 5104-5111). Amongst eubacteria, all organisms possess FolD, but some possess both FolD and Fhs (Paukert et al., Biochem. Biophys. Res. Commun., 1977, 77, 147-154). The advantage of possessing Fhs in addition to FolD was demonstrated by Sah et al. (Sah et al., J. Bacteriol., 2015, 197(4), 717-726). In the presence of formate and in anaerobic conditions of growth, predominant synthesis of N.sup.10-fTHF may occur via Fhs. In an jhs-supported E. coli folD deletion (.DELTA.folD+fhs) model, the enzyme 2-ketobutyrate formate-lyase/pyruvate formate-lyase (pflBtdcE) can produce N.sup.10-fTHF in the presence of formate (see FIG. 10B). N.sup.10-fTHF may then be converted to the other one-carbon metabolism intermediates (Whitehead et al., J. Bacteriol., 1988, 170, 9995-997).
[0162] According to this disclosure, when folD is knocked out and fhs is knocked in or expressed from an exogenously-derived nucleic acid, the microorganism strain should be able to grow after the redirection of the methanol oxidation into the modified THF-MC pathway as the former will act as a source of formate (FIG. 10B). Methanol will therefore become a growth critical intermediate by making it necessary for the synthesis of purine nucleotides, thymidylate, and initiator tRNAs. Accordingly, the cells reliant on methanol will revert to the production of more methanol, thereby pulling flux down the 7-AHA pathway and leading to high 7-AHA yield, titre, and productivity.
[0163] The described system may lead to increased yield, titre, and productivity through multiple modes. The described system mitigates the risk of carbon wastage in the system, which results in lower yield. By utilizing the carbon from the methanol byproduct of the BioH enzyme in the 7-AHA pathway, the described system reduces carbon wastage from typical methanol efflux. The described system may also mitigate risk of potential methanol toxicity. Therefore, since the host is dependent on methanol for growth, the accumulation of methanol, which is potentially deleterious to host viability, is decreased. Thus, decreasing methanol accumulation may also mitigate the risk of low 7-AHA yield. Furthermore, the described system may reduce production costs by mitigating the need for methanol removal during downstream processing of the product. As such, the described system has a potential improvement of about 15 to about 22% of the maximum theoretical yield in 7-AHA production, for example.
[0164] Thus, by increasing flux through the C7 building block biosynthesis pathway (e.g., the 7-AHA pathway) by requiring methanol for host growth, the biosynthesis of C7 building blocks is regulated.
[0165] The attenuation of certain enzymes may contribute to the regulation of biosynthesis of C7 building blocks. For example, a host may have attenuation of a biofunctional protein, classified, for example, under EC 1.5.1.5 and/or under EC 3.5.4.9. For example, and as described above, a host microorganism may have deletion, or knock out, of a bifunctional protein FolD (folD) (see UniProt Accession No. P24186, SEQ ID NO: 32; UniProt Accession No. B7LJI7, SEQ ID NO: 33; UniProt Accession No. Q32JK7, SEQ ID NO: 34).
[0166] A host microorganism may also have attenuation of a formate acetyltransferase and/or a pyruvate formate-lyase (PFL)-like enzyme, both of which may be classified under, for example, EC 2.3.1.54. For example, the host may have a deletion, or knock out, of formate acetyltransferase 1 (pflB) (see UniProt Accession No. P09373, SEQ ID NO: 35) and PFL-like enzyme TdcE (tdcE) (see UniProt Accession No. P42632, SEQ ID NO: 36). Formate acetyltransferase 1 synthesizes formate from pyruvate, and PFL-like enzyme TdcE catalyzes the cleavage of 2-ketobutyrate to propionyl-CoA and formate. By removing these two genes, the source of formate is removed. See FIGS. 10B and 11.
[0167] The host organism may also express one or more genes encoded in one or more exogenous nucleic acids, or genes may be knocked in to the host organism. For example, as described above, a host organism may express a formyltetrahydrofolate synthetase (Fhs), which is interchangeably referred to as a formate-tetrahydrofolate ligase (Fths), and is classified under, for example, EC 6.3.4.3. For example, the host may express one or more Fhs proteins set forth in SEQ ID NOs: 18-22 (see UniProt Accession Nos. Q07064. A8MIN 1, P131419, Q5XZD9, and Q251P8, respectively). A formyltetrahydrofolate synthetase may be knocked in or expressed from an exogenously-derived nucleic acid.
[0168] The host organism may also express an alcohol dehydrogenase (Adh), classified under, for example, EC 1.1.1.-, from one or more exogenous nucleic acids, or the alcohol dehydrogenase may be knocked in to the host organism. For example, the alcohol dehydrogenase may be a methanol dehydrogenase (Mdh), classified under, for example, EC 1.1.1.37. For example, the host may express an alcohol dehydrogenase as set forth in SEQ ID NO: 31 (see UniProt Accession No. Q46856). An alcohol dehydrogenase may then catalyze the conversion of methanol to formaldehyde. Formaldehyde can then be oxidized into formate. See FIG. 11.
[0169] To aid in oxidation of formaldehyde to formate, the host cell may express a S-(hydroxymethyl) glutathione dehydrogenase (e.g., FrmA), classified under, for example, EC 1.1.1.284, and/or a S-formylglutathione hydrolase (e.g., FrmB), classified under, for example, EC 3.1.2.12, from one or more exogenous nucleic acids, or may be knocked in to the host organism. For example, a host may express a FrmA protein as set forth in SEQ ID NOs: 23-26 (see UniProt Accession Nos. P25437. Q3Z550, A0A0M7MPD4, and W1AV69, respectively) and/or a FrmB protein as set forth in SEQ ID NOs: 27-30 (see UniProt Accession Nos. P51025, Q3Z551, A0A0M9J3Q3, and W1ATJ0, respectively). After formaldehyde spontaneously converts to H-hydroxymethylglutathione, S-(hydroxymethyl) glutathione dehydrogenase converts H-hydroxymethylglutathione to S-formylglutathione. S-formylglutathione hydrolase then converts S-formylglutathione to formate. See FIG. 11.
[0170] The described system may also lead to using methanol for cell growth. As described above, the modified THF-MC pathway requires methanol as described above, the engineered pathway requires methanol for the synthesis of purine nucleotides, thymidylate, and initiator tRNAs. As such, in this system, methanol is required for cell growth. As such, in the described system, cell growth is reliant on methanol.
Enzymes
[0171] Any of the enzymes described herein that can be used for production of one or more C7 building blocks, or in the regulation of the biosynthesis of C7 building blocks, can have at least 50%, at least 600/o, or at least 70% sequence identity or homology (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of the corresponding wild-type enzyme. It will be appreciated that the sequence identity can be determined on the basis of the mature enzyme (e.g., with any signal sequence removed).
[0172] A polypeptide having a certain percent (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of sequence identity with another sequence means that, when aligned, that percentage of bases or amino acid residues are the same in comparing the two sequences.
[0173] The percent identity and homology between two amino acid sequences can be determined as follows. First, the amino acid sequences are aligned using the BLAST 2 Sequences (Bl2seq) program from the stand-alone version of BLASTZ containing BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from www.fr.com/blast/ or the U.S. government's National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov). Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ. Bl2seq performs a comparison between two amino acid sequences using the BLASTP algorithm. To compare two amino acid sequences, the options of Bl2seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq-i c:\seq1.txt-j c:\seq2.txt-p blastp-o c:\output.txt. If the two compared sequences share homology (identity), then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology (identity), then the designated output file will not present aligned sequences. Similar procedures can be following for nucleic acid sequences except that blastn is used.
[0174] Once aligned, the number of matches is determined by counting the number of positions where an identical amino acid residue is presented in both sequences. The percent identity is determined by dividing the number of matches by the length of the full-length polypeptide amino acid sequence followed by multiplying the resulting value by 100. It is noted that the percent identity value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It also is noted that the length value will always be an integer.
[0175] When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution and this process results in "sequence homology" of, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 1988, 4, 11-17, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Cailf., USA). This alignment and the percent homology or identity can be determined using any suitable software program known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. (eds.) 1987, Supplement 30, section 7.7.18). Such programs may include the GCG Pileup program, FASTA (Pearson et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 2444-2448), and BLAST (BLAST Manual, Altschul et al., Nat'l Cent. Biotechnol. Inf., Nat'l Lib. Med. (NCIB NLM NIH), Bethesda, Md., and Altschul et al., NAR, 1997, 25, 3389-3402). Another alignment program is ALIGN Plus (Scientific and Educational Software, Pa.), using default parameters. Another sequence software program that finds use is the TFASTA Data Searching Program available in the Sequence Software Package Version 6.0 (Genetics Computer Group, University of Wisconsin, Madison, Wis.).
[0176] A conservative substitution is a substitution of one amino acid for another with similar characteristics. Conservative substitutions include substitutions within the following groups: valine, alanine, and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic, or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a non-conservative substitution is a substitution of one amino acid for another with dissimilar characteristics.
[0177] For example, a thioesterase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of an Escherichia coli thioesterase encoded by tesB (see GenBank Accession No. AAA24665.1, SEQ ID NO: 1).
[0178] For example, a carboxylate reductase described herein can have at least 70%/sequence identity or homology (e.g., at least 75%, at least 80%, at least 85%/0, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%/, or 100%) to the amino acid sequence of a Mycobacterium marinum (see Genbank Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmalis (see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), a Segniliparus rugosus (see Genbank Accession No. EFV11917.1, SEQ ID NO: 4), a Mycobacterium smegmatis (see Genbank Accession No. ABK75684.1, SEQ ID NO: 5), a Mycobacterium massiliense (see Genbank Accession No. EIV11143.1, SEQ ID NO: 6), or a Segniliparus rotundus (see Genbank Accession No. ADG98140.1, SEQ ID NO: 7) carboxylate reductase.
[0179] For example, a .omega.-transaminase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95/o, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of a Chromobacterium violaceum (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 8), a Pseudomonas aeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO: 9), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ ID NO: 10), a Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1, SEQ ID NO: 11), an Escherichia coli (see RefSeq Accession No. NP_417544.5, SEQ ID NO: 12), or a Vibrio fluvialis (see Genbank Accession No. AEA39183.1, SEQ ID NO: 13) .omega.-transaminase. Some of these a transaminases are diamine .omega.-transaminases.
[0180] For example, a phosphopantetheinyl transferase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Bacillus subtilis phosphopantetheinyl transferase (see RefSeq Accession No. WP_003234549.1, SEQ ID NO: 14) or a Nocardia sp. NRRL 5646 phosphopantetheinyl transferase (see Genbank Accession No. ABI83656.1, SEQ ID NO: 15).
[0181] For example, a malonyl-CoA methyltransferase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, 800/%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Bacillus cereus malonyl-CoA methyltransferase (see GenBank Accession No. AAS43086.1, SEQ ID NO: 16).
[0182] For example, a pimeloyl-ACP methyl ester esterase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichia coli pimeloyl-ACP methyl ester esterase (see GenBank Accession No. AAC76437.1, SEQ ID NO: 17).
[0183] For example, a formate-tetrahydrofolate ligase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of a Clostridium cylindrosporum (see UniProt Accession No. Q07064, SEQ ID NO: 18), an Alkaliphilus oremlandii (see UniProt Accession No. A8MINI, SEQ ID NO: 19), a Clostridium acidurici (see UniProt Accession No. P13419, SEQ ID NO: 20), a Eubacterium acidaminophilum (see UniProt Accession No. Q5XZD9, SEQ ID NO: 21), or a Desulfitobacterium hafniense (see UniProt Accession No. Q251P8, SEQ ID NO: 22) formate-tetrahydrofolate ligase.
[0184] For example, a S-(hydroxymethyl) glutathione dehydrogenase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of a Escherichia coli (see UniProt Accession No. P25437, SEQ ID NO: 23), a Shigella sonnei (see UniProt Accession No. Q3Z550, SEQ ID NO: 24), an Achromaobacter sp. (see UniProt Accession No. A0A0M7MPD4, SEQ ID NO: 25), or a Klebsiella pneumoniae (see UniProt Accession No. W1AV69, SEQ ID NO: 26) S-(hydroxymethyl) glutathione dehydrogenase.
[0185] For example, a S-formylgltuathione hydrolase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of an Escherichia coli (see Uni Prot Accession No. P51025, SEQ ID NO: 27), a Shigella sonnei (see UniProt Accession No. Q3Z551, SEQ ID NO: 28), an Achromobacter sp. (see UniProt Accession No. A0A0M9J3Q3, SEQ ID NO: 29), or a Klebsiella pneumoniae (see UniProt Accession No. W1ATJ0, SEQ ID NO: 30) S-formylglutathione hydrolase.
[0186] For example, an alcohol dehydrogenase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of an Escherichia coli (see UniProt Accession No. Q46856, SEQ ID NO: 31) alcohol dehydrogenase.
[0187] It will be appreciated that a number of nucleic acids can encode a polypeptide having a particular amino acid sequence. The degeneracy of the genetic code is well known to the art; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. For example, codons in the coding sequence for a given enzyme can be modified such that optimal expression in a particular species (e.g., bacterium or fungus) is obtained, using appropriate codon bias tables for that species.
[0188] Functional fragments of any of the enzymes described herein can also be used in the methods of the disclosure. The term "functional fragment" as used herein refers to a peptide fragment of a protein that has at least 25% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%; 98%; 99%; 100%; or even greater than 100%) of the activity of the corresponding mature, full-length, wild-type protein. The functional fragment can generally, but not always, be comprised of a continuous region of the protein, wherein the region has functional activity.
[0189] This disclosure also provides (i) functional variants of the enzymes used in the methods of the disclosure and (ii) functional variants of the functional fragments described above. Functional variants of the enzymes and functional fragments can contain additions, deletions, or substitutions relative to the corresponding wild-type sequences. Enzymes with substitutions will generally have not more than 50 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) amino acid substitutions (e.g., conservative substitutions). This applies to any of the enzymes described herein and functional fragments. A conservative substitution is a substitution of one amino acid for another with similar characteristics. Conservative substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a nonconservative substitution is a substitution of one amino acid for another with dissimilar characteristics.
[0190] Deletion variants can lack one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid segments (of two or more amino acids) or non-contiguous single amino acids. Additions (addition variants) include fusion proteins containing: (a) any of the enzymes described herein or a fragment thereof; and (b) internal or terminal (C or N) irrelevant or heterologous amino acid sequences. In the context of such fusion proteins, the term "heterologous amino acid sequences" refers to an amino acid sequence other than (a). A heterologous sequence can be, for example a sequence used for purification of the recombinant protein (e.g., FLAG, polyhistidine (e.g., hexahistidine (SEQ ID NO: 37)), hemagglutinin (HA), glutathione-S-transferase (GST), or maltosebinding protein (MBP)). Heterologous sequences also can be proteins useful as detectable markers, for example, luciferase, green fluorescent protein (GFP), or chloramphenicol acetyl transferase (CAT). In some embodiments, the fusion protein contains a signal sequence from another protein. In certain host cells (e.g., yeast host cells), expression and/or secretion of the target protein can be increased through use of a heterologous signal sequence. In some embodiments, the fusion protein can contain a carrier (e.g., KLH) useful, e.g., in eliciting an immune response for antibody generation) or ER or Golgi apparatus retention signals. Heterologous sequences can be of varying length and in some cases can be a longer sequences than the full-length target proteins to which the heterologous sequences are attached.
[0191] Engineered hosts can naturally express none or some (e.g., one or more, two or more, three or more, four or more, five or more, or six or more) of the enzymes of the pathways described herein. Thus, a pathway within an engineered host can include all exogenous enzymes, or can include both endogenous and exogenous enzymes. Endogenous genes of the engineered hosts also can be disrupted to prevent the formation of undesirable metabolites or prevent the loss of intermediates in the pathway through other enzymes acting on such intermediates. Engineered hosts can be referred to as recombinant hosts or recombinant host cells. As described herein recombinant hosts can include nucleic acids encoding one or more of a formate-tetrahydrofolate ligase, a S-(hydroxymethyl) glutathione dehydrogenase, a S-formylglutathione hydrolase, a methyltransferase, a synthase, .beta.-ketothiolase, a dehydratase, a hydratase, a dehydrogenase, a methylesterase, a thioesterase, a reversible CoA-ligase, a CoA-transferase, a reductase, deacetylase, N-acetyltransferase or a .omega.-transaminase as described in more detail below.
[0192] For example, the polynucleotides encoding the described polypeptides, and variants thereof, with the respective enzymatic activity, can be incorporated in a nucleic acid construct or vector. In some embodiments, the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide.
[0193] In addition, the production of one or more C7 building blocks can be performed in vitro using the isolated enzymes described herein, using a lysate (e.g., a cell lysate) from a host microorganism as a source of the enzymes, or using a plurality of lysates from different host microorganisms as the source of the enzymes.
Enzymes Generating the C7 Aliphatic Backbone for Conversion to C7 Building Blocks
[0194] As depicted in FIG. 1A, FIG. 1B, and FIG. 1C, a C7 aliphatic backbone for conversion to one or more C7 building blocks can be formed from malonyl-ACP, or acetyl-CoA and malonyl-CoA, via two cycles of methyl-ester shielded carbon chain elongation associated with biotin synthesis using either NADH or NADPH dependent enzymes.
[0195] In some embodiments, a methyl ester shielded carbon chain elongation associated with biotin biosynthesis route comprises using a malonyl-ACP O-methyltransferase to form a malonyl-ACP methyl ester, and then performing two cycles of carbon chain elongation using a .beta.-ketoacyl-ACP synthase, a 3-oxoacyl-ACP reductase, a 3-hydroxyacyl-ACP dehydratase, and an enoyl-ACP reductase. A pimeloyl-ACP methyl ester esterase can be used to cleave the resulting pimeloyl-ACP methyl ester.
[0196] In some embodiments, a methyl ester shielded carbon chain elongation route comprises using a malonyl-ACP O-methyltransferase to form a malonyl-CoA methyl ester, and then performing two cycles of carbon chain elongation using (i) a .beta.-ketothiolase or a .beta.-ketoacyl-ACP synthase, (ii) an acetoacetyl-CoA reductase, a 3-oxoacyl-ACP reductase, or a 3-hydroxybutyryl-CoA dehydrogenase, (iii) an enoyl-CoA hydratase, and (iv) a trans-2-enoyl-CoA reductase. A pimeloyl-ACP methyl ester esterase can be used to cleave the resulting pimeloyl-CoA methyl ester.
[0197] In some embodiments, a methyltransferase can be a malonyl-ACP O-methyltransferase classified, for example, under EC 2.1.1.197, such as, for example, the gene product of bioC from Bacillus cereus (see Genbank Accession No. AAS43086.1, SEQ ID NO: 16) (see, e.g., Lin, 2012, Biotin Synthesis in Escherichia coli, Ph.D. Dissertation, University of Illinois at Urbana-Champaign).
[0198] In some embodiments, a .beta.-ketothiolase may be classified, for example, under EC 2.3.1.16, such as, for example, the gene product of bktB. The .beta.-kelothiolase encoded by bktB from Cupriavidus necator accepts propanoyl-CoA and pentanoyl-CoA as substrates, forming the CoA-activated C7 aliphatic backbone (see, e.g., Haywood et al., FEMS Microbiology Letters, 1988, 52:91-96; Slater et al., J. Bacteriol., 1998, 180(8):1979-1987).
[0199] In some embodiments, a .beta.-ketoacyl-ACP synthase may be classified, for example, under EC 2.3.1.- (e.g., EC 2.3.1.41, EC 2.3.1.179, or EC 2.3.1.180), such as, for example, the gene product of fabB, fabF, or fabH.
[0200] In some embodiments, a 3-hydroxyacyl-CoA dehydrogenase may be classified, for example, under EC 1.1.1.35, such as, for example, the gene product of fadB, or classified under EC 1.1.1.157, such as, for example, the gene product of hbd (can be referred to as a 3-hydroxybutyryl-CoA dehydrogenase), or classified under EC 1.1.1.36, such as, for example, the gene product of phaB (see, e.g., Liu & Chen, Appl. Microbiol. Biotechnol., 2007, 76(5), 1153-1159; Shen et al., Appl. Environ. Microbiol., 2011, 77(9), 2905-2915; or Budde et al., J. Bacteriol., 2010, 192(20), 5319-5328).
[0201] In some embodiments, a 3-oxoacyl-CoA reductase may be, for example, classified under EC 1.1.1.100, such as, for example, the gene product of fabG (Budde et al., 2010, supra; Nomura et al., Appl. Environ. Microbiol., 2005, 71(8), 4297-4306).
[0202] In some embodiments, an enoyl-CoA hydratase may be classified, for example, under EC 4.2.1.17, such as the gene product of crt, or classified under EC 4.2.1.119, such as, for example, the gene product of phaJ (Shen et al., 2011, supra; or Fukui et al., J. Bacteriol., 1998, 180(3), 667-673).
[0203] In some embodiments, an enoyl-ACP dehydratase such as a 3-hydroxyacyl-ACP dehydratase may be classified, for example, under EC 4.2.1.59, such as, for example, the gene product of fabZ.
[0204] In some embodiments, a trans-2-enoyl-CoA reductase may be classified, for example, under EC 1.3.1.- (e.g., EC 1.3.1.38, EC 1.3.1.8, EC 1.3.1.44), such as, for example, the gene product of ter (Nishimaki et al., J. Biochem., 1984, 95, 1315-1321; Shen et al., 2011, supra) or tdter (Bond-Watts et al., Biochemistry, 2012, 51, 6827-6837).
[0205] In some embodiments, an enoyl-ACP reductase may be classified, for example, under EC 1.3.1.10, such as, for example, the gene product of fabL, or EC 1.3.1.9, such as, for example, the gene product of fabI.
[0206] In some embodiments, a pimeloyl-ACP methyl ester eslerase may be classified, for example, under EC 3.1.1.85, such as, for example, the gene product of bioH from E. coli. See Genbank Accession No. AAC76437.1 (SEQ ID NO: 17).
Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis of C7 Building Blocks
[0207] As depicted in FIG. 2, a terminal carboxyl group can be enzymatically formed using a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a CoA-transferase, or a reversible CoA-ligase.
[0208] In some embodiments, the second terminal carboxyl group leading to the synthesis of a C7 building block is enzymatically formed by a thioesterase classified, for example, under EC 3.1.2.-, such as, for example, the gene product of yciA, tesB (Genbank Accession No. AAA24665.1, SEQ ID NO: 1) or acot13 (see, e.g., Cantu et al., Protein Science, 2010, 19, 1281-1295; Zhuang et al., Biochemistry, 2008, 47(9), 2789-2796; or Naggert et al., J. Biol. Chem., 1991, 266(17), 11044-11050).
[0209] In some embodiments, the second terminal carboxyl group leading to the synthesis of a C7 building block is enzymatically formed by an acyl-ACP thioesterase classified, for example, under EC 3.1.2.-, such as, for example, the gene product of fatB or tesA. The acyl-ACP thioesterases encoded by Genbank Accession Nos. ABJ63754.1 and CCC78182.1 have C6-C8 chain length specificity (Jing et al., 2011, BMC Biochemistry, 12(44)).
[0210] In some embodiments, the second terminal carboxyl group leading to the synthesis of pimelic acid is enzymatically formed by an aldehyde dehydrogenase classified, for example, under EC 1.2.1.3 (see, for example, Guerrillot & Vandecasteele, Eur. J. Biochem., 1977, 81, 185-192).
[0211] In some embodiments, the second terminal carboxyl group leading to the synthesis of pimelic acid is enzymatically formed by a dehydrogenase classified, for example, under EC 1.2.1.-, such as, for example, a 6-orohexanoate dehydrogenase (e.g., the gene product of chnE from Acinetobacter sp.) or a 7-oxoheptanoate dehydrogenase (e.g., such as the gene product of thnG from Sphingomonas macrogolitabida). See, for example, Iwaki et al., Appl. Environ. Microbiol., 1999, 65(11), 5158-5162; or Lopez-Sanchez et al., Appl. Environ. Microbiol., 2010, 76(1), 110-118. For example, a 6-oxohexanoate dehydrogenase can be classified, for example, under EC 1.2.1.63. For example, a 7-oxoheptanoate dehydrogenase can be classified, for example, under EC 1.2.1.-.
[0212] In some embodiments, the second terminal carboxyl group leading to the synthesis of pimelic acid is enzymatically formed by a CoA-transferase (e.g., a glutaconate CoA-transferase) classified, for example, under EC 2.8.3.12, such as, for example, a CoA-transferase from Acidaminococcus fermentans. See, for example, Buckel et al., 1981, Eur. J. Biochem., 118:315-321.
[0213] In some embodiments, the second terminal carboxyl group leading to the synthesis of pimelic acid is enzymatically formed by a reversible CoA-ligase (e.g., a succinate-CoA ligase) classified, for example, under EC 6.2.1.5, such as, for example, a reversible CoA-ligase from Thermococcus kodakaraensis. See, for example, Shikata et al., 2007, J. Biol. Chem., 282(37):26963-26970.
Enzymes Generating the Terminal Amine Groups in the Biosynthesis of C7 Building Blocks
[0214] As depicted in FIG. 3 and FIG. 4, terminal amine groups can be enzymatically formed using a .omega.-transaminase or a deacetylase.
[0215] In some embodiments, the first or second terminal amine group leading to the synthesis of 7-aminoheptanoic acid is enzymatically formed by a .omega.-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82, such as, for example, that obtained from Chromobacterium violaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO: 8), Pseudomonas aeruginosa (Genbank Accession No. AAG08191.1, SEQ ID NO: 9), Pseudomonas syringae (Genbank Accession No. AAY39893.1, SEQ ID NO: 10), Rhodobacter sphaeroides (Genbank Accession No. ABA81135.1, SEQ ID NO: 11), Escherichia coli (RefSeq Accession No. NP_417544.5, SEQ ID NO: 12), Vibrio Fluvialis (Genbank Accession No. AEA39183.1, SEQ ID NO: 13), Streptomyces griseus, or Clostridium viride. Some of these .omega.-transaminases are diamine .omega.-transaminases (e.g., SEQ ID NO: 12). For example, the .omega.-transaminases classified, for example, under EC 2.6.1.29 or EC 2.6.1.82 may be diamine .omega.-transaminases.
[0216] The reversible .omega.-transaminase from Chromobacterium violaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO: 8) has demonstrated analogous activity accepting 6-aminohexanoic acid as amino donor, thus forming the first terminal amine group in adipate semialdehyde (Kaulmann et al., Enzyme and Microbial Technology, 2007, 41, 628-637).
[0217] The reversible 4-aminobubyrate:2-oxoglutarate transaminase from Streptomyces griseus has demonstrated analogous activity for the conversion of 6-aminohexanoate to adipate semialdehyde (Yonaha et al., Eur. J. Biochem., 1985, 146:101-106).
[0218] The reversible 5-aminovalerate transaminase from Clostridium viride has demonstrated analogous activity for the conversion of 6-aminohexanoate to adipate semialdehyde (Barker et al., J. Biol. Chem., 1987, 262(19), 8994-9003).
[0219] In some embodiments, a terminal amine group leading to the synthesis of 7-aminoheptanoate or heptamethylenediamine is enzymatically formed by a diamine .omega.-transaminase. For example, the second terminal amino group can be enzymatically formed by a diamine .omega.-transaminase classified, for example, under EC 2.6.1.29 or classified, for example, under EC 2.6.1.82, such as, for example, the gene product of ygjG from E. coli (RefSeq Accession No. NP_417544.5, SEQ ID NO: 12).
[0220] The gene product of ygiG accepts a broad range of diamine carbon chain length substrates, such as putrescine, cadaverine and spermidine (see, for example, Samsonova et al., BMC Microbiology, 2003, 3:2).
[0221] The diamine .omega.-transaminase from E. coli strain B has demonstrated activity for 1,7 diaminoheptane (Kim, The Journal of Chemistry, 1964, 239(3), 783-786).
[0222] In some embodiments, the second terminal amine group leading to the synthesis of heptamethylenediamine is enzymatically formed by a deacetylase such as acetylputrescine deacetylase classified, for example, under EC 3.5.1.62. The acetylputrescine deacetylase from Micrococcus luteus K-11 accepts a broad range of carbon chain length substrates, such as acetylputrescine, acetylcadaverine and N.sup.8.acetylspermidine (see, for example, Suzuki et al., 1986, BBA--General Subjects, 882(1): 140-142).
Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis of C7 Building Blocks
[0223] As depicted in FIG. 5 and FIG. 6, a terminal hydroxyl group can be enzymatically formed using an alcohol dehydrogenase.
[0224] In some embodiments, a terminal hydroxyl group leading to the synthesis of 1,7 heptanediol is enzymatically formed by an alcohol dehydrogenase classified, for example, under EC 1.1.1.- (e.g., 1, 2, 21, or 184), such as, for example, the gene product of YMR318C (classified, for example, under EC 1.1.1.2, see Genbank Accession No. CAA90836.1) (Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172), the gene product of yghD, the gene product of cpnD (Iwaki et al., 2002, Appl. Environ. Microbiol., 68(11):5671-5684), the gene product of gbd, or a 6-hydroxyhexanoate dehydrogenase classified, for example, under EC 1.1.1.258, such as, for example, the gene product of chnD (Iwaki et al., Appl. Environ. Microbiol., 1999, supra)
Biochemical Pathways
Pathways Using NADPH-Specific Enzymes to Pimeloyl-ACP as Central Precursor Leading to C7 Building Blocks
[0225] In some embodiments, pimeloyl-ACP is synthesized from the central precursor malonyl-ACP, by conversion of malonyl-ACP to malonyl-ACP methyl ester by a malonyl-CoA O-methyltransferase classified, for example, under EC 2.1.1.197, such as, for example, the gene product of bioC; followed by conversion with malonyl-ACP to 3-oxo-glutyryl-ACP methyl ester by a .beta.-ketoacyl-ACP synthase classified, for example, under EC 2.3.1.- (e.g., EC 2.3.1.41, EC 2.3.1.179 or EC 2.3.1.180), such as, for example, the gene product of fabB, fabF, or fabH; followed by conversion to 3-hydroxy-glutaryl-ACP methyl ester by a 3-oxoacyl-ACP reductase classified, for example, under EC 1.1.1.100, such as, for example, the gene product of fabG; followed by conversion to 2,3-dehydroglutaryl-ACP methyl ester by a 3-hydroxyacyl-ACP dehydratase classified, for example, under EC 4.2.1.59, such as, for example, the gene product of fabZ; followed by conversion to glutaryl-ACP methyl ester by an enoyl-ACP reductase classified, for example, under EC 1.3.1.10, such as, for example, the gene product of fabL; followed by conversion to 3-oxo-pimeloyl-ACP methyl ester by a .beta.-ketoacyl-ACP synthase classified, for example, under EC 2.3.1.- (e.g., EC 2.3.1.41 or EC 2.3.1.179), such as, for example, the gene product of fabB or fabF; followed by conversion to 3-hydroxy-pimeloyl-ACP methyl ester by a 3-oxoacyl-ACP reductase classified, for example, under EC 1.1.1.100, such as, for example, the gene product of fabG; followed by conversion to 2,3-dehydropimeloyl-ACP methyl ester by a 3-hydroxyacyl-ACP dehydratase classified, for example, under EC 4.2.1.59, such as, for example, the gene product of fabZ; followed by conversion to pimeloyl-ACP methyl ester by an enoyl-ACP reductase classified, for example, under EC 1.3.1.10, such as, for example, the gene product of fabL; followed by conversion to pimeloyl-ACP by a pimeloyl-ACP methyl ester esterase classified, for example, under EC 3.1.1.85, such as, for example, the gene product of bioH. See FIG. 1A.
Pathways Using NADPH-Specific Enzymes to Pimeloyl-CoA as Central Precursor Leading to C7 Building Blocks
[0226] In some embodiments, pimeloyl-CoA is synthesized from the central precursor malonyl-CoA, by conversion of malonyl-CoA to malonyl-CoA methyl ester by a maloyl-CoA O-methyltransferase classified, for example, under EC 2.1.1.197, such as, for example, the gene product of bioC; followed by conversion with acetyl-CoA to 3-oxo-glutaryl-CoA methyl ester by a .beta.-ketothiolase classified, for example, under EC 2.3.1.16, such as, for example, the gene product of bktB, or by conversion with malonoyl-CoA by a .beta.-ketoacyl-ACP synthase classified, for example, under EC 2.3.1.180, such as, for example, the gene product of fabH; followed by conversion to 3-hydroxy-glutaryl-CoA methyl ester by a 3-oxoacyl-ACP reductase classified, for example, under EC 1.1.1.100, such as, for example, the gene product of fabG, a 3-hydroxyacyl-CoA dehydrogenase classified, for example, under EC 1.1.1.157, such as, for example, the gene product of hbd, or an acetoacetyl-CoA reductase classified, for example, under EC 1.1.1.36 such as the gene product of phaB; followed by conversion to 2,3-dehydroglutaryl-CoA methyl ester by an enoyl-CoA hydratase classified, for example, under EC 4.2.1.119, such as, for example, the gene product of phaJ; followed by conversion to glutaryl-CoA methyl ester by a reductase classified, for example, under EC 1.3.1.-, such as, for example, an enoyl-ACP reductase classified under EC 1.3.1.10, such as, for example, the gene product of fabL or a trans-2-enoyl-CoA reductase classified, for example, under EC 1.3.1.38 or EC 1.3.1.8, such as, for example, the gene product of ter or idler; followed by conversion to 3-oxo-pimeloyl-CoA methyl ester by a .beta.-ketoacyl-ACP synthase classified, for example, under EC 2.3.1.- (e.g., EC 2.3.1.41 or EC 2.3.1.179), such as, for example, the gene product of fabB or fabF, or a .beta.-ketothiolase classified, for example, under EC 2.3.1.16, such as, for example, the gene product of bktB; followed by conversion to 3-hydroxy-pimeloyl-CoA methyl ester by a 3-oxoacyl-ACP reductase classified, for example, under EC 1.1.1.100, such as, for example, the gene product of fabG, a 3-hydroxyacyl-CoA dehydrogenase classified, for example, under EC 1.1.1.157, such as, for example, the gene product of hbd, or an acetoacetyl-CoA reductase classified, for example, under EC 1.1.1.36, such as, for example, the gene product of phaB; followed by conversion to 2,3-dehydropimeloyl-CoA methyl ester by an enoyl-CoA hydratase classified, for example, under EC 4.2.1.119, such as, for example, the gene product of phaJ; followed by conversion to pimeloyl-CoA methyl ester by a reductase classified, for example, under EC 1.3.1.-, such as, for example, an enoyl-ACP reductase classified, for example, under EC 1.3.1.10, such as the gene product of fabL, or a trans-2-enoyl-CoA reductase classified under, for example, EC 1.3.1.38 or EC 1.3.1.8, such as, for example, the gene product of ter or tdter; followed by conversion to pimeloyl-CoA by a pimeloyl-ACP methyl ester esterase classified, for example, under EC 3.1.1.85, such as, for example, the gene product of bioH. See FIG. 1B.
Pathways Using NADH-Specific Enzymes to Pimeloyl-CoA as Central Precursor Leading to C7 Building Blocks
[0227] In some embodiments, pimeloyl-CoA is synthesized from the central precursor, malonyl-CoA, by conversion of malonyl-CoA to malonyl-CoA methyl ester by a malonyl-CoA O-methyltransferase classified, for example, under EC 2.1.1.197, such as, for example, the gene product of bioC; followed by conversion with acetyl-CoA to 3-oxo-glutaryl-CoA methyl ester by a .beta.-ketothiolase classified, for example, under EC 2.3.1.16, such as, for example, the gene product of bktB or by conversion with malonyl-CoA by a fl-ketoacyl-ACP synthase classified, for example, under EC 2.3.1.180, such as, for example, the gene product of fabH; followed by conversion to 3-hydroxy-glutaryl-CoA methyl ester by a 3-hydroxyacyl-CoA dehydrogenase classified, for example, under EC 1.1.1.- (e.g., EC 1.1.1.35), such as, for example, the gene product of fadB; followed by conversion to 2,3-dehydroglutaryl-CoA methyl ester by an enoyl-CoA hydratase classified, for example, under EC 4.2.1.17, such as, for example, the gene product of crt; followed by conversion to glutaryl-CoA methyl ester by a trans-2-enoyl-CoA reductase classified, for example, under EC 1.3.1.44, such as, for example, the gene product of ter or tdlter or an enoyl-ACP reductase classified, for example, under EC 1.3.1.9, such as, for example, the gene product of fabI; followed by conversion to 3-oxopimeloyl-CoA methyl ester by a fl-ketoacyl-ACP synthase classified, for example, under EC 2.3.1.- (e.g., EC 2.3.1.41 or EC 2.3.1.179), such as, for example, the gene product of fabB or fabF or a .beta.-ketothiolase classified, for example, under EC 2.3.1.16, such as, for example, the gene product of bktB; followed by conversion to 3-hydroxy-pimeloyl-CoA methyl ester by a 3-hydroxyacyl-CoA dehydrogenase classified, for example, under EC 1.1.1.35, such as, for example, the gene product of fadB; followed by conversion to 2,3-dehydropimeloyl-CoA methyl ester by an enoyl-CoA hydratase classified, for example, under EC 4.2.1.17, such as, for example, the gene product of crt; followed by conversion to pimeloyl-CoA methyl ester by a trans-2-enoyl-CoA reductase classified, for example, under EC 1.3.1.44, such as, for example, the gene product of ter or tdter, or an enoyl-ACP reductase classified, for example, under EC 1.3.1.9, such as, for example, the gene product of fabI; followed by conversion to pimeloyl-CoA by a pimeloyl-ACP methyl ester esterase classified, for example, under EC 3.1.1.85, such as, for example, the gene product of bioH. See FIG. 1C.
Pathways Using Pimeloyl-CoA or Pimeloyl-ACP as Central Precursors to Pimelate
[0228] In some embodiments, pimelic acid is synthesized from the central precursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to pimelate semialdehyde by an acetylating aldehyde dehydrogenase classified, for example, under EC 1.2.1.10, such as, for example, the gene product of pduB or pduP (see, for example, Lan et al., 2013, Energy Environ. Sci., 6:2672-2681); followed by conversion to pimelic acid by a 7-oxoheptanoate dehydrogenase classified, for example, under EC 1.2.1.-, such as, for example, the gene product of thnG, a 6-oxohexanoate dehydrogenase classified, for example, under EC 1.2.1.-, such as, for example, the gene product of chnE, or an aldehyde dehydrogenase classified, for example, under EC 1.2.1.3. See FIG. 2.
[0229] In some embodiments, pimelic acid is synthesized from the central precursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to pimelate by a thioesterase classified, for example, under EC 3.1.2.-, such as, for example, the gene products of yciA, tesB (Genbank Accession No. AAA24665.1, SEQ ID NO: 1), or acot13. See FIG. 2.
[0230] In some embodiments, pimelic acid is synthesized from the central precursor, pimeloyl-ACP, by conversion of pimeloyl-ACP to pimelate by a thioesterase classified, for example, under EC 3.1.2.- such as the gene products encoded by Genbank Accession No. ABJ63754.1, Genbank Accession No. CCC78182.1, tesA or fatB. See FIG. 2.
[0231] In some embodiments, pimelate is synthesized from the central precursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to pimelate by a CoA-transferase such as a glutaconate CoA-transferase classified, for example, under EC 2.8.3.12. See FIG. 2.
[0232] In some embodiments, pimelate is synthesized from the central precursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to pimelate by a reversible CoA-ligase such as a reversible succinate-CoA ligase classified, for example, under EC 6.2.1.5. See FIG. 2.
[0233] In some embodiments, pimelate is synthesized from the central precursor, pimelate semialdehyde, by conversion of pimelate semialdehyde to pimelate by a 6-oxohexanoate dehydrogenase or a 7-oxoheptanoate dehydrogenase (classified, for example, under EC 1.2.1.-), such as the gene product of thnG or chnE, or an aldehyde dehydrogenase classified, for example, under EC 1.2.1.3. See FIG. 2.
Pathways Using Pimeloyl-CoA or Pimelate Semialdehyde as Central Precursor to 7-Aminoheptanoate
[0234] In some embodiments, 7-aminoheptanoate is synthesized from the central precursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to pimelate semialdehyde by an acetylating aldehyde dehydrogenase classified, for example, EC 1.2.1.10, such as, for example, the gene product of pduB or pduP; followed by conversion of pimelate semialdehyde to 7-aminoheptanoate by a .omega.-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82. See FIG. 3.
[0235] In some embodiments, 7-aminoheptanoate is synthesized from the central precursor, pimelate semialdehyde, by conversion of pimelate semialdehyde to 7-aminoheptanoate by a .omega.-transaminase (e.g., EC 2.6.1.18, EC 2.6.1.19, or EC 2.6.1.48). See FIG. 3.
[0236] In some embodiments, 7-aminoheptanoate is synthesized from the central precursor, pimelate, by conversion of pimelate to pimelate semialdehyde by a carboxylate reductase classified, for example, under EC 1.2.99.6, such as, for example, the gene product of car optionally in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp (RefSeq Accession No. WP_003234549.1, SEQ ID NO: 14) gene from Bacillus subtilis or npt (Genbank Accession No. ABI183656.1, SEQ ID NO: 15) gene from Nocardia) or the gene products of griC and griD from Streptomyces griseus (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion of pimelate semialdehyde to 7-aminoheptanoate by a .omega.-transaminase (e.g., EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, EC 2.6.1.29, or EC 2.6.1.82, such as SEQ ID NOs: 8-13). The carboxylate reductase can be obtained, for example, from Mycobacterium marinum (Genbank Accession No. ACC40567.1, SEQ ID NO: 2), Mycobacterium smegmatis (Genbank Accession No. ABK71854.1, SEQ ID NO: 3), Segniliparus rugosus (Genbank Accession No. EFV11917.1, SEQ ID NO: 4), Mycobacterium smegmatis (Genbank Accession No. ABK75684.1, SEQ ID NO: 5), Mycobacterium massiliense (Genbank Accession No. EIV11143.1, SEQ ID NO: 6), or Segniliparus rotundus (Genbank Accession No. ADG98140.1, SEQ ID NO: 7). See FIG. 3.
Pathway Using 7-Aminoheptanoate, 7-Hydroxyheptanoate, or Pimelate Semialdehyde as Central Precursor to Heptamethylenediamine
[0237] In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoate to 7-aminoheptanal by a carboxylate reductase classified, for example, under EC 1.2.99.6, such as, for example, the gene product of car (see above) optionally in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sf (RefSeq Accession No. WP_003234549.1, SEQ ID NO: 14) gene from Bacillus subtilis or npt (Genbank Accession No. ABI83656.1, SEQ ID NO: 15) gene from Nocardia) or the gene product of griC and grit); followed by conversion of 7-aminoheptanal to heptamethylenediamine by a .omega.-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82, such as SEQ ID NOs: 8-13 (see above). See FIG. 4.
[0238] The carboxylate reductase encoded by the gene product of car and the phosphopantetheine transferase enhancer npt or sfp has broad substrate specificity, including terminal difunctional C4 and C5 carboxylic acids (Venkitasubramanian et al., Enzyme and Microbial Technology, 2008, 42, 130-137).
[0239] In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-hydroxyheptanoate (which can be produced as described in FIG. 5), by conversion of 7-hydroxyheptanoate to 7-hydroxyheptanal by a carboxylate reductase classified, for example, under EC 1.2.99.6, such as, for example, the gene product of car (see above) optionally in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp, gene from Bacillus subtilis or npt gene from Nocardia) or the gene product of griC and griD (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion of 7-aminoheptanal to 7-aminoheptanol by a .omega.-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82, such as, for example, SEQ ID NOs: 8-13 (see above); followed by conversion to 7-aminoheptanal by an alcohol dehydrogenase classified, for example, under EC 1.1.1.- (e.g., EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184), such as, for example, the gene product of YMR318C (classified, for example, under EC 1.1.1.2, see Genbank Accession No. CAA90836.1) or yqhD (from E. coli, GenBank Accession No. AAA69178.1) (Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the protein having GenBank Accession No. CAA81612.1 (from Geobacillus stearothermophilus); followed by conversion to heptamethylenediamine by a .omega.-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82, such as, for example, SEQ ID NOs: 8-13 (see above). See FIG. 4.
[0240] In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoate to N7-acetyl-7-aminoheptanoate by a N-acetyltransferase such as a lysine N-acetyltransferase classified, for example, under EC 2.3.1.32; followed by conversion to N7-acetyl-7-aminoheptanal by a carboxylate reductase classified, for example, under EC 1.2.99.6, such as, for example, the gene product of car (see above) optionally in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sf gene from Bacillus subtilis or npt gene from Nocardia) or the gene product of griC and griD; followed by conversion to N7-acetyl-1,7-diaminoheptane by a .omega.-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, EC 2.6.1.46, or EC 2.6.1.82, such as, for example, SEQ ID NOs: 8-13 (see above); followed by conversion to heptamethylenediamine by an acetylputrescine deacylase classified, for example, under EC 3.5.1.62. See, FIG. 4.
[0241] In some embodiments, heptamethylenediamine is synthesized from the central precursor, pimelate semialdehyde, by conversion of pimelate semialdehyde to heptanedial by a carboxylate reductase classified, for example, under EC 1.2.99.6, such as, for example, the gene product of car (see above) optionally in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia) or the gene product of griC and griD; followed by conversion to 7-aminoheptanal by a co-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, or EC 2.6.1.48; followed by conversion to heptamethylenediamine by a .omega.-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, EC 2.6.1.46, or EC 2.6.1.82, such as, for example, SEQ ID NOs: 8-13, see above. See FIG. 4.
Pathways Using Pimelate or Pimelate Semialdehyde as Central Precursor to 1,7-Heptanediol
[0242] In some embodiments, 7-hydroxyheptanoate is synthesized from the central precursor pimelate by conversion of pimelate to pimelate semialdehyde by a carboxylate reductase classified, for example, under EC 1.2.99.6, such as, for example, the gene product of car (see above) optionally in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia) or the gene product of griC and griD; followed by conversion to 7-hydroxyheptanoate by a dehydrogenase classified, for example, under EC 1.1.1.-, such as, for example, a 6-hydroxyhexanoate dehydrogenase classified, for example, under EC 1.1.1.258, such as, for example, the gene from of chnD or a 5-hydroxypentanoate dehydrogenase classified, for example, under EC 1.1.1.-, such as, for example, the gene product of cpnD (see, for example, Iwaki et al., 2002, Appl. Environ. Microbiol., 68(11):5671-5684) or a 4-hydroxybutyrate dehydrogenase, such as the gene product of gbd. See FIG. 5. Pimelate semialdehyde also can be produced from pimeloyl-CoA using an acetylating aldehyde dehydrogenase as described above. See FIG. 5.
[0243] In some embodiments, 1,7 heptanediol is synthesized from the central precursor 7-hydroxyheptanoate by conversion of 7-hydroxyheptanoate to 7-hydroxyheptanal by a carboxylate reductase classified, for example, under EC 1.2.99.6, such as, for example, the gene product of car (see above) optionally in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia) or the gene product of griC and griD; followed by conversion of 7-hydroxyheptanal to 1,7 heptanediol by an alcohol dehydrogenase classified, for example, under EC 1.1.1.-, such as, for example, EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184, such as, for example, the gene product of YMR318C or yqhD (see, e.g., Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; or Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the protein having GenBank Accession No. CAA81612.1 (from Geobacillus stearothermophilus). See, FIG. 6.
Cultivation Strategy
[0244] In some embodiments, the cultivation strategy entails achieving an aerobic, anaerobic, or micro-aerobic cultivation condition.
[0245] In some embodiments, the cultivation strategy entails nutrient limitation such as nitrogen, phosphate, or oxygen limitation.
[0246] In some embodiments, the cultivation strategy for certain recombinant microorganism strains includes addition of specific nutrients or substrates. In some embodiments, the cultivation strategy for a recombinant microorganism strain may include addition of glycine and purine. In some embodiments, the cultivation strategy for a recombinant microorganism strain may include addition of formate. In some embodiments, the cultivation strategy for a recombinant microorganism strain may include addition of formaldehyde. In some embodiments, the cultivation strategy for a recombinant microorganism strain may include addition of methanol.
[0247] In some embodiments, a cell retention strategy using, for example, ceramic hollow fiber membranes can be employed to achieve and maintain a high cell density during either fed-batch or continuous fermentation.
[0248] In some embodiments, the principal carbon source fed to the fermentation in the synthesis of one or more C7 building blocks can derive from biological or non-biological feedstocks.
[0249] In some embodiments, the biological feedstock can be, can include, or can derive from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid and formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste.
[0250] The efficient catabolism of crude glycerol stemming from the production of biodiesel has been demonstrated in several microorganisms such as Escherichia coli, Cupriavidus necator, Pseudomonas oleavorans, Pseudomonas putida, and Yarrowia lipolytica (Lee et al., Appl. Biochem. Biotechnol., 2012, 166, 1801-1813; Yang et al., Biotechnology for Biofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotechnol., 2011, 90, 885-893).
[0251] The efficient catabolism of lignocellulosic-derived levulinic acid has been demonstrated in several organisms such as Cupriavidus necator and Pseudomonas putida in the synthesis of 3-hydroxyvalerate via the precursor propanoyl-CoA (Jaremko and Yu, Journal of Biotechnology, 2011, 155, 2011, 293-298; Martin and Prather, Journal of Biotechnology, 2009, 139, 61-67).
[0252] The efficient catabolism of lignin-derived aromatic compounds such as benzoate analogues has been demonstrated in several microorganisms such as Pseudomonas putida and Cupriavidus necator (Bugg et al., Current Opinion in Biotechnology, 2011, 22, 394-400; Perez-Pantoja et al., FEMS Microbiol. Rev., 2008, 32, 736-794).
[0253] The efficient utilization of agricultural waste, such as olive mill waste water has been demonstrated in several microorganisms, including Yarrowia lipolytica (Papanikolaou et al., Bioresour. Technol., 2008, 99(7), 2419-2428).
[0254] The efficient utilization of fermentable sugars such as monosaccharides and disaccharides derived from cellulosic, hemicellulosic, cane and beet molasses, cassava, corn and other agricultural sources has been demonstrated for several microorganism such as Escherichia coli, Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcus lactis (see, e.g., Hermann et al., Journal of Biotechnology, 2003, 104, 155-172; Wee et al., Food Technol. Biotechnol., 2006, 44(2), 163-172; Ohashi et al., Journal of Bioscience and Bioengineering, 1999, 87(5), 647-654).
[0255] The efficient utilization of furfural, derived from a variety of agricultural lignocellulosic sources, has been demonstrated for Cupriavidus necator (Li et al., Biodegradation, 2011, 22, 1215-1225).
[0256] In some embodiments, the non-biological feedstock can be or can derive from natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, benzoic acid, non-volatile residue (NVR), a caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.
[0257] The efficient catabolism of methanol has been demonstrated for the methylotrophic yeast Pichia pastoris.
[0258] The efficient catabolism of ethanol has been demonstrated for Clostridium kluyveri (Seedorf et al., Proc. Natl. Acad Sci. USA, 2008, 105(6) 2128-2133).
[0259] The efficient catabolism of CO.sub.2 and H.sub.2, which may be derived from natural gas and other chemical and petrochemical sources, has been demonstrated for Cupriavidus necator (Prybylski et al., Energy, Sustainability and Society, 2012, 2:11).
[0260] The efficient catabolism of syngas has been demonstrated for numerous microorganisms, such as Clostridium ljungdahlii and Clostridium autoethanogemim (Kopke et al., Applied and Environmental Microbiology, 2011, 77(15), 5467-5475).
[0261] The efficient catabolism of the non-volatile residue waste stream from cyclohexane processes has been demonstrated for numerous microorganisms, such as Delftia acidovorans and Cupriavidus necator (Ramsay et al., Applied and Environmental Microbiology, 1986, 52(1), 152-156).
[0262] In some embodiments, the host microorganism is a prokaryote. For example, the prokaryote can be a bacterium from the genus Escherichia such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogenum, or Clostridium kluyveri; from the genus Corynebacteria such as Corynebacterium glutamicum; from the genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans; from the genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida, or Pseudomonas oleavorans; from the genus Delftia such as Delftia acidovorans; from the genus Bacillus such as Bacillus subtillis; from the genus Lactobacillus such as Lactobacillus delbrueckii; or from the genus Lactococcus such as Lactococcus lactis. Such prokaryotes also can be a source of genes to construct recombinant host cells described herein that are capable of producing one or more C7 building blocks.
[0263] In some embodiments, the host microorganism is a eukaryote. For example, the eukaryote can be a filamentous fungus, e.g., one from the genus Aspergillus such as Aspergillus niger. Alternatively, the eukaryote can be a yeast, e.g., one from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris; or from the genus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkia such as Issathenkia orientalis; from the genus Debaryomyces such as Debaryomyces hansenii; from the genus Arxula such as Arxula adenoinivorans; or from the genus Kluyveromyces such as Kluyveromyces lactis. Such eukaryotes also can be a source of genes to construct recombinant host cells described herein that are capable of producing one or more C7 building blocks.
Metabolic Engineering
[0264] The present disclosure provides methods involving less than all the steps described for all the above pathways. Such methods can involve, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve or more of such steps. Where less than all the steps are included in such a method, the first, and in some embodiments the only, step can be any one of the steps listed.
[0265] Furthermore, recombinant hosts described herein can include any combination of the above enzymes such that one or more of the steps, e.g., one, two, three, four, five, six, seven, eight, nine, or ten or more of such steps, can be performed within a recombinant host. This disclosure provides host cells of any of the genera and species listed and genetically engineered to express one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, 11, or 12 or more) recombinant forms of any of the enzymes recited in the disclosure. Thus, for example, the host cells can contain exogenous nucleic acids encoding enzymes catalyzing one or more of the steps of any of the pathways described herein.
[0266] In addition, this disclosure recognizes that where enzymes have been described as accepting CoA-activated substrates, analogous enzyme activities associated with ACP-bound substrates exist that are not necessarily in the same enzyme class.
[0267] Also, this disclosure recognizes that where enzymes have been described accepting (R)-enantiomers of substrate, analogous enzyme activities associated with (S)-enantiomer substrates exist that are not necessarily in the same enzyme class.
[0268] This disclosure also recognizes that where an enzyme is shown to accept a particular co-factor, such as NADPH, or a co-substrate, such as acetyl-CoA, many enzymes are promiscuous in terms of accepting a number of different co-factors or co-substrates in catalyzing a particular enzyme activity. Also, this disclosure recognizes that where enzymes have high specificity for e.g., a particular co-factor such as NADH, an enzyme with similar or identical activity that has high specificity for the co-factor NADPH may be in a different enzyme class.
[0269] In some embodiments, the enzymes in the pathways outlined herein are the result of enzyme engineering via non-direct or rational enzyme design approaches with aims of improving activity, improving specificity, reducing feedback inhibition, reducing repression, improving enzyme solubility, changing stereo-specificity, or changing co-factor specificity.
[0270] In some embodiments, the enzymes in the pathways outlined herein can be gene dosed (i.e., overexpressed by having a plurality of copies of the gene in the host organism), into the resulting genetically modified organism via episomal or chromosomal integration approaches.
[0271] In some embodiments, genome-scale system biology techniques such as Flux Balance Analysis can be utilized to devise genome scale attenuation or knockout strategies for directing carbon flux to a C7 building block. Knock in and cloning strategies can likewise be devised to direct carbon flux to a C7 building block, as can combinations of attenuation, knock out, knock in, and cloning strategies.
[0272] Attenuation of the activity of a particular enzyme can occur through the inhibition of enzymatic activity itself and/or through a decrease in gene expression, including gene deletion/inactivation. Attenuation (decrease in gene expression) and knockout strategies include, but are not limited to: the use of transposons, homologous recombination (double cross-over approach), mutagenesis, non-homologous end-joining (NHEJ), Zinc Fingers (ZFs), Transcription-Activator-Like Effectors (TALEs), and the Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) sequences with CRISPR-Associated Protein 9 (Cas9) RNA interference (RNAi), double stranded RNA, and enzyme inhibitors. Cloning in strategies include, but are not limited to, inserting a nucleic acid of interest in a vector (e.g., a plasmid, bacteriophage, cosmid, or bacterial artificial chromosome) and inserting the vector into the host cell so that the host cell expresses the nucleic acid of interest. The terms "knocking in" and "cloning in" may both refer to introducing an exogenous nucleic acid into the host cells such that the host cell then expresses the exogenous nucleic acid.
[0273] In some embodiments, fluxomic, metabolomics, and transcriptomal data can be utilized to inform or support genome-scale system biology techniques, thereby devising genome scale attenuation or knockout strategies in directing carbon flux to a C7 building block.
[0274] In some embodiments, the host microorganism's tolerance to high concentrations of a C7 building block can be improved through continuous cultivation in a selective environment.
[0275] In some embodiments, the host microorganism's endogenous biochemical network can be attenuated or augmented to (1) ensure the intracellular availability of acetyl-CoA and malonyl-CoA, (2) create an NADH or NADPH imbalance that may only be balanced via the formation of one or more C7 building blocks, (3) prevent degradation of central metabolites and/or central precursors leading to and including one or more C7 building blocks, (4) ensure efficient efflux from the cell and/or (5) channel increased flux through the pathway leading to the C7 building block product(s).
[0276] In some embodiments requiring intracellular availability of acetyl-CoA for C7 building block synthesis, endogenous enzymes catalyzing the hydrolysis acetyl-CoA such as short-chain length thioesteraes can be attenuated in the host organism.
[0277] In some embodiments requiring condensation of acetyl-CoA and propanoyl-CoA for C7 building block synthesis, one or more endogenous .beta.-ketothiolases catalyzing the condensation of only acetyl-CoA to acetoacetyl-CoA such as the endogenous gene products of AtoB or phaA can be attenuated.
[0278] In some embodiments requiring the intracellular availability of acetyl-CoA for C7 building block synthesis, an endogenous phosphotransacetylase generating acetate such as pta can be attenuated (Shen et al., Appl. Environ. Microbiol., 2011, 77(9):2905-2915).
[0279] In some embodiments requiring the intracellular availability of acetyl-CoA for C7 building block synthesis, an endogenous gene in an acetate synthesis pathway encoding an acetate kinase, such as ack, can be attenuated.
[0280] In some embodiments requiring the intracellular availability of acetyl-CoA and NADH for C7 building block synthesis, an endogenous gene encoding an enzyme that catalyzes the degradation of pyruvate to lactate such as a lactate dehydrogenase encoded by IdhA can be attenuated (Shen et al., 2011, supra).
[0281] In some embodiments requiring the intracellular availability of acetyl-CoA and NADH for C7 building block synthesis, endogenous genes encoding enzymes, such as menaquinol-fumarate oxidoreductase, that catalyze the degradation of phosphoenolpyruvate to succinate such as frdBC can be attenuated (see, e.g., Shen et al., 2011, supra).
[0282] In some embodiments requiring the intracellular availability of acetyl-CoA and NADH for C7 building block synthesis, an endogenous gene encoding an enzyme that catalyzes the degradation of acetyl-CoA to ethanol such as the alcohol dehydrogenase encoded by adhE can be attenuated (Shen et al., 2011, supra).
[0283] In some embodiments, where pathways require excess NADH co-factor for C7 building block synthesis, a recombinant formate dehydrogenase gene can be overexpressed in the host organism (Shen et al., 2011, supra).
[0284] In some embodiments, where pathways require excess NADH or NADPH co-factor for C7 building block synthesis, an endogenous transhydrogenase dissipating the co-factor imbalance can be attenuated.
[0285] In some embodiments, an endogenous gene encoding an enzyme that catalyzes the degradation of pyruvate to ethanol such as pyruvate decarboxylate can be attenuated.
[0286] In some embodiments, an endogenous gene encoding an enzyme that catalyzes the generation of isobutanol such as a 2-oxoacid decarboxylase can be attenuated.
[0287] In some embodiments requiring the intracellular availability of acetyl-CoA for C7 building block synthesis, a recombinant acetyl-CoA synthetase such as the gene product of acs can be overexpressed in the microorganism (Satoh et al., J. Bioscience and Bioengineering, 2003, 95(4):335-341).
[0288] In some embodiments, carbon flux can be directed into the pentose phosphate cycle to increase the supply of NADPH by attenuating an endogenous glucose-6-phosphate isomerase (EC 5.3.1.9).
[0289] In some embodiments, carbon flux can be redirected into the pentose phosphate cycle to increase the supply of NADPH by overexpression a 6-phosphogluconate dehydrogenase and/or a transketolase (Lee et al., 2003, Biotechnology Progress, 19(5), 1444-1449).
[0290] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a gene such as udhA encoding a puridine nucleotide transhydrogenase can be overexpressed in the host organisms (Brigham et al., Advanced Biofuels and Bioproducts, 2012, Chapter 39, 1065-1090).
[0291] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 Building Block, a recombinant glyceraldehyde-3-phosphate-dehydrogenase gene such as gapN can be overexpressed in the host organisms (Brigham et al., 2012, supra).
[0292] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a recombinant malic enzyme gene such as maeA or maeB can be overexpressed in the host organisms (Brigham et al., 2012, supra).
[0293] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a recombinant glucose-6-phosphate dehydrogenase gene such as ztf can be overexpressed in the host organisms (Lim et al., J. Bioscience and Bioengineering, 2002, 93(6), 543-549).
[0294] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a recombinant fructose 1,6 diphosphatase gene such as fbp can be overexpressed in the host organisms (Becker et al., J. Biotechnol., 2007, 132:99-109).
[0295] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, endogenous triose phosphate isomerase (EC 5.3.1.1) can be attenuated.
[0296] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a recombinant glucose dehydrogenase such as the gene product of gdh can be overexpressed in the host organism (Satoh et al., J. Bioscience and Bioengineering, 2003, 95(4):335-341).
[0297] In some embodiments, endogenous enzymes facilitating the conversion of NADPH to NADH can be attenuated, such as the NADH generation cycle that may be generated via inter-conversion of glutamate dehydrogenases classified under EC 1.4.1.2 (NADH-specific) and EC 1.4.1.4 (NADPH-specific). For example, avoiding dissipation of an NADH imbalance towards C7 building blocks, a NADPH-specific glutamate dehydrogenase can be attenuated.
[0298] In some embodiments, an endogenous glutamate dehydrogenase (EC 1.4.1.3) that utilizes both NADH and NADPH as co-factors can be attenuated.
[0299] In some embodiments, a membrane-bound enoyl-CoA reductases can be solubilized via expression as a fusion protein to a small soluble protein such as a maltose binding protein (Gloerich et al., FEBS Letters, 2006, 580, 2092-2096).
[0300] In some embodiments using hosts that naturally accumulate polyhydroxyalkanoates, the endogenous polymer synthase enzymes can be attenuated in the host strain.
[0301] In some embodiments, a L-alanine dehydrogenase can be overexpressed in the host to regenerate L-alanine from pyruvate as an amino donor for .omega.-transaminase reactions.
[0302] In some embodiments, a L-glutamate dehydrogenase specific for the co-factor used to achieve co-factor imbalance can be overexpressed in the host to regenerate L-glutamate from 2-oxoglutarate as an amino donor for .omega.-transaminase reactions. For example, promoting dissipation of the NADH imbalance towards C7 building blocks, a NADH-specific glutamate dehydrogenase can be overexpressed.
[0303] In some embodiments, enzymes such as pimeloyl-CoA dehydrogenase classified under, EC 1.3.1.62; an acyl-CoA dehydrogenase classified, for example, under EC 1.3.8.7 or EC 1.3.8.1; and/or a glutaryl-CoA dehydrogenase classified, for example, under EC 1.3.8.6 that degrade central metabolites and/or central precursors leading to and including C7 building blocks can be attenuated.
[0304] In some embodiments, endogenous enzymes activating C7 building blocks via Coenzyme A esterification such as CoA-ligases (e.g., a pimeloyl-CoA synthetase) classified under, for example, EC 6.2.1.14 can be attenuated.
[0305] In some embodiments, a S-adenosylmethionine synthetase can be overexpressed in the host to generate S-Adenosyl-L-methionine as a co-factor for malonyl-ACP O-methyltransferase.
[0306] In some embodiments, an alcohol dehydrogenase, for example a methanol dehydrogenase, and a formaldehyde dehydrogenase can be overexpressed in the host to allow methanol catabolism via formate.
[0307] In some embodiments, where the production of formate requires methanol, formate acetyltransferase (e.g., formate acetyltransferase) classified under, for example, EC 2.3.1.54, and/or a pyruvate formate-lyase (PFL)-like enzyme (e.g., pyruvate formate-lyase (PFL)-like enzyme TdcE) classified under, for example, EC 2.3.1.54, can be attenuated.
[0308] In some embodiments, where the production of formate requires methanol, an exogenous S-(hydroxymethyl) glutathione dehydrogenase, classified under, for example, EC 1.1.1.284, and/or an exogenous S-formylglutathione hydrolase FrmB classified under, for example, EC 3.1.2.12, can be expressed in the host cell.
[0309] In some embodiments, where a modified tetrahydrofolate metabolic cycle requires formate, bifunctional protein FolD (folD) can be attenuated and an exogenous formate-tetrahydrofolate ligase (fhs), classified under, for example, EC 6.3.4.3, can be expressed in the host cell (Sah et al., J. Bacteriology, 2015, 197(4), 717-726).
[0310] In some embodiments, the efflux of a C7 building block across the cell membrane to the extracellular media can be enhanced or amplified by genetically engineering structural modifications to the cell membrane or increasing any associated transporter activity for a C7 building block.
[0311] The efflux of heptamethylenediamine can be enhanced or amplified by overexpressing broad substrate range multidrug transporters such as Blt from Bacillus subtilis (Woolridge et al., 1997, J. Biol. Chem., 272(14):8864-8866); AcrB and AcrD from Escherichia coli (Elkins & Nikaido, 2002, J. Bacteriol., 184(23), 6490-6499), NorA from Staphylococcus aereus (Ng et al., 1994, Antimicrob Agents Chemother, 38(6), 1345-1355), or Bmr from Bacillus subtilis (Neyfakh, 1992, Antimicrob Agents Chemother, 36(2), 484-485).
[0312] The efflux of 7-aminoheptanoate and heptamethylenediamine can be enhanced or amplified by overexpressing the solute transporters such as the lysE transporter from Corynebacterium glutamicum (Bellmann et al., 2001, Microbiology, 147, 1765-1774).
[0313] The efflux of pimelic acid can be enhanced or amplified by overexpressing a dicarboxylate transporter such as the SucE transporter from Corynebacterium glutamicum (Huhn et al., Appl. Microbiol. & Biotech., 89(2), 327-335).
Producing C7 Building Blocks Using a Recombinant Host
[0314] Typically, one or more C7 building blocks can be produced by providing a host microorganism and culturing the provided microorganism with a culture medium containing a suitable carbon source as described above. In general, the culture media and/or culture conditions can be such that the microorganisms grow to an adequate density and produce a C7 building block efficiently. For large-scale production processes, any method can be used such as those described elsewhere (Manual of Industrial Microbiology and Biotechnology, 2.sup.nd Edition, Editors: A. L. Demain and J. E. Davies, ASM Press; and Principles of Fermentation Technology, P. F. Stanbury and A. Whitaker, Pergamon). Briefly, a large tank (e.g., a 100 gallon, 200 gallon, or 500 gallon or more tank) containing an appropriate culture medium is inoculated with a particular microorganism. After inoculation, the microorganism is incubated to allow biomass to be produced. Once a desired biomass is reached, the broth containing the microorganisms can be transferred to a second tank. This second tank can be any size. For example, the second tank can be larger, smaller, or the same size as the first tank. Typically, the second tank is larger than the first such that additional culture medium can be added to the broth from the first tank. In addition, the culture medium within this second tank can be the same as, or different from, that used in the first tank.
[0315] Once transferred, the microorganisms can be incubated to allow for the production of a C7 building block. Once produced, any method can be used to isolate C7 building blocks. For example, C7 building blocks can be recovered selectively from the fermentation broth via adsorption processes. In the case of pimelic acid and 7-aminoheptanoic acid, the resulting eluate can be further concentrated via evaporation, crystallized via evaporative and/or cooling crystallization, and the crystals recovered via centrifugation. In the case of heptamethylenediamine and 1,7-heptanediol, distillation may be employed to achieve the desired product purity.
[0316] The invention is further described in the following non-limiting example.
Examples
[0317] Generating a strain that can utilize the methanol byproduct from the 7-AHA pathway as source of formate so that the methanol is an essential intermediate for purines and initiator tRNAs may be accomplished through the proposed cloning strategies discussed below and diagrammed in FIGS. 8A, 8B, and 9. FIGS. 8A, 8B, and 9 show the expected strains following appropriate knock out or knock in (left side) as well as the growth conditions for screening and the expected outcomes (right side). According to this disclosure, the ideal screen will be based on the viability of the host strain as well as pimelate production.
Example 1--Cloning Strategy 1
[0318] The cloning strategy involves starting with a "pimelate strain" (see FIG. 8A), where a pimelate host strain is a strain that has a pathway for producing pimelate or C7 building blocks (e.g., the 7-AHA pathway, see FIG. 7). The biosynthesis of C7 building blocks, such as 7-AHA, by recombinant host microorganisms has been described in U.S. Patent Publication Nos. 2014/0186904 and 2014/0242655. The host organism in the cloning strategy described in this example is E. coli.
[0319] The cloning strategy involves the knocking out folD to produce an fhs-supported E. coli folD deletion (.DELTA.folD+fhs) model. See FIG. 8A, strains 2 and 3. The .DELTA.folD/p-fhs strain and the .DELTA.folD::fhs strain, in which the strain is supported by a single copy of fhs, are described by Sah et al. (Sah et al., J. Bacteriol., 2015, 197(4), 717-726). The deletion of FolD in E. coli results in autotrophy for purines and glycine. To enable sufficient growth the media will be supplemented with purines and glycine, which PflB and TdcE convert to formate. The availability of formate then enables Fhs to convert THF to N.sup.10-formyltetrahydrofolate (N.sup.10-fTHF). N.sup.10-fTHF is important for the de novo pathway of purine nucleotide biosynthesis and formylation of the initiator tRNA (tRNAfMet) (FIGS. 10A and 10B).
[0320] The next strain, strain 4 (FIG. 8A, strain 4), to be engineered consists of deleting the pflB and tdcE genes from strain 3 which eliminates the source of formate. Knocking in the frmA and frmB genes into strain 4, would produce strain 5 (FIG. 8B, strain 5). The latter can then grow in media containing formaldehyde as FrmA and FrmB enable the spontaneous oxidation of formaldehyde into formate, through the organism's formaldehyde detoxification pathway linked to glutathione (GSH).
[0321] Strain 6 (FIG. 8B, strain 6), which is the final strain will be established by knocking in an alcohol dehydrogenase gene (adh). Methanol will accumulate after engineering a 7-AHA pathway producing 7-AHA from the central carbon metabolism into this strain. To enable the strain to utilize methanol effectively, the C1 substrate must be converted by unique enzymes and subsequently connected via intermediates to the central metabolism and thus converted into formate indispensable for the growth of the strain. Knocking in a heterologous aerobic adh gene enables the conversion of methanol to formaldehyde catalysed by Adh as well as subsequent oxidation of formaldehyde into formate. This strain can then grow, requiring methanol as a growth critical intermediate which is necessary for the synthesis of purine nucleotides, thymidylate, and tRNAfMet. This will ensure that as the strain grows, increased flux is channelled through the 7-AHA pathway to produce more 7-AHA.
Example 2--Cloning Strategy 2
[0322] The second cloning strategy also starts with a pimelate strain, as was discussed in Example 1. The host organism in the cloning strategy described in this example is E. coli.
[0323] The cloning strategy involves the knocking out folD to produce an fhs-supported E. coli foil) deletion (.DELTA.folD+fhs) model, as described above. See FIG. 9, strain 2. As described above, to enable sufficient growth of this strain the media must be supplemented with purines and glycine.
[0324] The next strain (FIG. 9, strain 3) to be engineered consists of knocking in the frmA and frmB genes from strain 2. Strain 3 can then grow in media containing formaldehyde as the frmA and frmB gene products enable the spontaneous oxidation of formaldehyde into formate, through the organism's formaldehyde detoxification pathway linked to glutathione (GSH).
[0325] The final strain, strain 4 (FIG. 9), is established by knocking in an alcohol dehydrogenase gene (adh). Knocking in a heterologous aerobic adh gene enables the conversion of methanol to formaldehyde catalysed by Adh as well as subsequent oxidation of formaldehyde into formate.
[0326] The proposed cloning strategy is further highlighted in Table 1.
TABLE-US-00001 TABLE 1 Proposed strains and projected outcomes Proposed strain Growth conditions Remarks E. coli pimelate strain (FIG. 9, Growth on glycerol strain 1) fhs-supported E. coli folD Growth on glycerol + A model discussed in Sah deletion (.DELTA.folD::fhs strain) formate + glycine (+purine) et al. (Sah et al., 2015). (FIG. 9, strain 2) No growth on glycerol Generation of E. coli .DELTA.folD strain having a single copy of fhs (.DELTA.folD::fhs) by subjecting the .DELTA.folD/p-fhs strain to curring of p-fhs. The deletion of folD in E. coli results in autotrophy for purines and glycine. To enable sufficient growth, media may be supplemented with purines and glycine .DELTA.folD::fhs::frmA::frmB (FIG. 9, Growth on glycerol + Introduction of frmA and strain 3) formaldehyde + glycine frmB enables oxidation of (+purine) formaldehyde to formate No growth on glycerol .DELTA.folD::fhs::mdh::frmA::frmB Growth on glycerol + Introduction of adh (FIG. 9, strain 4) methanol + glycine converts methanol to (+purine) formaldehyde which is then No growth on glycerol oxidised to formate
[0327] The systems described in the predictive examples have a potential improvement of about 15 to about 22% of the maximum theoretical yield in 7-AHA production.
Sequence CWU
1
SEQUENCE LISTING
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20 25 30
Ala Val Val Glu Ala Val Ser Asp Pro Ser Leu Ser Phe Thr Arg Tyr
35 40 45
Leu Asp Thr Leu Met Arg Gly Tyr Ala Glu Arg Pro Ala Leu Ala His
50 55 60
Arg Val Gly Ala Gly Tyr Glu Thr Ile Ser Tyr Gly Glu Leu Trp Ala
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Arg Val Gly Ala Ile Ala Ala Ala Trp Gln Ala Asp Gly Leu Ala Pro
85 90 95
Gly Asp Phe Val Ala Thr Val Gly Phe Thr Ser Pro Asp Tyr Val Ala
100 105 110
Val Asp Leu Ala Ala Ala Arg Ser Gly Leu Val Ser Val Pro Leu Gln
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Ala Gly Ala Ser Leu Ala Gln Leu Val Gly Ile Leu Glu Glu Thr Glu
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Ala Pro Leu Phe Glu Pro Ala Glu Gly Glu Asp Pro Leu Ala Leu Leu
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Ile Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly Ala Met Tyr Ser
245 250 255
Gln Arg Leu Val Ser Gln Leu Trp Gly Arg Thr Pro Val Val Pro Gly
260 265 270
Met Pro Asn Ile Ser Leu His Tyr Met Pro Leu Ser His Ser Tyr Gly
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Arg Ala Val Leu Ala Gly Ala Leu Ser Ala Gly Gly Thr Ala His Phe
290 295 300
Thr Ala Asn Ser Asp Leu Ser Thr Leu Phe Glu Asp Ile Ala Leu Ala
305 310 315 320
Arg Pro Thr Phe Leu Ala Leu Val Pro Arg Val Cys Glu Met Leu Phe
325 330 335
Gln Glu Ser Gln Arg Gly Gln Asp Val Ala Glu Leu Arg Glu Arg Val
340 345 350
Leu Gly Gly Arg Leu Leu Val Ala Val Cys Gly Ser Ala Pro Leu Ser
355 360 365
Pro Glu Met Arg Ala Phe Met Glu Glu Val Leu Gly Phe Pro Leu Leu
370 375 380
Asp Gly Tyr Gly Ser Thr Glu Ala Leu Gly Val Met Arg Asn Gly Ile
385 390 395 400
Ile Gln Arg Pro Pro Val Ile Asp Tyr Lys Leu Val Asp Val Pro Glu
405 410 415
Leu Gly Tyr Arg Thr Thr Asp Lys Pro Tyr Pro Arg Gly Glu Leu Cys
420 425 430
Ile Arg Ser Thr Ser Leu Ile Ser Gly Tyr Tyr Lys Arg Pro Glu Ile
435 440 445
Thr Ala Glu Val Phe Asp Ala Gln Gly Tyr Tyr Lys Thr Gly Asp Val
450 455 460
Met Ala Glu Ile Ala Pro Asp His Leu Val Tyr Val Asp Arg Ser Lys
465 470 475 480
Asn Val Leu Lys Leu Ser Gln Gly Glu Phe Val Ala Val Ala Lys Leu
485 490 495
Glu Ala Ala Tyr Gly Thr Ser Pro Tyr Val Lys Gln Ile Phe Val Tyr
500 505 510
Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala Val Val Val Pro Asn Ala
515 520 525
Glu Val Leu Gly Ala Arg Asp Gln Glu Glu Ala Lys Pro Leu Ile Ala
530 535 540
Ala Ser Leu Gln Lys Ile Ala Lys Glu Ala Gly Leu Gln Ser Tyr Glu
545 550 555 560
Val Pro Arg Asp Phe Leu Ile Glu Thr Glu Pro Phe Thr Thr Gln Asn
565 570 575
Gly Leu Leu Ser Glu Val Gly Lys Leu Leu Arg Pro Lys Leu Lys Ala
580 585 590
Arg Tyr Gly Glu Ala Leu Glu Ala Arg Tyr Asp Glu Ile Ala His Gly
595 600 605
Gln Ala Asp Glu Leu Arg Ala Leu Arg Asp Gly Ala Gly Gln Arg Pro
610 615 620
Val Val Glu Thr Val Val Arg Ala Ala Val Ala Ile Ser Gly Ser Glu
625 630 635 640
Gly Ala Glu Val Gly Pro Glu Ala Asn Phe Ala Asp Leu Gly Gly Asp
645 650 655
Ser Leu Ser Ala Leu Ser Leu Ala Asn Leu Leu His Asp Val Phe Glu
660 665 670
Val Glu Val Pro Val Arg Ile Ile Ile Gly Pro Thr Ala Ser Leu Ala
675 680 685
Gly Ile Ala Lys His Ile Glu Ala Glu Arg Ala Gly Ala Ser Ala Pro
690 695 700
Thr Ala Ala Ser Val His Gly Ala Gly Ala Thr Arg Ile Arg Ala Ser
705 710 715 720
Glu Leu Thr Leu Glu Lys Phe Leu Pro Glu Asp Leu Leu Ala Ala Ala
725 730 735
Lys Gly Leu Pro Ala Ala Asp Gln Val Arg Thr Val Leu Leu Thr Gly
740 745 750
Ala Asn Gly Trp Leu Gly Arg Phe Leu Ala Leu Glu Gln Leu Glu Arg
755 760 765
Leu Ala Arg Ser Gly Gln Asp Gly Gly Lys Leu Ile Cys Leu Val Arg
770 775 780
Gly Lys Asp Ala Ala Ala Ala Arg Arg Arg Ile Glu Glu Thr Leu Gly
785 790 795 800
Thr Asp Pro Ala Leu Ala Ala Arg Phe Ala Glu Leu Ala Glu Gly Arg
805 810 815
Leu Glu Val Val Pro Gly Asp Val Gly Glu Pro Lys Phe Gly Leu Asp
820 825 830
Asp Ala Ala Trp Asp Arg Leu Ala Glu Glu Val Asp Val Ile Val His
835 840 845
Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr His Gln Leu Phe Gly
850 855 860
Pro Asn Val Val Gly Thr Ala Glu Ile Ile Arg Leu Ala Ile Thr Ala
865 870 875 880
Lys Arg Lys Pro Val Thr Tyr Leu Ser Thr Val Ala Val Ala Ala Gly
885 890 895
Val Glu Pro Ser Ser Phe Glu Glu Asp Gly Asp Ile Arg Ala Val Val
900 905 910
Pro Glu Arg Pro Leu Gly Asp Gly Tyr Ala Asn Gly Tyr Gly Asn Ser
915 920 925
Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Glu Leu Val Gly
930 935 940
Leu Pro Val Ala Val Phe Arg Ser Asp Met Ile Leu Ala His Thr Arg
945 950 955 960
Tyr Thr Gly Gln Leu Asn Val Pro Asp Gln Phe Thr Arg Leu Val Leu
965 970 975
Ser Leu Leu Ala Thr Gly Ile Ala Pro Lys Ser Phe Tyr Gln Gln Gly
980 985 990
Ala Ala Gly Glu Arg Gln Arg Ala His Tyr Asp Gly Ile Pro Val Asp
995 1000 1005
Phe Thr Ala Glu Ala Ile Thr Thr Leu Gly Ala Glu Pro Ser Trp
1010 1015 1020
Phe Asp Gly Gly Ala Gly Phe Arg Ser Phe Asp Val Phe Asn Pro
1025 1030 1035
His His Asp Gly Val Gly Leu Asp Glu Phe Val Asp Trp Leu Ile
1040 1045 1050
Glu Ala Gly His Pro Ile Ser Arg Ile Asp Asp His Lys Glu Trp
1055 1060 1065
Phe Ala Arg Phe Glu Thr Ala Val Arg Gly Leu Pro Glu Ala Gln
1070 1075 1080
Arg Gln His Ser Leu Leu Pro Leu Leu Arg Ala Tyr Ser Phe Pro
1085 1090 1095
His Pro Pro Val Asp Gly Ser Val Tyr Pro Thr Gly Lys Phe Gln
1100 1105 1110
Gly Ala Val Lys Ala Ala Gln Val Gly Ser Asp His Asp Val Pro
1115 1120 1125
His Leu Gly Lys Ala Leu Ile Val Lys Tyr Ala Asp Asp Leu Lys
1130 1135 1140
Ala Leu Gly Leu Leu
1145
<210> SEQ ID NO 5
<211> LENGTH: 1168
<212> TYPE: PRT
<213> ORGANISM: Mycobacterium smegmatis
<400> SEQUENCE: 5
Met Thr Ile Glu Thr Arg Glu Asp Arg Phe Asn Arg Arg Ile Asp His
1 5 10 15
Leu Phe Glu Thr Asp Pro Gln Phe Ala Ala Ala Arg Pro Asp Glu Ala
20 25 30
Ile Ser Ala Ala Ala Ala Asp Pro Glu Leu Arg Leu Pro Ala Ala Val
35 40 45
Lys Gln Ile Leu Ala Gly Tyr Ala Asp Arg Pro Ala Leu Gly Lys Arg
50 55 60
Ala Val Glu Phe Val Thr Asp Glu Glu Gly Arg Thr Thr Ala Lys Leu
65 70 75 80
Leu Pro Arg Phe Asp Thr Ile Thr Tyr Arg Gln Leu Ala Gly Arg Ile
85 90 95
Gln Ala Val Thr Asn Ala Trp His Asn His Pro Val Asn Ala Gly Asp
100 105 110
Arg Val Ala Ile Leu Gly Phe Thr Ser Val Asp Tyr Thr Thr Ile Asp
115 120 125
Ile Ala Leu Leu Glu Leu Gly Ala Val Ser Val Pro Leu Gln Thr Ser
130 135 140
Ala Pro Val Ala Gln Leu Gln Pro Ile Val Ala Glu Thr Glu Pro Lys
145 150 155 160
Val Ile Ala Ser Ser Val Asp Phe Leu Ala Asp Ala Val Ala Leu Val
165 170 175
Glu Ser Gly Pro Ala Pro Ser Arg Leu Val Val Phe Asp Tyr Ser His
180 185 190
Glu Val Asp Asp Gln Arg Glu Ala Phe Glu Ala Ala Lys Gly Lys Leu
195 200 205
Ala Gly Thr Gly Val Val Val Glu Thr Ile Thr Asp Ala Leu Asp Arg
210 215 220
Gly Arg Ser Leu Ala Asp Ala Pro Leu Tyr Val Pro Asp Glu Ala Asp
225 230 235 240
Pro Leu Thr Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro Lys
245 250 255
Gly Ala Met Tyr Pro Glu Ser Lys Thr Ala Thr Met Trp Gln Ala Gly
260 265 270
Ser Lys Ala Arg Trp Asp Glu Thr Leu Gly Val Met Pro Ser Ile Thr
275 280 285
Leu Asn Phe Met Pro Met Ser His Val Met Gly Arg Gly Ile Leu Cys
290 295 300
Ser Thr Leu Ala Ser Gly Gly Thr Ala Tyr Phe Ala Ala Arg Ser Asp
305 310 315 320
Leu Ser Thr Phe Leu Glu Asp Leu Ala Leu Val Arg Pro Thr Gln Leu
325 330 335
Asn Phe Val Pro Arg Ile Trp Asp Met Leu Phe Gln Glu Tyr Gln Ser
340 345 350
Arg Leu Asp Asn Arg Arg Ala Glu Gly Ser Glu Asp Arg Ala Glu Ala
355 360 365
Ala Val Leu Glu Glu Val Arg Thr Gln Leu Leu Gly Gly Arg Phe Val
370 375 380
Ser Ala Leu Thr Gly Ser Ala Pro Ile Ser Ala Glu Met Lys Ser Trp
385 390 395 400
Val Glu Asp Leu Leu Asp Met His Leu Leu Glu Gly Tyr Gly Ser Thr
405 410 415
Glu Ala Gly Ala Val Phe Ile Asp Gly Gln Ile Gln Arg Pro Pro Val
420 425 430
Ile Asp Tyr Lys Leu Val Asp Val Pro Asp Leu Gly Tyr Phe Ala Thr
435 440 445
Asp Arg Pro Tyr Pro Arg Gly Glu Leu Leu Val Lys Ser Glu Gln Met
450 455 460
Phe Pro Gly Tyr Tyr Lys Arg Pro Glu Ile Thr Ala Glu Met Phe Asp
465 470 475 480
Glu Asp Gly Tyr Tyr Arg Thr Gly Asp Ile Val Ala Glu Leu Gly Pro
485 490 495
Asp His Leu Glu Tyr Leu Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
500 505 510
Gln Gly Glu Phe Val Thr Val Ser Lys Leu Glu Ala Val Phe Gly Asp
515 520 525
Ser Pro Leu Val Arg Gln Ile Tyr Val Tyr Gly Asn Ser Ala Arg Ser
530 535 540
Tyr Leu Leu Ala Val Val Val Pro Thr Glu Glu Ala Leu Ser Arg Trp
545 550 555 560
Asp Gly Asp Glu Leu Lys Ser Arg Ile Ser Asp Ser Leu Gln Asp Ala
565 570 575
Ala Arg Ala Ala Gly Leu Gln Ser Tyr Glu Ile Pro Arg Asp Phe Leu
580 585 590
Val Glu Thr Thr Pro Phe Thr Leu Glu Asn Gly Leu Leu Thr Gly Ile
595 600 605
Arg Lys Leu Ala Arg Pro Lys Leu Lys Ala His Tyr Gly Glu Arg Leu
610 615 620
Glu Gln Leu Tyr Thr Asp Leu Ala Glu Gly Gln Ala Asn Glu Leu Arg
625 630 635 640
Glu Leu Arg Arg Asn Gly Ala Asp Arg Pro Val Val Glu Thr Val Ser
645 650 655
Arg Ala Ala Val Ala Leu Leu Gly Ala Ser Val Thr Asp Leu Arg Ser
660 665 670
Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser
675 680 685
Phe Ser Asn Leu Leu His Glu Ile Phe Asp Val Asp Val Pro Val Gly
690 695 700
Val Ile Val Ser Pro Ala Thr Asp Leu Ala Gly Val Ala Ala Tyr Ile
705 710 715 720
Glu Gly Glu Leu Arg Gly Ser Lys Arg Pro Thr Tyr Ala Ser Val His
725 730 735
Gly Arg Asp Ala Thr Glu Val Arg Ala Arg Asp Leu Ala Leu Gly Lys
740 745 750
Phe Ile Asp Ala Lys Thr Leu Ser Ala Ala Pro Gly Leu Pro Arg Ser
755 760 765
Gly Thr Glu Ile Arg Thr Val Leu Leu Thr Gly Ala Thr Gly Phe Leu
770 775 780
Gly Arg Tyr Leu Ala Leu Glu Trp Leu Glu Arg Met Asp Leu Val Asp
785 790 795 800
Gly Lys Val Ile Cys Leu Val Arg Ala Arg Ser Asp Asp Glu Ala Arg
805 810 815
Ala Arg Leu Asp Ala Thr Phe Asp Thr Gly Asp Ala Thr Leu Leu Glu
820 825 830
His Tyr Arg Ala Leu Ala Ala Asp His Leu Glu Val Ile Ala Gly Asp
835 840 845
Lys Gly Glu Ala Asp Leu Gly Leu Asp His Asp Thr Trp Gln Arg Leu
850 855 860
Ala Asp Thr Val Asp Leu Ile Val Asp Pro Ala Ala Leu Val Asn His
865 870 875 880
Val Leu Pro Tyr Ser Gln Met Phe Gly Pro Asn Ala Leu Gly Thr Ala
885 890 895
Glu Leu Ile Arg Ile Ala Leu Thr Thr Thr Ile Lys Pro Tyr Val Tyr
900 905 910
Val Ser Thr Ile Gly Val Gly Gln Gly Ile Ser Pro Glu Ala Phe Val
915 920 925
Glu Asp Ala Asp Ile Arg Glu Ile Ser Ala Thr Arg Arg Val Asp Asp
930 935 940
Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu
945 950 955 960
Leu Arg Glu Ala His Asp Trp Cys Gly Leu Pro Val Ser Val Phe Arg
965 970 975
Cys Asp Met Ile Leu Ala Asp Thr Thr Tyr Ser Gly Gln Leu Asn Leu
980 985 990
Pro Asp Met Phe Thr Arg Leu Met Leu Ser Leu Val Ala Thr Gly Ile
995 1000 1005
Ala Pro Gly Ser Phe Tyr Glu Leu Asp Ala Asp Gly Asn Arg Gln
1010 1015 1020
Arg Ala His Tyr Asp Gly Leu Pro Val Glu Phe Ile Ala Glu Ala
1025 1030 1035
Ile Ser Thr Ile Gly Ser Gln Val Thr Asp Gly Phe Glu Thr Phe
1040 1045 1050
His Val Met Asn Pro Tyr Asp Asp Gly Ile Gly Leu Asp Glu Tyr
1055 1060 1065
Val Asp Trp Leu Ile Glu Ala Gly Tyr Pro Val His Arg Val Asp
1070 1075 1080
Asp Tyr Ala Thr Trp Leu Ser Arg Phe Glu Thr Ala Leu Arg Ala
1085 1090 1095
Leu Pro Glu Arg Gln Arg Gln Ala Ser Leu Leu Pro Leu Leu His
1100 1105 1110
Asn Tyr Gln Gln Pro Ser Pro Pro Val Cys Gly Ala Met Ala Pro
1115 1120 1125
Thr Asp Arg Phe Arg Ala Ala Val Gln Asp Ala Lys Ile Gly Pro
1130 1135 1140
Asp Lys Asp Ile Pro His Val Thr Ala Asp Val Ile Val Lys Tyr
1145 1150 1155
Ile Ser Asn Leu Gln Met Leu Gly Leu Leu
1160 1165
<210> SEQ ID NO 6
<211> LENGTH: 1185
<212> TYPE: PRT
<213> ORGANISM: Mycobacterium massiliense
<400> SEQUENCE: 6
Met Thr Asn Glu Thr Asn Pro Gln Gln Glu Gln Leu Ser Arg Arg Ile
1 5 10 15
Glu Ser Leu Arg Glu Ser Asp Pro Gln Phe Arg Ala Ala Gln Pro Asp
20 25 30
Pro Ala Val Ala Glu Gln Val Leu Arg Pro Gly Leu His Leu Ser Glu
35 40 45
Ala Ile Ala Ala Leu Met Thr Gly Tyr Ala Glu Arg Pro Ala Leu Gly
50 55 60
Glu Arg Ala Arg Glu Leu Val Ile Asp Gln Asp Gly Arg Thr Thr Leu
65 70 75 80
Arg Leu Leu Pro Arg Phe Asp Thr Thr Thr Tyr Gly Glu Leu Trp Ser
85 90 95
Arg Thr Thr Ser Val Ala Ala Ala Trp His His Asp Ala Thr His Pro
100 105 110
Val Lys Ala Gly Asp Leu Val Ala Thr Leu Gly Phe Thr Ser Ile Asp
115 120 125
Tyr Thr Val Leu Asp Leu Ala Ile Met Ile Leu Gly Gly Val Ala Val
130 135 140
Pro Leu Gln Thr Ser Ala Pro Ala Ser Gln Trp Thr Thr Ile Leu Ala
145 150 155 160
Glu Ala Glu Pro Asn Thr Leu Ala Val Ser Ile Glu Leu Ile Gly Ala
165 170 175
Ala Met Glu Ser Val Arg Ala Thr Pro Ser Ile Lys Gln Val Val Val
180 185 190
Phe Asp Tyr Thr Pro Glu Val Asp Asp Gln Arg Glu Ala Phe Glu Ala
195 200 205
Ala Ser Thr Gln Leu Ala Gly Thr Gly Ile Ala Leu Glu Thr Leu Asp
210 215 220
Ala Val Ile Ala Arg Gly Ala Ala Leu Pro Ala Ala Pro Leu Tyr Ala
225 230 235 240
Pro Ser Ala Gly Asp Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly
245 250 255
Ser Thr Gly Ala Pro Lys Gly Ala Met His Ser Glu Asn Ile Val Arg
260 265 270
Arg Trp Trp Ile Arg Glu Asp Val Met Ala Gly Thr Glu Asn Leu Pro
275 280 285
Met Ile Gly Leu Asn Phe Met Pro Met Ser His Ile Met Gly Arg Gly
290 295 300
Thr Leu Thr Ser Thr Leu Ser Thr Gly Gly Thr Gly Tyr Phe Ala Ala
305 310 315 320
Ser Ser Asp Met Ser Thr Leu Phe Glu Asp Met Glu Leu Ile Arg Pro
325 330 335
Thr Ala Leu Ala Leu Val Pro Arg Val Cys Asp Met Val Phe Gln Arg
340 345 350
Phe Gln Thr Glu Val Asp Arg Arg Leu Ala Ser Gly Asp Thr Ala Ser
355 360 365
Ala Glu Ala Val Ala Ala Glu Val Lys Ala Asp Ile Arg Asp Asn Leu
370 375 380
Phe Gly Gly Arg Val Ser Ala Val Met Val Gly Ser Ala Pro Leu Ser
385 390 395 400
Glu Glu Leu Gly Glu Phe Ile Glu Ser Cys Phe Glu Leu Asn Leu Thr
405 410 415
Asp Gly Tyr Gly Ser Thr Glu Ala Gly Met Val Phe Arg Asp Gly Ile
420 425 430
Val Gln Arg Pro Pro Val Ile Asp Tyr Lys Leu Val Asp Val Pro Glu
435 440 445
Leu Gly Tyr Phe Ser Thr Asp Lys Pro His Pro Arg Gly Glu Leu Leu
450 455 460
Leu Lys Thr Asp Gly Met Phe Leu Gly Tyr Tyr Lys Arg Pro Glu Val
465 470 475 480
Thr Ala Ser Val Phe Asp Ala Asp Gly Phe Tyr Met Thr Gly Asp Ile
485 490 495
Val Ala Glu Leu Ala His Asp Asn Ile Glu Ile Ile Asp Arg Arg Asn
500 505 510
Asn Val Leu Lys Leu Ser Gln Gly Glu Phe Val Ala Val Ala Thr Leu
515 520 525
Glu Ala Glu Tyr Ala Asn Ser Pro Val Val His Gln Ile Tyr Val Tyr
530 535 540
Gly Ser Ser Glu Arg Ser Tyr Leu Leu Ala Val Val Val Pro Thr Pro
545 550 555 560
Glu Ala Val Ala Ala Ala Lys Gly Asp Ala Ala Ala Leu Lys Thr Thr
565 570 575
Ile Ala Asp Ser Leu Gln Asp Ile Ala Lys Glu Ile Gln Leu Gln Ser
580 585 590
Tyr Glu Val Pro Arg Asp Phe Ile Ile Glu Pro Gln Pro Phe Thr Gln
595 600 605
Gly Asn Gly Leu Leu Thr Gly Ile Ala Lys Leu Ala Arg Pro Asn Leu
610 615 620
Lys Ala His Tyr Gly Pro Arg Leu Glu Gln Met Tyr Ala Glu Ile Ala
625 630 635 640
Glu Gln Gln Ala Ala Glu Leu Arg Ala Leu His Gly Val Asp Pro Asp
645 650 655
Lys Pro Ala Leu Glu Thr Val Leu Lys Ala Ala Gln Ala Leu Leu Gly
660 665 670
Val Ser Ser Ala Glu Leu Ala Ala Asp Ala His Phe Thr Asp Leu Gly
675 680 685
Gly Asp Ser Leu Ser Ala Leu Ser Phe Ser Asp Leu Leu Arg Asp Ile
690 695 700
Phe Ala Val Glu Val Pro Val Gly Val Ile Val Ser Ala Ala Asn Asp
705 710 715 720
Leu Gly Gly Val Ala Lys Phe Val Asp Glu Gln Arg His Ser Gly Gly
725 730 735
Thr Arg Pro Thr Ala Glu Thr Val His Gly Ala Gly His Thr Glu Ile
740 745 750
Arg Ala Ala Asp Leu Thr Leu Asp Lys Phe Ile Asp Glu Ala Thr Leu
755 760 765
His Ala Ala Pro Ser Leu Pro Lys Ala Ala Gly Ile Pro His Thr Val
770 775 780
Leu Leu Thr Gly Ser Asn Gly Tyr Leu Gly His Tyr Leu Ala Leu Glu
785 790 795 800
Trp Leu Glu Arg Leu Asp Lys Thr Asp Gly Lys Leu Ile Val Ile Val
805 810 815
Arg Gly Lys Asn Ala Glu Ala Ala Tyr Gly Arg Leu Glu Glu Ala Phe
820 825 830
Asp Thr Gly Asp Thr Glu Leu Leu Ala His Phe Arg Ser Leu Ala Asp
835 840 845
Lys His Leu Glu Val Leu Ala Gly Asp Ile Gly Asp Pro Asn Leu Gly
850 855 860
Leu Asp Ala Asp Thr Trp Gln Arg Leu Ala Asp Thr Val Asp Val Ile
865 870 875 880
Val His Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr Asn Gln Leu
885 890 895
Phe Gly Pro Asn Val Val Gly Thr Ala Glu Ile Ile Lys Leu Ala Ile
900 905 910
Thr Thr Lys Ile Lys Pro Val Thr Tyr Leu Ser Thr Val Ala Val Ala
915 920 925
Ala Tyr Val Asp Pro Thr Thr Phe Asp Glu Glu Ser Asp Ile Arg Leu
930 935 940
Ile Ser Ala Val Arg Pro Ile Asp Asp Gly Tyr Ala Asn Gly Tyr Gly
945 950 955 960
Asn Ala Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu
965 970 975
Cys Gly Leu Pro Val Ala Val Phe Arg Ser Asp Met Ile Leu Ala His
980 985 990
Ser Arg Tyr Thr Gly Gln Leu Asn Val Pro Asp Gln Phe Thr Arg Leu
995 1000 1005
Ile Leu Ser Leu Ile Ala Thr Gly Ile Ala Pro Gly Ser Phe Tyr
1010 1015 1020
Gln Ala Gln Thr Thr Gly Glu Arg Pro Leu Ala His Tyr Asp Gly
1025 1030 1035
Leu Pro Gly Asp Phe Thr Ala Glu Ala Ile Thr Thr Leu Gly Thr
1040 1045 1050
Gln Val Pro Glu Gly Ser Glu Gly Phe Val Thr Tyr Asp Cys Val
1055 1060 1065
Asn Pro His Ala Asp Gly Ile Ser Leu Asp Asn Phe Val Asp Trp
1070 1075 1080
Leu Ile Glu Ala Gly Tyr Pro Ile Ala Arg Ile Asp Asn Tyr Thr
1085 1090 1095
Glu Trp Phe Thr Arg Phe Asp Thr Ala Ile Arg Gly Leu Ser Glu
1100 1105 1110
Lys Gln Lys Gln His Ser Leu Leu Pro Leu Leu His Ala Phe Glu
1115 1120 1125
Gln Pro Ser Ala Ala Glu Asn His Gly Val Val Pro Ala Lys Arg
1130 1135 1140
Phe Gln His Ala Val Gln Ala Ala Gly Ile Gly Pro Val Gly Gln
1145 1150 1155
Asp Gly Thr Thr Asp Ile Pro His Leu Ser Arg Arg Leu Ile Val
1160 1165 1170
Lys Tyr Ala Lys Asp Leu Glu Gln Leu Gly Leu Leu
1175 1180 1185
<210> SEQ ID NO 7
<211> LENGTH: 1186
<212> TYPE: PRT
<213> ORGANISM: Segniliparus rotundus
<400> SEQUENCE: 7
Met Thr Gln Ser His Thr Gln Gly Pro Gln Ala Ser Ala Ala His Ser
1 5 10 15
Arg Leu Ala Arg Arg Ala Ala Glu Leu Leu Ala Thr Asp Pro Gln Ala
20 25 30
Ala Ala Thr Leu Pro Asp Pro Glu Val Val Arg Gln Ala Thr Arg Pro
35 40 45
Gly Leu Arg Leu Ala Glu Arg Val Asp Ala Ile Leu Ser Gly Tyr Ala
50 55 60
Asp Arg Pro Ala Leu Gly Gln Arg Ser Phe Gln Thr Val Lys Asp Pro
65 70 75 80
Ile Thr Gly Arg Ser Ser Val Glu Leu Leu Pro Thr Phe Asp Thr Ile
85 90 95
Thr Tyr Arg Glu Leu Arg Glu Arg Ala Thr Ala Ile Ala Ser Asp Leu
100 105 110
Ala His His Pro Gln Ala Pro Ala Lys Pro Gly Asp Phe Leu Ala Ser
115 120 125
Ile Gly Phe Ile Ser Val Asp Tyr Val Ala Ile Asp Ile Ala Gly Val
130 135 140
Phe Ala Gly Leu Thr Ala Val Pro Leu Gln Thr Gly Ala Thr Leu Ala
145 150 155 160
Thr Leu Thr Ala Ile Thr Ala Glu Thr Ala Pro Thr Leu Phe Ala Ala
165 170 175
Ser Ile Glu His Leu Pro Thr Ala Val Asp Ala Val Leu Ala Thr Pro
180 185 190
Ser Val Arg Arg Leu Leu Val Phe Asp Tyr Arg Ala Gly Ser Asp Glu
195 200 205
Asp Arg Glu Ala Val Glu Ala Ala Lys Arg Lys Ile Ala Asp Ala Gly
210 215 220
Ser Ser Val Leu Val Asp Val Leu Asp Glu Val Ile Ala Arg Gly Lys
225 230 235 240
Ser Ala Pro Lys Ala Pro Leu Pro Pro Ala Thr Asp Ala Gly Asp Asp
245 250 255
Ser Leu Ser Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro Lys
260 265 270
Gly Ala Met Tyr Pro Glu Arg Asn Val Ala His Phe Trp Gly Gly Val
275 280 285
Trp Ala Ala Ala Phe Asp Glu Asp Ala Ala Pro Pro Val Pro Ala Ile
290 295 300
Asn Ile Thr Phe Leu Pro Leu Ser His Val Ala Ser Arg Leu Ser Leu
305 310 315 320
Met Pro Thr Leu Ala Arg Gly Gly Leu Met His Phe Val Ala Lys Ser
325 330 335
Asp Leu Ser Thr Leu Phe Glu Asp Leu Lys Leu Ala Arg Pro Thr Asn
340 345 350
Leu Phe Leu Val Pro Arg Val Val Glu Met Leu Tyr Gln His Tyr Gln
355 360 365
Ser Glu Leu Asp Arg Arg Gly Val Gln Asp Gly Thr Arg Glu Ala Glu
370 375 380
Ala Val Lys Asp Asp Leu Arg Thr Gly Leu Leu Gly Gly Arg Ile Leu
385 390 395 400
Thr Ala Gly Phe Gly Ser Ala Pro Leu Ser Ala Glu Leu Ala Gly Phe
405 410 415
Ile Glu Ser Leu Leu Gln Ile His Leu Val Asp Gly Tyr Gly Ser Thr
420 425 430
Glu Ala Gly Pro Val Trp Arg Asp Gly Tyr Leu Val Lys Pro Pro Val
435 440 445
Thr Asp Tyr Lys Leu Ile Asp Val Pro Glu Leu Gly Tyr Phe Ser Thr
450 455 460
Asp Ser Pro His Pro Arg Gly Glu Leu Ala Ile Lys Thr Gln Thr Ile
465 470 475 480
Leu Pro Gly Tyr Tyr Lys Arg Pro Glu Thr Thr Ala Glu Val Phe Asp
485 490 495
Glu Asp Gly Phe Tyr Leu Thr Gly Asp Val Val Ala Gln Ile Gly Pro
500 505 510
Glu Gln Phe Ala Tyr Val Asp Arg Arg Lys Asn Val Leu Lys Leu Ser
515 520 525
Gln Gly Glu Phe Val Thr Leu Ala Lys Leu Glu Ala Ala Tyr Ser Ser
530 535 540
Ser Pro Leu Val Arg Gln Leu Phe Val Tyr Gly Ser Ser Glu Arg Ser
545 550 555 560
Tyr Leu Leu Ala Val Ile Val Pro Thr Pro Asp Ala Leu Lys Lys Phe
565 570 575
Gly Val Gly Glu Ala Ala Lys Ala Ala Leu Gly Glu Ser Leu Gln Lys
580 585 590
Ile Ala Arg Asp Glu Gly Leu Gln Ser Tyr Glu Val Pro Arg Asp Phe
595 600 605
Ile Ile Glu Thr Asp Pro Phe Thr Val Glu Asn Gly Leu Leu Ser Asp
610 615 620
Ala Arg Lys Ser Leu Arg Pro Lys Leu Lys Glu His Tyr Gly Glu Arg
625 630 635 640
Leu Glu Ala Met Tyr Lys Glu Leu Ala Asp Gly Gln Ala Asn Glu Leu
645 650 655
Arg Asp Ile Arg Arg Gly Val Gln Gln Arg Pro Thr Leu Glu Thr Val
660 665 670
Arg Arg Ala Ala Ala Ala Met Leu Gly Ala Ser Ala Ala Glu Ile Lys
675 680 685
Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu
690 695 700
Thr Phe Ser Asn Phe Leu His Asp Leu Phe Glu Val Asp Val Pro Val
705 710 715 720
Gly Val Ile Val Ser Ala Ala Asn Thr Leu Gly Ser Val Ala Glu His
725 730 735
Ile Asp Ala Gln Leu Ala Gly Gly Arg Ala Arg Pro Thr Phe Ala Thr
740 745 750
Val His Gly Lys Gly Ser Thr Thr Ile Lys Ala Ser Asp Leu Thr Leu
755 760 765
Asp Lys Phe Ile Asp Glu Gln Thr Leu Glu Ala Ala Lys His Leu Pro
770 775 780
Lys Pro Ala Asp Pro Pro Arg Thr Val Leu Leu Thr Gly Ala Asn Gly
785 790 795 800
Trp Leu Gly Arg Phe Leu Ala Leu Glu Trp Leu Glu Arg Leu Ala Pro
805 810 815
Ala Gly Gly Lys Leu Ile Thr Ile Val Arg Gly Lys Asp Ala Ala Gln
820 825 830
Ala Lys Ala Arg Leu Asp Ala Ala Tyr Glu Ser Gly Asp Pro Lys Leu
835 840 845
Ala Gly His Tyr Gln Asp Leu Ala Ala Thr Thr Leu Glu Val Leu Ala
850 855 860
Gly Asp Phe Ser Glu Pro Arg Leu Gly Leu Asp Glu Ala Thr Trp Asn
865 870 875 880
Arg Leu Ala Asp Glu Val Asp Phe Ile Ser His Pro Gly Ala Leu Val
885 890 895
Asn His Val Leu Pro Tyr Asn Gln Leu Phe Gly Pro Asn Val Ala Gly
900 905 910
Val Ala Glu Ile Ile Lys Leu Ala Ile Thr Thr Arg Ile Lys Pro Val
915 920 925
Thr Tyr Leu Ser Thr Val Ala Val Ala Ala Gly Val Glu Pro Ser Ala
930 935 940
Leu Asp Glu Asp Gly Asp Ile Arg Thr Val Ser Ala Glu Arg Ser Val
945 950 955 960
Asp Glu Gly Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Gly Gly Glu
965 970 975
Val Leu Leu Arg Glu Ala His Asp Arg Thr Gly Leu Pro Val Arg Val
980 985 990
Phe Arg Ser Asp Met Ile Leu Ala His Gln Lys Tyr Thr Gly Gln Val
995 1000 1005
Asn Ala Thr Asp Gln Phe Thr Arg Leu Val Gln Ser Leu Leu Ala
1010 1015 1020
Thr Gly Leu Ala Pro Lys Ser Phe Tyr Glu Leu Asp Ala Gln Gly
1025 1030 1035
Asn Arg Gln Arg Ala His Tyr Asp Gly Ile Pro Val Asp Phe Thr
1040 1045 1050
Ala Glu Ser Ile Thr Thr Leu Gly Gly Asp Gly Leu Glu Gly Tyr
1055 1060 1065
Arg Ser Tyr Asn Val Phe Asn Pro His Arg Asp Gly Val Gly Leu
1070 1075 1080
Asp Glu Phe Val Asp Trp Leu Ile Glu Ala Gly His Pro Ile Thr
1085 1090 1095
Arg Ile Asp Asp Tyr Asp Gln Trp Leu Ser Arg Phe Glu Thr Ser
1100 1105 1110
Leu Arg Gly Leu Pro Glu Ser Lys Arg Gln Ala Ser Val Leu Pro
1115 1120 1125
Leu Leu His Ala Phe Ala Arg Pro Gly Pro Ala Val Asp Gly Ser
1130 1135 1140
Pro Phe Arg Asn Thr Val Phe Arg Thr Asp Val Gln Lys Ala Lys
1145 1150 1155
Ile Gly Ala Glu His Asp Ile Pro His Leu Gly Lys Ala Leu Val
1160 1165 1170
Leu Lys Tyr Ala Asp Asp Ile Lys Gln Leu Gly Leu Leu
1175 1180 1185
<210> SEQ ID NO 8
<211> LENGTH: 459
<212> TYPE: PRT
<213> ORGANISM: Chromobacterium violaceum
<400> SEQUENCE: 8
Met Gln Lys Gln Arg Thr Thr Ser Gln Trp Arg Glu Leu Asp Ala Ala
1 5 10 15
His His Leu His Pro Phe Thr Asp Thr Ala Ser Leu Asn Gln Ala Gly
20 25 30
Ala Arg Val Met Thr Arg Gly Glu Gly Val Tyr Leu Trp Asp Ser Glu
35 40 45
Gly Asn Lys Ile Ile Asp Gly Met Ala Gly Leu Trp Cys Val Asn Val
50 55 60
Gly Tyr Gly Arg Lys Asp Phe Ala Glu Ala Ala Arg Arg Gln Met Glu
65 70 75 80
Glu Leu Pro Phe Tyr Asn Thr Phe Phe Lys Thr Thr His Pro Ala Val
85 90 95
Val Glu Leu Ser Ser Leu Leu Ala Glu Val Thr Pro Ala Gly Phe Asp
100 105 110
Arg Val Phe Tyr Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Met Ile
115 120 125
Arg Met Val Arg Arg Tyr Trp Asp Val Gln Gly Lys Pro Glu Lys Lys
130 135 140
Thr Leu Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr Ile Gly Gly
145 150 155 160
Ala Ser Leu Gly Gly Met Lys Tyr Met His Glu Gln Gly Asp Leu Pro
165 170 175
Ile Pro Gly Met Ala His Ile Glu Gln Pro Trp Trp Tyr Lys His Gly
180 185 190
Lys Asp Met Thr Pro Asp Glu Phe Gly Val Val Ala Ala Arg Trp Leu
195 200 205
Glu Glu Lys Ile Leu Glu Ile Gly Ala Asp Lys Val Ala Ala Phe Val
210 215 220
Gly Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro Pro Ala Thr
225 230 235 240
Tyr Trp Pro Glu Ile Glu Arg Ile Cys Arg Lys Tyr Asp Val Leu Leu
245 250 255
Val Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Trp Phe
260 265 270
Gly His Gln His Phe Gly Phe Gln Pro Asp Leu Phe Thr Ala Ala Lys
275 280 285
Gly Leu Ser Ser Gly Tyr Leu Pro Ile Gly Ala Val Phe Val Gly Lys
290 295 300
Arg Val Ala Glu Gly Leu Ile Ala Gly Gly Asp Phe Asn His Gly Phe
305 310 315 320
Thr Tyr Ser Gly His Pro Val Cys Ala Ala Val Ala His Ala Asn Val
325 330 335
Ala Ala Leu Arg Asp Glu Gly Ile Val Gln Arg Val Lys Asp Asp Ile
340 345 350
Gly Pro Tyr Met Gln Lys Arg Trp Arg Glu Thr Phe Ser Arg Phe Glu
355 360 365
His Val Asp Asp Val Arg Gly Val Gly Met Val Gln Ala Phe Thr Leu
370 375 380
Val Lys Asn Lys Ala Lys Arg Glu Leu Phe Pro Asp Phe Gly Glu Ile
385 390 395 400
Gly Thr Leu Cys Arg Asp Ile Phe Phe Arg Asn Asn Leu Ile Met Arg
405 410 415
Ala Cys Gly Asp His Ile Val Ser Ala Pro Pro Leu Val Met Thr Arg
420 425 430
Ala Glu Val Asp Glu Met Leu Ala Val Ala Glu Arg Cys Leu Glu Glu
435 440 445
Phe Glu Gln Thr Leu Lys Ala Arg Gly Leu Ala
450 455
<210> SEQ ID NO 9
<211> LENGTH: 468
<212> TYPE: PRT
<213> ORGANISM: Pseudomonas aeruginosa
<400> SEQUENCE: 9
Met Asn Ala Arg Leu His Ala Thr Ser Pro Leu Gly Asp Ala Asp Leu
1 5 10 15
Val Arg Ala Asp Gln Ala His Tyr Met His Gly Tyr His Val Phe Asp
20 25 30
Asp His Arg Val Asn Gly Ser Leu Asn Ile Ala Ala Gly Asp Gly Ala
35 40 45
Tyr Ile Tyr Asp Thr Ala Gly Asn Arg Tyr Leu Asp Ala Val Gly Gly
50 55 60
Met Trp Cys Thr Asn Ile Gly Leu Gly Arg Glu Glu Met Ala Arg Thr
65 70 75 80
Val Ala Glu Gln Thr Arg Leu Leu Ala Tyr Ser Asn Pro Phe Cys Asp
85 90 95
Met Ala Asn Pro Arg Ala Ile Glu Leu Cys Arg Lys Leu Ala Glu Leu
100 105 110
Ala Pro Gly Asp Leu Asp His Val Phe Leu Thr Thr Gly Gly Ser Thr
115 120 125
Ala Val Asp Thr Ala Ile Arg Leu Met His Tyr Tyr Gln Asn Cys Arg
130 135 140
Gly Lys Arg Ala Lys Lys His Val Ile Thr Arg Ile Asn Ala Tyr His
145 150 155 160
Gly Ser Thr Phe Leu Gly Met Ser Leu Gly Gly Lys Ser Ala Asp Arg
165 170 175
Pro Ala Glu Phe Asp Phe Leu Asp Glu Arg Ile His His Leu Ala Cys
180 185 190
Pro Tyr Tyr Tyr Arg Ala Pro Glu Gly Leu Gly Glu Ala Glu Phe Leu
195 200 205
Asp Gly Leu Val Asp Glu Phe Glu Arg Lys Ile Leu Glu Leu Gly Ala
210 215 220
Asp Arg Val Gly Ala Phe Ile Ser Glu Pro Val Phe Gly Ser Gly Gly
225 230 235 240
Val Ile Val Pro Pro Ala Gly Tyr His Arg Arg Met Trp Glu Leu Cys
245 250 255
Gln Arg Tyr Asp Val Leu Tyr Ile Ser Asp Glu Val Val Thr Ser Phe
260 265 270
Gly Arg Leu Gly His Phe Phe Ala Ser Gln Ala Val Phe Gly Val Gln
275 280 285
Pro Asp Ile Ile Leu Thr Ala Lys Gly Leu Thr Ser Gly Tyr Gln Pro
290 295 300
Leu Gly Ala Cys Ile Phe Ser Arg Arg Ile Trp Glu Val Ile Ala Glu
305 310 315 320
Pro Asp Lys Gly Arg Cys Phe Ser His Gly Phe Thr Tyr Ser Gly His
325 330 335
Pro Val Ala Cys Ala Ala Ala Leu Lys Asn Ile Glu Ile Ile Glu Arg
340 345 350
Glu Gly Leu Leu Ala His Ala Asp Glu Val Gly Arg Tyr Phe Glu Glu
355 360 365
Arg Leu Gln Ser Leu Arg Asp Leu Pro Ile Val Gly Asp Val Arg Gly
370 375 380
Met Arg Phe Met Ala Cys Val Glu Phe Val Ala Asp Lys Ala Ser Lys
385 390 395 400
Ala Leu Phe Pro Glu Ser Leu Asn Ile Gly Glu Trp Val His Leu Arg
405 410 415
Ala Gln Lys Arg Gly Leu Leu Val Arg Pro Ile Val His Leu Asn Val
420 425 430
Met Ser Pro Pro Leu Ile Leu Thr Arg Glu Gln Val Asp Thr Val Val
435 440 445
Arg Val Leu Arg Glu Ser Ile Glu Glu Thr Val Glu Asp Leu Val Arg
450 455 460
Ala Gly His Arg
465
<210> SEQ ID NO 10
<211> LENGTH: 454
<212> TYPE: PRT
<213> ORGANISM: Pseudomonas syringae
<400> SEQUENCE: 10
Met Ser Ala Asn Asn Pro Gln Thr Leu Glu Trp Gln Ala Leu Ser Ser
1 5 10 15
Glu His His Leu Ala Pro Phe Ser Asp Tyr Lys Gln Leu Lys Glu Lys
20 25 30
Gly Pro Arg Ile Ile Thr Arg Ala Glu Gly Val Tyr Leu Trp Asp Ser
35 40 45
Glu Gly Asn Lys Ile Leu Asp Gly Met Ser Gly Leu Trp Cys Val Ala
50 55 60
Ile Gly Tyr Gly Arg Glu Glu Leu Ala Asp Ala Ala Ser Lys Gln Met
65 70 75 80
Arg Glu Leu Pro Tyr Tyr Asn Leu Phe Phe Gln Thr Ala His Pro Pro
85 90 95
Val Leu Glu Leu Ala Lys Ala Ile Ser Asp Ile Ala Pro Glu Gly Met
100 105 110
Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Gly Asn Asp Thr Met
115 120 125
Leu Arg Met Val Arg His Tyr Trp Ala Leu Lys Gly Gln Pro Asn Lys
130 135 140
Lys Thr Ile Ile Ser Arg Val Asn Gly Tyr His Gly Ser Thr Val Ala
145 150 155 160
Gly Ala Ser Leu Gly Gly Met Thr Tyr Met His Glu Gln Gly Asp Leu
165 170 175
Pro Ile Pro Gly Val Val His Ile Pro Gln Pro Tyr Trp Phe Gly Glu
180 185 190
Gly Gly Asp Met Thr Pro Asp Glu Phe Gly Ile Trp Ala Ala Glu Gln
195 200 205
Leu Glu Lys Lys Ile Leu Glu Leu Gly Val Glu Asn Val Gly Ala Phe
210 215 220
Ile Ala Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro Pro Asp
225 230 235 240
Ser Tyr Trp Pro Lys Ile Lys Glu Ile Leu Ser Arg Tyr Asp Ile Leu
245 250 255
Phe Ala Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Ser Glu Trp
260 265 270
Phe Gly Ser Asp Phe Tyr Gly Leu Arg Pro Asp Met Met Thr Ile Ala
275 280 285
Lys Gly Leu Thr Ser Gly Tyr Val Pro Met Gly Gly Leu Ile Val Arg
290 295 300
Asp Glu Ile Val Ala Val Leu Asn Glu Gly Gly Asp Phe Asn His Gly
305 310 315 320
Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu Glu Asn
325 330 335
Ile Arg Ile Leu Arg Glu Glu Lys Ile Val Glu Arg Val Arg Ser Glu
340 345 350
Thr Ala Pro Tyr Leu Gln Lys Arg Leu Arg Glu Leu Ser Asp His Pro
355 360 365
Leu Val Gly Glu Val Arg Gly Val Gly Leu Leu Gly Ala Ile Glu Leu
370 375 380
Val Lys Asp Lys Thr Thr Arg Glu Arg Tyr Thr Asp Lys Gly Ala Gly
385 390 395 400
Met Ile Cys Arg Thr Phe Cys Phe Asp Asn Gly Leu Ile Met Arg Ala
405 410 415
Val Gly Asp Thr Met Ile Ile Ala Pro Pro Leu Val Ile Ser Phe Ala
420 425 430
Gln Ile Asp Glu Leu Val Glu Lys Ala Arg Thr Cys Leu Asp Leu Thr
435 440 445
Leu Ala Val Leu Gln Gly
450
<210> SEQ ID NO 11
<211> LENGTH: 467
<212> TYPE: PRT
<213> ORGANISM: Rhodobacter sphaeroides
<400> SEQUENCE: 11
Met Thr Arg Asn Asp Ala Thr Asn Ala Ala Gly Ala Val Gly Ala Ala
1 5 10 15
Met Arg Asp His Ile Leu Leu Pro Ala Gln Glu Met Ala Lys Leu Gly
20 25 30
Lys Ser Ala Gln Pro Val Leu Thr His Ala Glu Gly Ile Tyr Val His
35 40 45
Thr Glu Asp Gly Arg Arg Leu Ile Asp Gly Pro Ala Gly Met Trp Cys
50 55 60
Ala Gln Val Gly Tyr Gly Arg Arg Glu Ile Val Asp Ala Met Ala His
65 70 75 80
Gln Ala Met Val Leu Pro Tyr Ala Ser Pro Trp Tyr Met Ala Thr Ser
85 90 95
Pro Ala Ala Arg Leu Ala Glu Lys Ile Ala Thr Leu Thr Pro Gly Asp
100 105 110
Leu Asn Arg Ile Phe Phe Thr Thr Gly Gly Ser Thr Ala Val Asp Ser
115 120 125
Ala Leu Arg Phe Ser Glu Phe Tyr Asn Asn Val Leu Gly Arg Pro Gln
130 135 140
Lys Lys Arg Ile Ile Val Arg Tyr Asp Gly Tyr His Gly Ser Thr Ala
145 150 155 160
Leu Thr Ala Ala Cys Thr Gly Arg Thr Gly Asn Trp Pro Asn Phe Asp
165 170 175
Ile Ala Gln Asp Arg Ile Ser Phe Leu Ser Ser Pro Asn Pro Arg His
180 185 190
Ala Gly Asn Arg Ser Gln Glu Ala Phe Leu Asp Asp Leu Val Gln Glu
195 200 205
Phe Glu Asp Arg Ile Glu Ser Leu Gly Pro Asp Thr Ile Ala Ala Phe
210 215 220
Leu Ala Glu Pro Ile Leu Ala Ser Gly Gly Val Ile Ile Pro Pro Ala
225 230 235 240
Gly Tyr His Ala Arg Phe Lys Ala Ile Cys Glu Lys His Asp Ile Leu
245 250 255
Tyr Ile Ser Asp Glu Val Val Thr Gly Phe Gly Arg Cys Gly Glu Trp
260 265 270
Phe Ala Ser Glu Lys Val Phe Gly Val Val Pro Asp Ile Ile Thr Phe
275 280 285
Ala Lys Gly Val Thr Ser Gly Tyr Val Pro Leu Gly Gly Leu Ala Ile
290 295 300
Ser Glu Ala Val Leu Ala Arg Ile Ser Gly Glu Asn Ala Lys Gly Ser
305 310 315 320
Trp Phe Thr Asn Gly Tyr Thr Tyr Ser Asn Gln Pro Val Ala Cys Ala
325 330 335
Ala Ala Leu Ala Asn Ile Glu Leu Met Glu Arg Glu Gly Ile Val Asp
340 345 350
Gln Ala Arg Glu Met Ala Asp Tyr Phe Ala Ala Ala Leu Ala Ser Leu
355 360 365
Arg Asp Leu Pro Gly Val Ala Glu Thr Arg Ser Val Gly Leu Val Gly
370 375 380
Cys Val Gln Cys Leu Leu Asp Pro Thr Arg Ala Asp Gly Thr Ala Glu
385 390 395 400
Asp Lys Ala Phe Thr Leu Lys Ile Asp Glu Arg Cys Phe Glu Leu Gly
405 410 415
Leu Ile Val Arg Pro Leu Gly Asp Leu Cys Val Ile Ser Pro Pro Leu
420 425 430
Ile Ile Ser Arg Ala Gln Ile Asp Glu Met Val Ala Ile Met Arg Gln
435 440 445
Ala Ile Thr Glu Val Ser Ala Ala His Gly Leu Thr Ala Lys Glu Pro
450 455 460
Ala Ala Val
465
<210> SEQ ID NO 12
<211> LENGTH: 459
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli
<400> SEQUENCE: 12
Met Asn Arg Leu Pro Ser Ser Ala Ser Ala Leu Ala Cys Ser Ala His
1 5 10 15
Ala Leu Asn Leu Ile Glu Lys Arg Thr Leu Asp His Glu Glu Met Lys
20 25 30
Ala Leu Asn Arg Glu Val Ile Glu Tyr Phe Lys Glu His Val Asn Pro
35 40 45
Gly Phe Leu Glu Tyr Arg Lys Ser Val Thr Ala Gly Gly Asp Tyr Gly
50 55 60
Ala Val Glu Trp Gln Ala Gly Ser Leu Asn Thr Leu Val Asp Thr Gln
65 70 75 80
Gly Gln Glu Phe Ile Asp Cys Leu Gly Gly Phe Gly Ile Phe Asn Val
85 90 95
Gly His Arg Asn Pro Val Val Val Ser Ala Val Gln Asn Gln Leu Ala
100 105 110
Lys Gln Pro Leu His Ser Gln Glu Leu Leu Asp Pro Leu Arg Ala Met
115 120 125
Leu Ala Lys Thr Leu Ala Ala Leu Thr Pro Gly Lys Leu Lys Tyr Ser
130 135 140
Phe Phe Cys Asn Ser Gly Thr Glu Ser Val Glu Ala Ala Leu Lys Leu
145 150 155 160
Ala Lys Ala Tyr Gln Ser Pro Arg Gly Lys Phe Thr Phe Ile Ala Thr
165 170 175
Ser Gly Ala Phe His Gly Lys Ser Leu Gly Ala Leu Ser Ala Thr Ala
180 185 190
Lys Ser Thr Phe Arg Lys Pro Phe Met Pro Leu Leu Pro Gly Phe Arg
195 200 205
His Val Pro Phe Gly Asn Ile Glu Ala Met Arg Thr Ala Leu Asn Glu
210 215 220
Cys Lys Lys Thr Gly Asp Asp Val Ala Ala Val Ile Leu Glu Pro Ile
225 230 235 240
Gln Gly Glu Gly Gly Val Ile Leu Pro Pro Pro Gly Tyr Leu Thr Ala
245 250 255
Val Arg Lys Leu Cys Asp Glu Phe Gly Ala Leu Met Ile Leu Asp Glu
260 265 270
Val Gln Thr Gly Met Gly Arg Thr Gly Lys Met Phe Ala Cys Glu His
275 280 285
Glu Asn Val Gln Pro Asp Ile Leu Cys Leu Ala Lys Ala Leu Gly Gly
290 295 300
Gly Val Met Pro Ile Gly Ala Thr Ile Ala Thr Glu Glu Val Phe Ser
305 310 315 320
Val Leu Phe Asp Asn Pro Phe Leu His Thr Thr Thr Phe Gly Gly Asn
325 330 335
Pro Leu Ala Cys Ala Ala Ala Leu Ala Thr Ile Asn Val Leu Leu Glu
340 345 350
Gln Asn Leu Pro Ala Gln Ala Glu Gln Lys Gly Asp Met Leu Leu Asp
355 360 365
Gly Phe Arg Gln Leu Ala Arg Glu Tyr Pro Asp Leu Val Gln Glu Ala
370 375 380
Arg Gly Lys Gly Met Leu Met Ala Ile Glu Phe Val Asp Asn Glu Ile
385 390 395 400
Gly Tyr Asn Phe Ala Ser Glu Met Phe Arg Gln Arg Val Leu Val Ala
405 410 415
Gly Thr Leu Asn Asn Ala Lys Thr Ile Arg Ile Glu Pro Pro Leu Thr
420 425 430
Leu Thr Ile Glu Gln Cys Glu Leu Val Ile Lys Ala Ala Arg Lys Ala
435 440 445
Leu Ala Ala Met Arg Val Ser Val Glu Glu Ala
450 455
<210> SEQ ID NO 13
<211> LENGTH: 453
<212> TYPE: PRT
<213> ORGANISM: Vibrio fluvialis
<400> SEQUENCE: 13
Met Asn Lys Pro Gln Ser Trp Glu Ala Arg Ala Glu Thr Tyr Ser Leu
1 5 10 15
Tyr Gly Phe Thr Asp Met Pro Ser Leu His Gln Arg Gly Thr Val Val
20 25 30
Val Thr His Gly Glu Gly Pro Tyr Ile Val Asp Val Asn Gly Arg Arg
35 40 45
Tyr Leu Asp Ala Asn Ser Gly Leu Trp Asn Met Val Ala Gly Phe Asp
50 55 60
His Lys Gly Leu Ile Asp Ala Ala Lys Ala Gln Tyr Glu Arg Phe Pro
65 70 75 80
Gly Tyr His Ala Phe Phe Gly Arg Met Ser Asp Gln Thr Val Met Leu
85 90 95
Ser Glu Lys Leu Val Glu Val Ser Pro Phe Asp Ser Gly Arg Val Phe
100 105 110
Tyr Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr Met Val Lys Met Leu
115 120 125
Trp Phe Leu His Ala Ala Glu Gly Lys Pro Gln Lys Arg Lys Ile Leu
130 135 140
Thr Arg Trp Asn Ala Tyr His Gly Val Thr Ala Val Ser Ala Ser Met
145 150 155 160
Thr Gly Lys Pro Tyr Asn Ser Val Phe Gly Leu Pro Leu Pro Gly Phe
165 170 175
Val His Leu Thr Cys Pro His Tyr Trp Arg Tyr Gly Glu Glu Gly Glu
180 185 190
Thr Glu Glu Gln Phe Val Ala Arg Leu Ala Arg Glu Leu Glu Glu Thr
195 200 205
Ile Gln Arg Glu Gly Ala Asp Thr Ile Ala Gly Phe Phe Ala Glu Pro
210 215 220
Val Met Gly Ala Gly Gly Val Ile Pro Pro Ala Lys Gly Tyr Phe Gln
225 230 235 240
Ala Ile Leu Pro Ile Leu Arg Lys Tyr Asp Ile Pro Val Ile Ser Asp
245 250 255
Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Asn Thr Trp Gly Cys Val
260 265 270
Thr Tyr Asp Phe Thr Pro Asp Ala Ile Ile Ser Ser Lys Asn Leu Thr
275 280 285
Ala Gly Phe Phe Pro Met Gly Ala Val Ile Leu Gly Pro Glu Leu Ser
290 295 300
Lys Arg Leu Glu Thr Ala Ile Glu Ala Ile Glu Glu Phe Pro His Gly
305 310 315 320
Phe Thr Ala Ser Gly His Pro Val Gly Cys Ala Ile Ala Leu Lys Ala
325 330 335
Ile Asp Val Val Met Asn Glu Gly Leu Ala Glu Asn Val Arg Arg Leu
340 345 350
Ala Pro Arg Phe Glu Glu Arg Leu Lys His Ile Ala Glu Arg Pro Asn
355 360 365
Ile Gly Glu Tyr Arg Gly Ile Gly Phe Met Trp Ala Leu Glu Ala Val
370 375 380
Lys Asp Lys Ala Ser Lys Thr Pro Phe Asp Gly Asn Leu Ser Val Ser
385 390 395 400
Glu Arg Ile Ala Asn Thr Cys Thr Asp Leu Gly Leu Ile Cys Arg Pro
405 410 415
Leu Gly Gln Ser Val Val Leu Cys Pro Pro Phe Ile Leu Thr Glu Ala
420 425 430
Gln Met Asp Glu Met Phe Asp Lys Leu Glu Lys Ala Leu Asp Lys Val
435 440 445
Phe Ala Glu Val Ala
450
<210> SEQ ID NO 14
<211> LENGTH: 224
<212> TYPE: PRT
<213> ORGANISM: Bacillus subtilis
<400> SEQUENCE: 14
Met Lys Ile Tyr Gly Ile Tyr Met Asp Arg Pro Leu Ser Gln Glu Glu
1 5 10 15
Asn Glu Arg Phe Met Ser Phe Ile Ser Pro Glu Lys Arg Glu Lys Cys
20 25 30
Arg Arg Phe Tyr His Lys Glu Asp Ala His Arg Thr Leu Leu Gly Asp
35 40 45
Val Leu Val Arg Ser Val Ile Ser Arg Gln Tyr Gln Leu Asp Lys Ser
50 55 60
Asp Ile Arg Phe Ser Thr Gln Glu Tyr Gly Lys Pro Cys Ile Pro Asp
65 70 75 80
Leu Pro Asp Ala His Phe Asn Ile Ser His Ser Gly Arg Trp Val Ile
85 90 95
Cys Ala Phe Asp Ser Gln Pro Ile Gly Ile Asp Ile Glu Lys Thr Lys
100 105 110
Pro Ile Ser Leu Glu Ile Ala Lys Arg Phe Phe Ser Lys Thr Glu Tyr
115 120 125
Ser Asp Leu Leu Ala Lys Asp Lys Asp Glu Gln Thr Asp Tyr Phe Tyr
130 135 140
His Leu Trp Ser Met Lys Glu Ser Phe Ile Lys Gln Glu Gly Lys Gly
145 150 155 160
Leu Ser Leu Pro Leu Asp Ser Phe Ser Val Arg Leu His Gln Asp Gly
165 170 175
Gln Val Ser Ile Glu Leu Pro Asp Ser His Ser Pro Cys Tyr Ile Lys
180 185 190
Thr Tyr Glu Val Asp Pro Gly Tyr Lys Met Ala Val Cys Ala Ala His
195 200 205
Pro Asp Phe Pro Glu Asp Ile Thr Met Val Ser Tyr Glu Glu Leu Leu
210 215 220
<210> SEQ ID NO 15
<211> LENGTH: 222
<212> TYPE: PRT
<213> ORGANISM: Nocardia sp. NRRL 5646
<400> SEQUENCE: 15
Met Ile Glu Thr Ile Leu Pro Ala Gly Val Glu Ser Ala Glu Leu Leu
1 5 10 15
Glu Tyr Pro Glu Asp Leu Lys Ala His Pro Ala Glu Glu His Leu Ile
20 25 30
Ala Lys Ser Val Glu Lys Arg Arg Arg Asp Phe Ile Gly Ala Arg His
35 40 45
Cys Ala Arg Leu Ala Leu Ala Glu Leu Gly Glu Pro Pro Val Ala Ile
50 55 60
Gly Lys Gly Glu Arg Gly Ala Pro Ile Trp Pro Arg Gly Val Val Gly
65 70 75 80
Ser Leu Thr His Cys Asp Gly Tyr Arg Ala Ala Ala Val Ala His Lys
85 90 95
Met Arg Phe Arg Ser Ile Gly Ile Asp Ala Glu Pro His Ala Thr Leu
100 105 110
Pro Glu Gly Val Leu Asp Ser Val Ser Leu Pro Pro Glu Arg Glu Trp
115 120 125
Leu Lys Thr Thr Asp Ser Ala Leu His Leu Asp Arg Leu Leu Phe Cys
130 135 140
Ala Lys Glu Ala Thr Tyr Lys Ala Trp Trp Pro Leu Thr Ala Arg Trp
145 150 155 160
Leu Gly Phe Glu Glu Ala His Ile Thr Phe Glu Ile Glu Asp Gly Ser
165 170 175
Ala Asp Ser Gly Asn Gly Thr Phe His Ser Glu Leu Leu Val Pro Gly
180 185 190
Gln Thr Asn Asp Gly Gly Thr Pro Leu Leu Ser Phe Asp Gly Arg Trp
195 200 205
Leu Ile Ala Asp Gly Phe Ile Leu Thr Ala Ile Ala Tyr Ala
210 215 220
<210> SEQ ID NO 16
<211> LENGTH: 269
<212> TYPE: PRT
<213> ORGANISM: Bacillus cereus
<400> SEQUENCE: 16
Met Ile Asn Lys Thr Leu Leu Gln Lys Arg Phe Asn Val Ala Ala Val
1 5 10 15
Ser Tyr Asp Gln Tyr Ala Asn Val Gln Lys Lys Met Ala His Ser Leu
20 25 30
Leu Ser Thr Leu Asn Arg Arg Tyr Ser Thr Asn Ser Ser Ile Arg Ile
35 40 45
Leu Glu Leu Gly Cys Gly Thr Gly Tyr Val Thr Glu Gln Leu Ser Asn
50 55 60
Leu Phe Pro Lys Ala Gln Ile Thr Ala Ile Asp Phe Ala Glu Ser Met
65 70 75 80
Ile Ala Val Ala Lys Thr Arg Gln Asn Val Asn Asn Val Thr Phe Tyr
85 90 95
Cys Glu Asp Ile Glu Arg Leu Arg Leu Glu Glu Thr Tyr Asp Val Ile
100 105 110
Ile Ser Asn Ala Thr Phe Gln Trp Leu Asn Asp Leu Lys Gln Val Ile
115 120 125
Thr Asn Leu Phe Arg His Leu Ser Ile Glu Gly Ile Leu Leu Phe Ser
130 135 140
Thr Phe Gly Gln Glu Thr Phe Gln Glu Leu His Ala Ser Phe Gln Arg
145 150 155 160
Ala Lys Glu Glu Lys Asn Ile Gln Asn Glu Thr Ser Ile Gly Gln Arg
165 170 175
Phe Tyr Ser Lys Asn Gln Leu Arg His Ile Cys Glu Ile Glu Thr Gly
180 185 190
Asp Val His Val Ser Glu Thr Cys Tyr Ile Glu Arg Phe Thr Glu Val
195 200 205
Arg Glu Phe Leu His Ser Ile Arg Lys Val Gly Ala Thr Asn Ser Asn
210 215 220
Glu Glu Ser Tyr Cys Gln Ser Pro Ser Leu Phe Arg Ala Met Leu Arg
225 230 235 240
Ile Tyr Glu Arg Asp Phe Thr Gly Asn Glu Gly Ile Met Ala Thr Tyr
245 250 255
His Ala Leu Phe Val His Ile Thr Lys Glu Gly Lys Arg
260 265
<210> SEQ ID NO 17
<211> LENGTH: 256
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli
<400> SEQUENCE: 17
Met Asn Asn Ile Trp Trp Gln Thr Lys Gly Gln Gly Asn Val His Leu
1 5 10 15
Val Leu Leu His Gly Trp Gly Leu Asn Ala Glu Val Trp Arg Cys Ile
20 25 30
Asp Glu Glu Leu Ser Ser His Phe Thr Leu His Leu Val Asp Leu Pro
35 40 45
Gly Phe Gly Arg Ser Arg Gly Phe Gly Ala Leu Ser Leu Ala Asp Met
50 55 60
Ala Glu Ala Val Leu Gln Gln Ala Pro Asp Lys Ala Ile Trp Leu Gly
65 70 75 80
Trp Ser Leu Gly Gly Leu Val Ala Ser Gln Ile Ala Leu Thr His Pro
85 90 95
Glu Arg Val Gln Ala Leu Val Thr Val Ala Ser Ser Pro Cys Phe Ser
100 105 110
Ala Arg Asp Glu Trp Pro Gly Ile Lys Pro Asp Val Leu Ala Gly Phe
115 120 125
Gln Gln Gln Leu Ser Asp Asp Phe Gln Arg Thr Val Glu Arg Phe Leu
130 135 140
Ala Leu Gln Thr Met Gly Thr Glu Thr Ala Arg Gln Asp Ala Arg Ala
145 150 155 160
Leu Lys Lys Thr Val Leu Ala Leu Pro Met Pro Glu Val Asp Val Leu
165 170 175
Asn Gly Gly Leu Glu Ile Leu Lys Thr Val Asp Leu Arg Gln Pro Leu
180 185 190
Gln Asn Val Ser Met Pro Phe Leu Arg Leu Tyr Gly Tyr Leu Asp Gly
195 200 205
Leu Val Pro Arg Lys Val Val Pro Met Leu Asp Lys Leu Trp Pro His
210 215 220
Ser Glu Ser Tyr Ile Phe Ala Lys Ala Ala His Ala Pro Phe Ile Ser
225 230 235 240
His Pro Ala Glu Phe Cys His Leu Leu Val Ala Leu Lys Gln Arg Val
245 250 255
<210> SEQ ID NO 18
<211> LENGTH: 556
<212> TYPE: PRT
<213> ORGANISM: Clostridium cylindrosporum
<400> SEQUENCE: 18
Met Lys Thr Asp Val Gln Ile Ala Gln Glu Ala Gln Met Lys Pro Ile
1 5 10 15
Thr Glu Val Ala Asn Tyr Leu Gly Ile Gln Asp Asp Glu Leu Glu Leu
20 25 30
Tyr Gly Lys Tyr Lys Ala Lys Val Ser Leu Asp Val Leu Glu Arg Gln
35 40 45
Lys Asp Lys Glu Asp Ala Lys Leu Val Leu Val Thr Ala Ile Asn Pro
50 55 60
Thr Pro Ala Gly Glu Gly Lys Thr Thr Thr Asn Val Gly Leu Ser Met
65 70 75 80
Gly Leu Asn Lys Ile Gly Lys Arg Thr Ile Thr Ala Leu Arg Glu Pro
85 90 95
Ser Leu Gly Pro Cys Phe Gly Val Lys Gly Gly Ala Ala Gly Gly Gly
100 105 110
Tyr Ala Gln Val Val Pro Met Asp Asp Ile Asn Leu His Phe Thr Gly
115 120 125
Asp Phe His Ala Ile Thr Ser Ala His Asn Leu Leu Ala Ala Leu Leu
130 135 140
Asp Asn His Leu His Gln Gly Asn Ala Leu Asn Ile Asn Pro Lys Lys
145 150 155 160
Ile Val Trp Lys Arg Val Ile Asp Met Asn Asp Arg Ser Leu Arg Asn
165 170 175
Val Ile Ile Gly Leu Gly Gly Asn Gly Asp Gly Phe Val Arg Gln Ala
180 185 190
Gln Phe Asp Ile Thr Val Ala Ser Glu Ile Met Ala Ile Leu Cys Leu
195 200 205
Ala Thr Ser Met Ser Asp Leu Lys Glu Arg Leu Ser Lys Met Ile Val
210 215 220
Ala Tyr Ala Lys Asp Gly Ser Ala Val Thr Ala Gly Gln Leu Glu Ala
225 230 235 240
Thr Gly Ala Met Ala Leu Leu Leu Lys Asp Ala Val Lys Pro Asn Leu
245 250 255
Val Gln Thr Leu Glu Asn Thr Pro Ala Phe Ile His Gly Gly Pro Phe
260 265 270
Ala Asn Ile Ala His Gly Cys Asn Ser Val Leu Ala Thr Lys Val Ala
275 280 285
Leu Lys Leu Ala Asp Tyr Val Val Thr Glu Gly Gly Phe Gly Ala Asp
290 295 300
Leu Gly Ala Glu Lys Phe Phe Asp Ile Lys Ser Arg Phe Ala Gly Leu
305 310 315 320
Lys Pro Asn Cys Asp Val Ser Val Ala Thr Val Arg Ala Leu Lys Met
325 330 335
Asn Gly Gly Val Pro Lys Thr Glu Leu Ala Ala Glu Asn Val Glu Ala
340 345 350
Val Lys Lys Gly Val Ala Asn Leu Glu Arg His Ile Glu Asn Val Ala
355 360 365
Lys Phe Gly Val Pro Ala Val Val Ala Ile Asn Lys Phe Pro Leu Asp
370 375 380
Thr Glu Ala Glu Leu Lys Ala Val Glu Asp Ala Cys Asn Ala Lys Gly
385 390 395 400
Ala Asp Val Val Leu Ser Asp Val Trp Ala Asn Gly Gly Glu Gly Gly
405 410 415
Val Glu Met Ala Lys Lys Val Val Glu Ile Cys Glu Lys Asn Glu Ala
420 425 430
Asn Phe Ala Pro Leu Tyr Asp Val Asn Leu Ser Ile Pro Glu Lys Ile
435 440 445
Glu Lys Ile Ala Thr Thr Ile Tyr Arg Ala Asp Gly Val Asp Phe Thr
450 455 460
Ser Asp Cys Lys Lys Gln Ile Ala Glu Leu Glu Lys Leu Gly Leu Asp
465 470 475 480
Lys Met Pro Ile Cys Met Ala Lys Thr Gln Tyr Ser Phe Ser Asp Asp
485 490 495
Pro Thr Leu Leu Gly Ala Pro Thr Gly Phe Arg Ile Thr Val Arg Glu
500 505 510
Val Arg Val Ser Ala Gly Ala Gly Phe Ile Val Ala Leu Thr Gly Asn
515 520 525
Met Met Thr Met Pro Gly Leu Pro Lys Val Pro Ala Ala Asn Gly Met
530 535 540
Asp Ile Leu Glu Ser Gly Glu Ile Ile Gly Leu Ser
545 550 555
<210> SEQ ID NO 19
<211> LENGTH: 556
<212> TYPE: PRT
<213> ORGANISM: Alkaliphilus oremlandii (strain OhILAs)
<400> SEQUENCE: 19
Met Lys Thr Asp Val Gln Ile Ala Gln Glu Ala Lys Met Leu Pro Ile
1 5 10 15
Ala Asp Ile Ala Ala Gly Leu Gly Ile Gln Asp Asp Glu Leu Glu Leu
20 25 30
Tyr Gly Lys Tyr Lys Ala Lys Val Ser Leu Asp Val Phe Asp Arg Leu
35 40 45
Lys Asp Lys Pro Asp Gly Lys Leu Ile Leu Val Thr Ala Ile Asn Pro
50 55 60
Thr Pro Ala Gly Glu Gly Lys Thr Thr Thr Asn Val Gly Leu Ser Met
65 70 75 80
Gly Leu Asn Lys Ile Gly Lys Lys Thr Ile Thr Ala Leu Arg Glu Pro
85 90 95
Ser Leu Gly Pro Asn Phe Gly Val Lys Gly Gly Ala Ala Gly Gly Gly
100 105 110
Tyr Ala Gln Val Val Pro Met Glu Asp Ile Asn Leu His Phe Thr Gly
115 120 125
Asp Ile His Ala Ile Thr Thr Ala His Asn Leu Leu Ala Ala Leu Leu
130 135 140
Asp Asn His Leu His Gln Gly Asn Lys Leu Asn Ile Asp Ser Arg Arg
145 150 155 160
Ile Val Trp Arg Arg Val Leu Asp Met Asn Asp Arg Ala Leu Arg Asn
165 170 175
Thr Val Ile Gly Leu Gly Ser Arg Gly Asp Gly Val Pro Arg Gln Asp
180 185 190
Gly Phe Asp Ile Thr Val Ala Ser Glu Ile Met Ala Ile Leu Cys Leu
195 200 205
Ser Asn Ser Leu Glu Asp Leu Lys Asp Arg Ile Ser Arg Met Val Val
210 215 220
Ala Tyr Asn Leu Asp Asn Gln Pro Ile Thr Val Asn Asp Leu Glu Ala
225 230 235 240
Thr Gly Ala Leu Ser Leu Leu Leu Lys Asp Ala Ile Lys Pro Asn Leu
245 250 255
Val Gln Thr Leu Glu Asn Thr Pro Ala Phe Ile His Gly Gly Pro Phe
260 265 270
Ala Asn Ile Ala His Gly Cys Asn Ser Val Leu Ala Thr Lys Leu Gly
275 280 285
Leu Lys Leu Ala Asp Tyr Val Val Thr Glu Ala Gly Phe Gly Ala Asp
290 295 300
Leu Gly Ala Glu Lys Phe Phe Asp Ile Lys Cys Arg Phe Ala Gly Leu
305 310 315 320
Lys Pro Asp Cys Ala Val Ile Val Ala Thr Val Arg Ala Leu Lys Asn
325 330 335
His Gly Gly Val Pro Lys Ala Glu Leu Asn Asn Glu Asn Leu Glu Ala
340 345 350
Leu Glu Lys Gly Tyr Arg Asn Leu Glu Lys His Ile Glu Asn Val Gln
355 360 365
Lys Phe Gly Val Pro Ala Val Val Ala Ile Asn Lys Phe Pro Thr Asp
370 375 380
Thr Glu Ala Glu Leu Asn Phe Leu Arg Lys His Cys Ala Glu Met Gly
385 390 395 400
Ala Glu Val Val Leu Ser Asp Val Trp Ala Asn Gly Gly Asp Gly Gly
405 410 415
Ile Glu Met Ala Lys Lys Val Val Glu Val Val Glu Ser Lys Glu Ser
420 425 430
Asn Phe Lys Pro Leu Tyr Asp Val Asn Ala Ser Ile Val Glu Lys Ile
435 440 445
Asn Thr Ile Ala Lys Glu Val Tyr Gly Ala Asp Gly Val Asp Phe Thr
450 455 460
Lys Ser Ala Gln Thr Gln Ile Lys Lys Tyr Glu Asp Leu Gly Leu Asp
465 470 475 480
Lys Met Pro Ile Cys Met Ala Lys Thr Gln Tyr Ser Leu Ser Asp Asp
485 490 495
Pro Ser Leu Ile Gly Arg Pro Ser Gly Phe Arg Ile Thr Val Lys Glu
500 505 510
Ile Arg Leu Ser Ala Gly Ala Gly Phe Leu Val Ala Leu Thr Gly Asp
515 520 525
Ile Met Val Met Pro Gly Leu Pro Lys Val Pro Ala Ala Asn His Met
530 535 540
Asp Ile Leu Glu Ser Gly Glu Ile Ile Gly Leu Phe
545 550 555
<210> SEQ ID NO 20
<211> LENGTH: 556
<212> TYPE: PRT
<213> ORGANISM: Clostridium acidurici
<400> SEQUENCE: 20
Met Lys Thr Asp Ile Gln Ile Ala Gln Glu Ala Gln Met Lys His Ile
1 5 10 15
Lys Asp Val Ala Glu Leu Ile Asp Ile His Glu Asp Asp Leu Glu Leu
20 25 30
Tyr Gly Lys Tyr Lys Ala Lys Val Ser Leu Asp Val Leu Asp Gln Leu
35 40 45
Lys Asp Lys Pro Asp Gly Lys Leu Val Leu Val Thr Ala Ile Asn Pro
50 55 60
Thr Pro Ala Gly Glu Gly Lys Thr Thr Thr Asn Ile Gly Leu Ser Met
65 70 75 80
Gly Leu Asn Lys Leu Gly Lys Lys Thr Ser Thr Ala Leu Arg Glu Pro
85 90 95
Ser Leu Gly Pro Ser Phe Gly Val Lys Gly Gly Ala Ala Gly Gly Gly
100 105 110
Tyr Ala Gln Val Val Pro Met Ala Asp Ile Asn Leu His Phe Thr Gly
115 120 125
Asp Phe His Ala Ile Thr Ser Ala His Ser Leu Leu Ala Ala Leu Val
130 135 140
Asp Asn His Leu His His Gly Asn Ala Leu Arg Ile Asp Thr Asn Arg
145 150 155 160
Ile Val Trp Lys Arg Val Val Asp Met Asn Asp Arg Ala Leu Arg Lys
165 170 175
Ile Val Val Gly Leu Gly Gly Lys Ala Gln Gly Ile Thr Arg Glu Asp
180 185 190
Gly Phe Asp Ile Thr Val Ala Ser Glu Ile Met Ala Ile Leu Cys Leu
195 200 205
Ala Asn Asp Arg Glu Asp Leu Lys Glu Arg Leu Gly Asn Met Val Val
210 215 220
Ala Tyr Asn Val Asp Gly Asp Ala Val Arg Ala Lys Asp Leu Glu Ala
225 230 235 240
Gln Gly Ala Leu Thr Leu Ile Leu Lys Asp Ala Ile Asn Pro Asn Ile
245 250 255
Val Gln Thr Leu Glu Asn Thr Pro Ala Phe Ile His Gly Gly Pro Phe
260 265 270
Ala Asn Ile Ala His Gly Cys Asn Ser Val Leu Ala Thr Lys Leu Ala
275 280 285
Leu Lys Thr Gly Asp Tyr Ala Val Thr Glu Ala Gly Phe Gly Ala Asp
290 295 300
Leu Gly Ala Glu Lys Phe Phe Asp Ile Lys Cys Arg Tyr Ala Gly Leu
305 310 315 320
Asn Pro Asp Val Ala Val Ile Val Ala Thr Val Arg Ala Leu Lys Met
325 330 335
His Gly Gly Val Ala Lys Glu Asp Leu Gly Thr Glu Asn Leu Asp Ala
340 345 350
Leu Ala Lys Gly Met Thr Asn Leu Glu Arg His Ile Glu Asn Val Ala
355 360 365
Lys Phe Gly Val Pro Ser Val Val Ala Ile Asn Ala Phe Pro Thr Asp
370 375 380
Thr Glu Ala Glu Lys Gln Leu Val Phe Asp Lys Cys Lys Glu Met Gly
385 390 395 400
Val Asp Val Ala Ile Ser Asp Val Phe Ala Lys Gly Gly Asp Gly Gly
405 410 415
Val Glu Leu Ala Gln Lys Val Ile Asp Val Cys Glu Asn Lys Lys Ser
420 425 430
Asp Phe Lys Val Leu Tyr Asp Val Glu Glu Ser Ile Pro Glu Lys Ile
435 440 445
Thr Lys Ile Ala Lys Glu Ile Tyr Arg Ala Asp Lys Val Asn Phe Ser
450 455 460
Lys Ala Ala Lys Lys Gln Ile Ala Glu Leu Glu Lys Leu Gly Leu Asp
465 470 475 480
Lys Leu Pro Ile Cys Met Ala Lys Thr Gln Tyr Ser Phe Ser Asp Asp
485 490 495
Pro Ala Leu Leu Gly Ala Pro Glu Gly Phe Glu Leu Thr Ile Arg Asp
500 505 510
Leu Glu Leu Ala Ala Gly Ala Gly Phe Ile Val Ala Leu Thr Gly Asp
515 520 525
Ile Met Arg Met Pro Gly Leu Pro Lys Val Pro Ala Ala Asn Arg Met
530 535 540
Asp Val Leu Pro Asn Gly Glu Ile Ile Gly Leu Phe
545 550 555
<210> SEQ ID NO 21
<211> LENGTH: 556
<212> TYPE: PRT
<213> ORGANISM: Eubacterium acidaminophilum
<400> SEQUENCE: 21
Met Lys Thr Asp Val Gln Ile Ala Gln Glu Ala Lys Met Leu Pro Ile
1 5 10 15
Met Glu Val Ala Lys Gln Ile Gly Leu Gly Glu Asp Asp Ile Glu Leu
20 25 30
Tyr Gly Lys Tyr Lys Ala Lys Ile Ser Leu Asp Val Tyr Lys Arg Leu
35 40 45
Ala Asp Lys Pro Asp Gly Lys Leu Val Leu Val Thr Ala Ile Asn Pro
50 55 60
Thr Pro Ala Gly Glu Gly Lys Thr Thr Thr Asn Val Gly Leu Ser Met
65 70 75 80
Gly Leu Asn Lys Ile Gly Lys Lys Thr Ile Thr Ala Leu Asn Glu Pro
85 90 95
Ser Leu Gly Pro Cys Phe Gly Val Lys Gly Gly Ala Ala Gly Gly Gly
100 105 110
Tyr Ala Gln Val Val Pro Met Asp Asp Ile Asn Leu His Phe Thr Gly
115 120 125
Asp Ile His Ala Ile Thr Thr Ala His Asn Leu Leu Ala Ala Leu Met
130 135 140
Asp Asn His Ile Lys Gln Gly Asn Ala Leu Gly Ile Asp Ile Asn Lys
145 150 155 160
Ile Thr Trp Lys Arg Val Leu Asp Met Asn Asp Arg Ala Leu Arg Asp
165 170 175
Ile Val Ile Gly Leu Gly Gly Thr Ala Asn Gly Ile Pro Arg Gln Asp
180 185 190
Gly Phe Asp Ile Thr Val Ala Ser Glu Ile Met Ala Ile Met Cys Leu
195 200 205
Ala Thr Ser Leu Ser Asp Leu Lys Asp Arg Leu Ser Arg Met Ile Val
210 215 220
Gly Tyr Thr Ser Arg Arg Leu Ala Val Thr Ala Asp Ser Leu Thr Leu
225 230 235 240
Arg Gly Ala Leu Ala Leu Leu Leu Lys Asp Ala Leu Lys Pro Asn Leu
245 250 255
Val Gln Thr Leu Glu Asn Thr Pro Ala Ile Ile His Gly Gly Pro Phe
260 265 270
Ala Asn Ile Ala His Gly Cys Asn Ser Val Thr Thr Thr Lys Thr Ala
275 280 285
Leu Lys Ile Ala Asp Tyr Val Val Thr Glu Ala Gly Phe Gly Ala Asp
290 295 300
Leu Gly Ala Glu Lys Phe Phe Asp Ile Lys Cys Arg Phe Ala Asp Leu
305 310 315 320
Lys Pro Asp Val Ala Val Ile Val Ala Thr Val Arg Ala Leu Lys Asn
325 330 335
His Gly Gly Val Ala Lys Ala Asn Leu Gly Ala Glu Asn Met Lys Ala
340 345 350
Leu Glu Asp Gly Phe Gly Asn Leu Glu Arg His Ile Glu Asn Val His
355 360 365
Lys Phe Gly Val Pro Ala Val Val Ala Ile Asn Ala Phe Pro Thr Asp
370 375 380
Thr Glu Lys Glu Leu Lys Phe Val Glu Asp Ala Cys Arg Lys Leu Gly
385 390 395 400
Ala Asp Val Val Leu Ser Glu Val Trp Ala Lys Gly Gly Glu Gly Gly
405 410 415
Val Glu Leu Ala Lys Lys Val Val Glu Val Thr Glu Lys Gly Ala Ala
420 425 430
Lys Phe Lys Pro Leu Tyr Pro Ala Glu Met Pro Leu Lys Gln Lys Ile
435 440 445
Glu Thr Ile Ala Lys Glu Ile Tyr Arg Ala Asp Gly Val Glu Phe Ser
450 455 460
Ala Lys Ala Ser Lys Glu Leu Asp Lys Phe Glu Lys Leu Gly Phe Gly
465 470 475 480
Asn Leu Pro Ile Cys Val Ala Lys Thr Gln Tyr Ser Phe Ser Asp Asn
485 490 495
Pro Asn Leu Lys Gly Ala Pro Lys Gly Phe Thr Val Ser Val Ser Asn
500 505 510
Ala Arg Ile Ser Ala Gly Ala Gly Phe Ile Val Val Leu Thr Gly Asp
515 520 525
Ile Met Thr Met Pro Gly Leu Pro Lys Val Pro Ala Ala Asn His Met
530 535 540
Asp Val Leu Glu Ser Gly Glu Ile Val Gly Leu Phe
545 550 555
<210> SEQ ID NO 22
<211> LENGTH: 556
<212> TYPE: PRT
<213> ORGANISM: Desulfitobacterium hafniense (strain Y51)
<400> SEQUENCE: 22
Met Lys Thr Asp Ile Glu Ile Ala Gln Glu Ala Thr Met Lys Pro Ile
1 5 10 15
Thr Glu Ile Ala Gln Gly Leu Asp Leu Leu Glu Asp Glu Ile Glu Leu
20 25 30
Tyr Gly Lys Tyr Lys Ala Lys Val Asn Phe Ser Ala Trp Glu Arg Leu
35 40 45
Lys Asp Lys Pro Asp Ala Lys Leu Ile Leu Val Thr Ala Ile Asn Pro
50 55 60
Thr Pro Ala Gly Glu Gly Lys Thr Thr Thr Thr Val Gly Leu Gly Gln
65 70 75 80
Ala Met Ser Lys Ile Gly Lys Asn Ala Met Ile Ala Leu Arg Glu Pro
85 90 95
Ser Leu Gly Pro Cys Phe Gly Val Lys Gly Gly Ala Ala Gly Gly Gly
100 105 110
Tyr Ala Gln Val Val Pro Met Glu Asp Ile Asn Leu His Phe Thr Gly
115 120 125
Asp Phe His Ala Ile Thr Ser Thr His Asn Leu Leu Ala Ala Leu Leu
130 135 140
Asp Asn His Ile Gln Gln Gly Asn Leu Leu Asn Ile Asp Pro Arg Gln
145 150 155 160
Ile Val Phe Arg Arg Val Met Asp Met Asn Asp Arg Ala Leu Arg Lys
165 170 175
Ile Val Ile Gly Leu Gly Gly Arg Thr Glu Gly Ile Pro Arg Glu Asn
180 185 190
Gly Phe Asp Ile Thr Val Ala Ser Glu Ile Met Ala Ile Leu Cys Leu
195 200 205
Ala Lys Asp Leu Met Asp Leu Lys Glu Arg Phe Gly Arg Ile Val Val
210 215 220
Ala Tyr Thr Tyr Asp Gly Lys Ala Ile Thr Ala His Asp Leu Glu Ala
225 230 235 240
Glu Gly Ala Met Ala Leu Leu Met Lys Asp Ala Ile Lys Pro Asn Leu
245 250 255
Val Gln Thr Leu Glu Asn Thr Pro Val Phe Ile His Gly Gly Pro Phe
260 265 270
Ala Asn Ile Ala His Gly Cys Asn Ser Val Val Ala Thr Arg Met Ala
275 280 285
Met Lys Leu Ala Asp Tyr Val Ile Thr Glu Ala Gly Phe Gly Ala Asp
290 295 300
Leu Gly Ala Glu Lys Phe Tyr Asp Leu Lys Cys Arg Phe Ala Glu Leu
305 310 315 320
Lys Pro Ala Ala Thr Val Ile Val Ala Thr Val Arg Ala Leu Lys Met
325 330 335
Asn Gly Gly Val Ala Lys Glu Asp Leu Gly Pro Glu Asn Leu Glu Ala
340 345 350
Leu Ala Lys Gly Ile Val Asn Leu Glu Lys His Ile Glu Asn Ile Gly
355 360 365
Lys Phe Gly Val Pro Ala Val Val Ala Ile Asn Arg Phe Pro Thr Asp
370 375 380
Thr Asp Ala Glu Leu Glu Phe Val Ala Glu Arg Cys Arg Gln Leu Gly
385 390 395 400
Ala Glu Phe Ala Leu Ser Glu Val Phe Thr Lys Gly Gly Glu Gly Gly
405 410 415
Ile Glu Leu Ala Lys Ala Val Leu Asn Ile Val Asp Asn Lys Glu Ser
420 425 430
Asn Phe His Val Leu Tyr Glu Leu Asp Leu Pro Ile Ala Lys Lys Ile
435 440 445
Glu Thr Ile Cys Lys Glu Val Tyr Gly Ala Asp Gly Val Asn Phe Thr
450 455 460
Lys Glu Ala Leu Thr Ser Met Lys Lys Tyr Glu Glu Leu Gly Tyr Gly
465 470 475 480
Gln Leu Pro Ile Cys Met Ala Lys Thr Gln Tyr Ser Leu Thr Asp Asp
485 490 495
Gln Asn Val Leu Gly Arg Pro Ser Gly Phe Thr Ile Thr Val Arg Glu
500 505 510
Leu Arg Leu Ser Ala Gly Ala Gly Phe Leu Val Ala Ile Thr Gly Ala
515 520 525
Ile Met Thr Met Pro Gly Leu Pro Lys Arg Pro Ala Ala Leu Arg Met
530 535 540
Asp Ile Asp Ala Ala Gly Arg Ile Thr Gly Leu Phe
545 550 555
<210> SEQ ID NO 23
<211> LENGTH: 369
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli (strain K12)
<400> SEQUENCE: 23
Met Lys Ser Arg Ala Ala Val Ala Phe Ala Pro Gly Lys Pro Leu Glu
1 5 10 15
Ile Val Glu Ile Asp Val Ala Pro Pro Lys Lys Gly Glu Val Leu Ile
20 25 30
Lys Val Thr His Thr Gly Val Cys His Thr Asp Ala Phe Thr Leu Ser
35 40 45
Gly Asp Asp Pro Glu Gly Val Phe Pro Val Val Leu Gly His Glu Gly
50 55 60
Ala Gly Val Val Val Glu Val Gly Glu Gly Val Thr Ser Val Lys Pro
65 70 75 80
Gly Asp His Val Ile Pro Leu Tyr Thr Ala Glu Cys Gly Glu Cys Glu
85 90 95
Phe Cys Arg Ser Gly Lys Thr Asn Leu Cys Val Ala Val Arg Glu Thr
100 105 110
Gln Gly Lys Gly Leu Met Pro Asp Gly Thr Thr Arg Phe Ser Tyr Asn
115 120 125
Gly Gln Pro Leu Tyr His Tyr Met Gly Cys Ser Thr Phe Ser Glu Tyr
130 135 140
Thr Val Val Ala Glu Val Ser Leu Ala Lys Ile Asn Pro Glu Ala Asn
145 150 155 160
His Glu His Val Cys Leu Leu Gly Cys Gly Val Thr Thr Gly Ile Gly
165 170 175
Ala Val His Asn Thr Ala Lys Val Gln Pro Gly Asp Ser Val Ala Val
180 185 190
Phe Gly Leu Gly Ala Ile Gly Leu Ala Val Val Gln Gly Ala Arg Gln
195 200 205
Ala Lys Ala Gly Arg Ile Ile Ala Ile Asp Thr Asn Pro Lys Lys Phe
210 215 220
Asp Leu Ala Arg Arg Phe Gly Ala Thr Asp Cys Ile Asn Pro Asn Asp
225 230 235 240
Tyr Asp Lys Pro Ile Lys Asp Val Leu Leu Asp Ile Asn Lys Trp Gly
245 250 255
Ile Asp His Thr Phe Glu Cys Ile Gly Asn Val Asn Val Met Arg Ala
260 265 270
Ala Leu Glu Ser Ala His Arg Gly Trp Gly Gln Ser Val Ile Ile Gly
275 280 285
Val Ala Val Ala Gly Gln Glu Ile Ser Thr Arg Pro Phe Gln Leu Val
290 295 300
Thr Gly Arg Val Trp Lys Gly Ser Ala Phe Gly Gly Val Lys Gly Arg
305 310 315 320
Ser Gln Leu Pro Gly Met Val Glu Asp Ala Met Lys Gly Asp Ile Asp
325 330 335
Leu Glu Pro Phe Val Thr His Thr Met Ser Leu Asp Glu Ile Asn Asp
340 345 350
Ala Phe Asp Leu Met His Glu Gly Lys Ser Ile Arg Thr Val Ile Arg
355 360 365
Tyr
<210> SEQ ID NO 24
<211> LENGTH: 369
<212> TYPE: PRT
<213> ORGANISM: Shigella sonnei (strain Ss046)
<400> SEQUENCE: 24
Met Lys Ser Arg Ala Ala Val Ala Phe Ala Pro Gly Lys Pro Leu Glu
1 5 10 15
Ile Val Glu Ile Asp Val Ala Pro Pro Lys Lys Gly Glu Val Leu Ile
20 25 30
Lys Val Thr His Thr Gly Val Cys His Thr Asp Ala Phe Thr Leu Ser
35 40 45
Gly Asp Asp Pro Glu Gly Val Phe Pro Val Val Leu Gly His Glu Gly
50 55 60
Ala Gly Val Val Val Glu Val Gly Glu Gly Val Thr Ser Val Lys Pro
65 70 75 80
Gly Asp His Val Ile Pro Leu Tyr Thr Ala Glu Cys Gly Glu Cys Glu
85 90 95
Phe Cys Arg Ser Gly Lys Thr Asn Leu Cys Val Ala Val Arg Glu Thr
100 105 110
Gln Gly Lys Gly Leu Met Pro Asp Gly Thr Thr Arg Phe Ser Tyr Asn
115 120 125
Gly Gln Pro Leu Tyr His Tyr Met Gly Cys Ser Thr Phe Ser Glu Tyr
130 135 140
Thr Val Val Ala Glu Val Ser Leu Ala Lys Ile Asn Pro Glu Ala Asn
145 150 155 160
His Glu His Val Cys Leu Leu Gly Cys Gly Val Thr Thr Gly Ile Gly
165 170 175
Ala Val His Asn Thr Ala Lys Val Gln Pro Gly Asp Ser Val Ala Val
180 185 190
Phe Gly Leu Gly Ala Ile Gly Leu Ala Val Val Gln Gly Ala Arg Gln
195 200 205
Ala Lys Ala Gly Arg Ile Ile Ala Ile Asp Thr Asn Pro Lys Lys Phe
210 215 220
Asp Leu Ala Arg Arg Phe Gly Ala Thr Asp Cys Ile Asn Pro Asn Asp
225 230 235 240
Tyr Asp Lys Pro Ile Lys Asp Val Leu Leu Asp Ile Asn Lys Trp Gly
245 250 255
Ile Asp His Thr Phe Glu Cys Ile Gly Asn Val Asn Val Met Arg Ala
260 265 270
Ala Leu Glu Ser Ala His Arg Gly Trp Gly Gln Ser Val Ile Ile Gly
275 280 285
Val Ala Gly Ser Gly Gln Glu Ile Ser Thr Arg Pro Phe Gln Leu Val
290 295 300
Thr Gly Arg Val Trp Lys Gly Ser Ala Phe Gly Gly Val Lys Gly Arg
305 310 315 320
Ser Gln Leu Pro Gly Met Val Glu Asp Ala Met Lys Gly Asp Ile Asp
325 330 335
Leu Glu Pro Phe Val Thr His Thr Met Ser Leu Asp Glu Ile Asn Asp
340 345 350
Ala Phe Asp Leu Met His Glu Gly Lys Ser Ile Arg Thr Val Ile Arg
355 360 365
Tyr
<210> SEQ ID NO 25
<211> LENGTH: 369
<212> TYPE: PRT
<213> ORGANISM: Achromobacter sp.
<400> SEQUENCE: 25
Met Lys Ser Arg Ala Ala Val Ala Phe Ala Pro Gly Lys Pro Leu Glu
1 5 10 15
Ile Val Glu Ile Asp Val Ala Pro Pro Lys Lys Gly Glu Val Leu Ile
20 25 30
Lys Val Thr His Thr Gly Val Cys His Thr Asp Ala Phe Thr Leu Ser
35 40 45
Gly Asp Asp Pro Glu Gly Val Phe Pro Val Val Leu Gly His Glu Gly
50 55 60
Ala Gly Val Val Val Glu Val Gly Glu Gly Val Thr Ser Val Lys Pro
65 70 75 80
Gly Asp His Val Ile Pro Leu Tyr Thr Ala Glu Cys Gly Glu Cys Glu
85 90 95
Phe Cys Arg Ser Gly Lys Thr Asn Leu Cys Val Ala Val Arg Glu Thr
100 105 110
Gln Gly Lys Gly Leu Met Pro Asp Gly Thr Thr Arg Phe Ser Tyr Asn
115 120 125
Gly Gln Pro Leu Tyr His Tyr Met Gly Cys Ser Thr Phe Ser Glu Tyr
130 135 140
Thr Val Val Ala Glu Val Ser Leu Ala Lys Ile Asn Pro Glu Ala Asn
145 150 155 160
His Glu His Val Cys Leu Leu Gly Cys Gly Val Thr Thr Gly Ile Gly
165 170 175
Ala Val His Asn Thr Ala Lys Val Gln Pro Gly Asp Ser Val Ala Val
180 185 190
Phe Gly Leu Gly Ala Ile Gly Leu Ala Val Val Gln Gly Ala Arg Gln
195 200 205
Ala Lys Ala Gly Arg Ile Ile Ala Ile Asp Thr Asn Pro Lys Lys Phe
210 215 220
Asp Leu Ala Arg Arg Phe Gly Ala Thr Asp Cys Ile Asn Pro Asn Asp
225 230 235 240
Tyr Asp Lys Pro Ile Lys Asp Val Leu Leu Asp Ile Asn Lys Trp Gly
245 250 255
Ile Asp His Thr Phe Glu Cys Ile Gly Asn Val Asn Val Met Arg Ala
260 265 270
Ala Leu Glu Ser Ala His Arg Gly Trp Gly Gln Ser Val Ile Ile Gly
275 280 285
Val Ala Gly Ala Gly Gln Glu Ile Ser Thr Arg Pro Phe Gln Leu Val
290 295 300
Thr Gly Arg Val Trp Lys Gly Ser Ala Phe Gly Gly Val Lys Gly Arg
305 310 315 320
Ser Gln Leu Pro Gly Met Val Glu Asp Ala Met Lys Gly Asp Ile Asp
325 330 335
Leu Glu Pro Phe Val Thr His Thr Met Ser Leu Asp Glu Ile Asn Asp
340 345 350
Ala Phe Asp Leu Met His Glu Gly Lys Ser Ile Arg Thr Val Ile Arg
355 360 365
Tyr
<210> SEQ ID NO 26
<211> LENGTH: 369
<212> TYPE: PRT
<213> ORGANISM: Klebsiella pneumoniae IS22
<400> SEQUENCE: 26
Met Lys Ser Arg Ala Ala Val Ala Phe Ala Pro Gly Lys Pro Leu Glu
1 5 10 15
Ile Val Glu Ile Asp Val Ala Pro Pro Lys Lys Gly Glu Val Leu Ile
20 25 30
Lys Val Thr His Thr Gly Val Cys His Thr Asp Ala Phe Thr Leu Ser
35 40 45
Gly Asp Asp Pro Glu Gly Val Phe Pro Val Val Leu Gly His Glu Gly
50 55 60
Ala Gly Val Val Val Glu Val Gly Glu Gly Val Thr Ser Val Lys Pro
65 70 75 80
Gly Asp His Val Ile Pro Leu Tyr Thr Ala Glu Cys Gly Glu Cys Glu
85 90 95
Phe Cys Arg Ser Gly Lys Thr Asn Leu Cys Val Ala Val Arg Glu Thr
100 105 110
Gln Gly Lys Gly Leu Met Pro Asp Gly Thr Thr Arg Phe Ser Tyr Asn
115 120 125
Gly Gln Pro Leu Tyr His Tyr Met Gly Cys Ser Thr Phe Ser Glu Tyr
130 135 140
Thr Val Val Ala Glu Val Ser Leu Ala Lys Ile Asn Pro Glu Ala Asn
145 150 155 160
His Glu His Val Cys Leu Leu Gly Cys Gly Val Thr Thr Gly Ile Gly
165 170 175
Ala Val His Asn Thr Ala Lys Val Gln Pro Gly Asp Ser Val Ala Val
180 185 190
Phe Gly Leu Gly Ala Ile Gly Leu Ala Val Val Gln Gly Ala Arg Gln
195 200 205
Ala Lys Ala Gly Arg Ile Ile Ala Ile Asp Thr Asn Pro Lys Lys Phe
210 215 220
Asp Leu Ala Arg Arg Phe Gly Ala Thr Asp Cys Ile Asn Pro Asn Asp
225 230 235 240
Tyr Asp Lys Pro Ile Lys Asp Val Leu Leu Asp Ile Asn Lys Trp Gly
245 250 255
Ile Asp His Thr Phe Glu Cys Ile Gly Asn Val Asn Val Met Arg Ala
260 265 270
Ala Leu Glu Ser Ala His Arg Gly Trp Gly Gln Ser Val Ile Ile Gly
275 280 285
Val Ala Gly Ala Gly Gln Glu Ile Ser Thr Arg Pro Phe Gln Leu Val
290 295 300
Thr Gly Arg Val Trp Lys Gly Ser Ala Phe Gly Gly Val Lys Gly Arg
305 310 315 320
Ser Gln Leu Pro Gly Met Val Glu Asp Ala Met Lys Gly Asp Ile Asp
325 330 335
Leu Glu Pro Phe Val Thr His Thr Met Ser Leu Asp Glu Ile Asn Asp
340 345 350
Ala Phe Asp Leu Met His Glu Gly Lys Ser Ile Arg Thr Val Ile Arg
355 360 365
Tyr
<210> SEQ ID NO 27
<211> LENGTH: 277
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli (strain K12)
<400> SEQUENCE: 27
Met Glu Leu Ile Glu Lys His Val Ser Phe Gly Gly Trp Gln Asn Met
1 5 10 15
Tyr Arg His Tyr Ser Gln Ser Leu Lys Cys Glu Met Asn Val Gly Val
20 25 30
Tyr Leu Pro Pro Lys Ala Ala Asn Glu Lys Leu Pro Val Leu Tyr Trp
35 40 45
Leu Ser Gly Leu Thr Cys Asn Glu Gln Asn Phe Ile Thr Lys Ser Gly
50 55 60
Met Gln Arg Tyr Ala Ala Glu His Asn Ile Ile Val Val Ala Pro Asp
65 70 75 80
Thr Ser Pro Arg Gly Ser His Val Ala Asp Ala Asp Arg Tyr Asp Leu
85 90 95
Gly Gln Gly Ala Gly Phe Tyr Leu Asn Ala Thr Gln Ala Pro Trp Asn
100 105 110
Glu His Tyr Lys Met Tyr Asp Tyr Ile Arg Asn Glu Leu Pro Asp Leu
115 120 125
Val Met His His Phe Pro Ala Thr Ala Lys Lys Ser Ile Ser Gly His
130 135 140
Ser Met Gly Gly Leu Gly Ala Leu Val Leu Ala Leu Arg Asn Pro Asp
145 150 155 160
Glu Tyr Val Ser Val Ser Ala Phe Ser Pro Ile Val Ser Pro Ser Gln
165 170 175
Val Pro Trp Gly Gln Gln Ala Phe Ala Ala Tyr Leu Ala Glu Asn Lys
180 185 190
Asp Ala Trp Leu Asp Tyr Asp Pro Val Ser Leu Ile Ser Gln Gly Gln
195 200 205
Arg Val Ala Glu Ile Met Val Asp Gln Gly Leu Ser Asp Asp Phe Tyr
210 215 220
Ala Glu Gln Leu Arg Thr Pro Asn Leu Glu Lys Ile Cys Gln Glu Met
225 230 235 240
Asn Ile Lys Thr Leu Ile Arg Tyr His Glu Gly Tyr Asp His Ser Tyr
245 250 255
Tyr Phe Val Ser Ser Phe Ile Gly Glu His Ile Ala Tyr His Ala Asn
260 265 270
Lys Leu Asn Met Arg
275
<210> SEQ ID NO 28
<211> LENGTH: 277
<212> TYPE: PRT
<213> ORGANISM: Shigella sonnei (strain Ss046)
<400> SEQUENCE: 28
Met Glu Leu Ile Glu Lys His Ala Ser Phe Gly Gly Trp Gln Asn Val
1 5 10 15
Tyr Arg His Tyr Ser Gln Ser Leu Lys Cys Glu Met Asn Val Gly Val
20 25 30
Tyr Leu Pro Pro Lys Ala Ala Asn Glu Lys Leu Pro Val Leu Tyr Trp
35 40 45
Leu Ser Gly Leu Thr Cys Asn Glu Gln Asn Phe Ile Thr Lys Ser Gly
50 55 60
Met Gln Arg Tyr Ala Ala Glu His Asn Ile Ile Val Val Ala Pro Asp
65 70 75 80
Thr Ser Pro Arg Gly Ser His Val Ala Asp Ala Asp Arg Tyr Asp Leu
85 90 95
Gly Gln Gly Ala Gly Phe Tyr Leu Asn Ala Thr Gln Ala Pro Trp Asn
100 105 110
Glu His Tyr Lys Met Tyr Asp Tyr Ile Arg Asn Glu Leu Pro Asp Leu
115 120 125
Val Met Gln His Phe Pro Ala Thr Thr Arg Lys Ser Ile Ser Gly His
130 135 140
Ser Met Gly Gly Leu Gly Ala Leu Val Leu Ala Leu Arg Asn Pro Asp
145 150 155 160
Glu Tyr Val Ser Val Ser Ala Phe Ser Pro Ile Val Ser Pro Ser Gln
165 170 175
Val Pro Trp Gly Gln Gln Ala Phe Ala Ala Tyr Leu Gly Glu Asn Lys
180 185 190
Asp Ala Trp Leu Asp Tyr Asp Pro Val Ser Leu Ile Ser Gln Gly Gln
195 200 205
Arg Val Ala Glu Ile Met Val Asp Gln Gly Leu Ser Asp Asp Phe Tyr
210 215 220
Ala Glu Gln Leu Arg Thr Pro Asn Leu Glu Lys Ile Cys Gln Glu Met
225 230 235 240
Asn Ile Lys Thr Leu Ile Arg Tyr His Glu Gly Tyr Asp His Ser Tyr
245 250 255
Tyr Phe Val Ser Ser Phe Ile Gly Glu His Ile Ala Tyr His Ala Asn
260 265 270
Lys Leu Asn Met Arg
275
<210> SEQ ID NO 29
<211> LENGTH: 277
<212> TYPE: PRT
<213> ORGANISM: Achromobacter sp.
<400> SEQUENCE: 29
Met Glu Leu Ile Glu Lys His Val Ser Phe Gly Gly Trp Gln Asn Val
1 5 10 15
Tyr Arg His Tyr Ser Gln Ser Leu Lys Cys Glu Met Asn Val Gly Val
20 25 30
Tyr Leu Pro Pro Lys Ala Ala Asn Glu Lys Leu Pro Val Leu Tyr Trp
35 40 45
Leu Ser Gly Leu Thr Cys Asn Glu Gln Asn Phe Ile Thr Lys Ser Gly
50 55 60
Met Gln Arg Tyr Ala Ala Glu His Asn Ile Ile Val Val Ala Pro Asp
65 70 75 80
Thr Ser Pro Arg Gly Ser His Val Ala Asp Ala Asp Arg Tyr Asp Leu
85 90 95
Gly Gln Gly Ala Gly Phe Tyr Leu Asn Ala Thr Gln Ala Pro Trp Asn
100 105 110
Glu His Tyr Lys Met Tyr Asp Tyr Ile Arg Asn Glu Leu Pro Asp Leu
115 120 125
Val Met His His Phe Pro Ala Thr Ala Lys Lys Ser Ile Ser Gly His
130 135 140
Ser Met Gly Gly Leu Gly Ala Leu Val Leu Ala Leu Arg Asn Pro Asp
145 150 155 160
Glu Tyr Val Ser Val Ser Ala Phe Ser Pro Ile Val Ser Pro Ser Gln
165 170 175
Val Pro Trp Gly Gln Gln Ala Phe Ala Ala Tyr Leu Ala Glu Asn Lys
180 185 190
Asp Ala Trp Leu Asp Tyr Asp Pro Val Ser Leu Ile Ser Gln Gly Gln
195 200 205
Arg Val Ala Glu Ile Met Val Asp Gln Gly Leu Ser Asp Asp Phe Tyr
210 215 220
Ala Glu Gln Leu Arg Thr Pro Asn Leu Glu Lys Ile Cys Gln Glu Met
225 230 235 240
Asn Ile Lys Thr Leu Ile Arg Tyr His Glu Gly Tyr Asp His Ser Tyr
245 250 255
Tyr Phe Val Ser Ser Phe Ile Gly Glu His Ile Ala Tyr His Ala Asn
260 265 270
Lys Leu Asn Met Arg
275
<210> SEQ ID NO 30
<211> LENGTH: 277
<212> TYPE: PRT
<213> ORGANISM: Klebsiella pneumoniae IS22
<400> SEQUENCE: 30
Met Glu Leu Ile Glu Lys His Ala Ser Phe Gly Gly Trp Gln Asn Val
1 5 10 15
Tyr Arg His Tyr Ser Gln Ser Leu Lys Cys Glu Met Asn Val Gly Val
20 25 30
Tyr Leu Pro Pro Lys Ala Ala Asn Glu Lys Leu Pro Val Leu Tyr Trp
35 40 45
Leu Ser Gly Leu Thr Cys Asn Glu Gln Asn Phe Ile Thr Lys Ser Gly
50 55 60
Met Gln Arg Tyr Ala Ala Glu His Asn Ile Ile Val Val Ala Pro Asp
65 70 75 80
Thr Ser Pro Arg Gly Ser His Val Ala Asp Ala Asp Arg Tyr Asp Leu
85 90 95
Gly Gln Gly Ala Gly Phe Tyr Leu Asn Ala Thr Gln Ala Pro Trp Asn
100 105 110
Glu His Tyr Lys Met Tyr Asp Tyr Ile Arg Asn Glu Leu Pro Asp Leu
115 120 125
Val Met His His Phe Pro Ala Thr Ala Lys Lys Ser Ile Ser Gly His
130 135 140
Ser Met Gly Gly Leu Gly Ala Leu Val Leu Ala Leu Arg Asn Pro Asp
145 150 155 160
Glu Tyr Val Ser Val Ser Ala Phe Ser Pro Ile Val Ser Pro Ser Gln
165 170 175
Val Pro Trp Gly Gln Gln Ala Phe Ala Ala Tyr Leu Ala Glu Asn Lys
180 185 190
Asp Ala Trp Leu Asp Tyr Asp Pro Val Ser Leu Ile Ser Gln Gly Gln
195 200 205
Arg Val Ala Glu Ile Met Val Asp Gln Gly Leu Ser Asp Asp Phe Tyr
210 215 220
Ala Glu Gln Leu Arg Thr Pro Asn Leu Glu Lys Ile Cys Gln Glu Met
225 230 235 240
Asn Ile Lys Thr Leu Ile Arg Tyr His Glu Gly Tyr Asp His Ser Tyr
245 250 255
Tyr Phe Val Ser Ser Phe Ile Gly Glu His Ile Ala Tyr His Ala Asn
260 265 270
Lys Leu Asn Met Arg
275
<210> SEQ ID NO 31
<211> LENGTH: 387
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli (strain K12)
<400> SEQUENCE: 31
Met Asn Asn Phe Asn Leu His Thr Pro Thr Arg Ile Leu Phe Gly Lys
1 5 10 15
Gly Ala Ile Ala Gly Leu Arg Glu Gln Ile Pro His Asp Ala Arg Val
20 25 30
Leu Ile Thr Tyr Gly Gly Gly Ser Val Lys Lys Thr Gly Val Leu Asp
35 40 45
Gln Val Leu Asp Ala Leu Lys Gly Met Asp Val Leu Glu Phe Gly Gly
50 55 60
Ile Glu Pro Asn Pro Ala Tyr Glu Thr Leu Met Asn Ala Val Lys Leu
65 70 75 80
Val Arg Glu Gln Lys Val Thr Phe Leu Leu Ala Val Gly Gly Gly Ser
85 90 95
Val Leu Asp Gly Thr Lys Phe Ile Ala Ala Ala Ala Asn Tyr Pro Glu
100 105 110
Asn Ile Asp Pro Trp His Ile Leu Gln Thr Gly Gly Lys Glu Ile Lys
115 120 125
Ser Ala Ile Pro Met Gly Cys Val Leu Thr Leu Pro Ala Thr Gly Ser
130 135 140
Glu Ser Asn Ala Gly Ala Val Ile Ser Arg Lys Thr Thr Gly Asp Lys
145 150 155 160
Gln Ala Phe His Ser Ala His Val Gln Pro Val Phe Ala Val Leu Asp
165 170 175
Pro Val Tyr Thr Tyr Thr Leu Pro Pro Arg Gln Val Ala Asn Gly Val
180 185 190
Val Asp Ala Phe Val His Thr Val Glu Gln Tyr Val Thr Lys Pro Val
195 200 205
Asp Ala Lys Ile Gln Asp Arg Phe Ala Glu Gly Ile Leu Leu Thr Leu
210 215 220
Ile Glu Asp Gly Pro Lys Ala Leu Lys Glu Pro Glu Asn Tyr Asp Val
225 230 235 240
Arg Ala Asn Val Met Trp Ala Ala Thr Gln Ala Leu Asn Gly Leu Ile
245 250 255
Gly Ala Gly Val Pro Gln Asp Trp Ala Thr His Met Leu Gly His Glu
260 265 270
Leu Thr Ala Met His Gly Leu Asp His Ala Gln Thr Leu Ala Ile Val
275 280 285
Leu Pro Ala Leu Trp Asn Glu Lys Arg Asp Thr Lys Arg Ala Lys Leu
290 295 300
Leu Gln Tyr Ala Glu Arg Val Trp Asn Ile Thr Glu Gly Ser Asp Asp
305 310 315 320
Glu Arg Ile Asp Ala Ala Ile Ala Ala Thr Arg Asn Phe Phe Glu Gln
325 330 335
Leu Gly Val Pro Thr His Leu Ser Asp Tyr Gly Leu Asp Gly Ser Ser
340 345 350
Ile Pro Ala Leu Leu Lys Lys Leu Glu Glu His Gly Met Thr Gln Leu
355 360 365
Gly Glu Asn His Asp Ile Thr Leu Asp Val Ser Arg Arg Ile Tyr Glu
370 375 380
Ala Ala Arg
385
<210> SEQ ID NO 32
<211> LENGTH: 288
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli (strain K12)
<400> SEQUENCE: 32
Met Ala Ala Lys Ile Ile Asp Gly Lys Thr Ile Ala Gln Gln Val Arg
1 5 10 15
Ser Glu Val Ala Gln Lys Val Gln Ala Arg Ile Ala Ala Gly Leu Arg
20 25 30
Ala Pro Gly Leu Ala Val Val Leu Val Gly Ser Asn Pro Ala Ser Gln
35 40 45
Ile Tyr Val Ala Ser Lys Arg Lys Ala Cys Glu Glu Val Gly Phe Val
50 55 60
Ser Arg Ser Tyr Asp Leu Pro Glu Thr Thr Ser Glu Ala Glu Leu Leu
65 70 75 80
Glu Leu Ile Asp Thr Leu Asn Ala Asp Asn Thr Ile Asp Gly Ile Leu
85 90 95
Val Gln Leu Pro Leu Pro Ala Gly Ile Asp Asn Val Lys Val Leu Glu
100 105 110
Arg Ile His Pro Asp Lys Asp Val Asp Gly Phe His Pro Tyr Asn Val
115 120 125
Gly Arg Leu Cys Gln Arg Ala Pro Arg Leu Arg Pro Cys Thr Pro Arg
130 135 140
Gly Ile Val Thr Leu Leu Glu Arg Tyr Asn Ile Asp Thr Phe Gly Leu
145 150 155 160
Asn Ala Val Val Ile Gly Ala Ser Asn Ile Val Gly Arg Pro Met Ser
165 170 175
Met Glu Leu Leu Leu Ala Gly Cys Thr Thr Thr Val Thr His Arg Phe
180 185 190
Thr Lys Asn Leu Arg His His Val Glu Asn Ala Asp Leu Leu Ile Val
195 200 205
Ala Val Gly Lys Pro Gly Phe Ile Pro Gly Asp Trp Ile Lys Glu Gly
210 215 220
Ala Ile Val Ile Asp Val Gly Ile Asn Arg Leu Glu Asn Gly Lys Val
225 230 235 240
Val Gly Asp Val Val Phe Glu Asp Ala Ala Lys Arg Ala Ser Tyr Ile
245 250 255
Thr Pro Val Pro Gly Gly Val Gly Pro Met Thr Val Ala Thr Leu Ile
260 265 270
Glu Asn Thr Leu Gln Ala Cys Val Glu Tyr His Asp Pro Gln Asp Glu
275 280 285
<210> SEQ ID NO 33
<211> LENGTH: 288
<212> TYPE: PRT
<213> ORGANISM: Escherichia fergusonii (strain ATCC
35469/DSM 13698/CDC 0568-73)
<400> SEQUENCE: 33
Met Ala Ala Lys Ile Ile Asp Gly Lys Thr Ile Ala Gln Gln Val Arg
1 5 10 15
Ser Glu Val Ala Gln Lys Val Gln Ala Arg Val Ala Ala Gly Leu Arg
20 25 30
Ala Pro Gly Leu Ala Val Val Leu Val Gly Ser Asn Pro Ala Ser Gln
35 40 45
Ile Tyr Val Ala Ser Lys Arg Lys Ala Cys Glu Glu Val Gly Phe Val
50 55 60
Ser Arg Ser Tyr Asp Leu Pro Glu Thr Thr Ser Glu Ala Glu Leu Leu
65 70 75 80
Glu Leu Ile Asp Val Leu Asn Ala Asp Asn Thr Ile Asp Gly Ile Leu
85 90 95
Val Gln Leu Pro Leu Pro Ala Gly Ile Asp Asn Val Lys Val Leu Glu
100 105 110
Arg Ile His Pro Asp Lys Asp Val Asp Gly Phe His Pro Tyr Asn Val
115 120 125
Gly Arg Leu Cys Gln Arg Ala Pro Arg Leu Arg Pro Cys Thr Pro Arg
130 135 140
Gly Ile Val Thr Leu Leu Glu Arg Tyr Asn Ile Asp Thr Phe Gly Leu
145 150 155 160
Asn Ala Val Val Ile Gly Ala Ser Asn Ile Val Gly Arg Pro Met Ser
165 170 175
Met Glu Leu Leu Leu Ala Gly Cys Thr Thr Thr Val Thr His Arg Phe
180 185 190
Thr Lys Asn Leu Arg His His Val Glu Asn Ala Asp Leu Leu Ile Val
195 200 205
Ala Val Gly Lys Pro Gly Phe Ile Pro Gly Asp Trp Ile Lys Glu Gly
210 215 220
Ala Ile Val Ile Asp Val Gly Ile Asn Arg Leu Glu Asn Gly Lys Val
225 230 235 240
Val Gly Asp Val Val Phe Glu Asp Ala Ala Lys Arg Ala Ser Tyr Ile
245 250 255
Thr Pro Val Pro Gly Gly Val Gly Pro Met Thr Val Ala Thr Leu Ile
260 265 270
Glu Asn Thr Leu Gln Ala Cys Val Glu Tyr His Asp Pro Gln Asp Glu
275 280 285
<210> SEQ ID NO 34
<211> LENGTH: 288
<212> TYPE: PRT
<213> ORGANISM: Shigella dysenteriae serotype 1 (strain Sd197)
<400> SEQUENCE: 34
Met Ala Ala Lys Ile Ile Asp Gly Lys Thr Ile Ala Gln Gln Val Arg
1 5 10 15
Ser Glu Val Ala Gln Lys Val Gln Ala Arg Ile Ala Ala Gly Leu Arg
20 25 30
Ala Pro Gly Leu Ala Val Val Leu Val Gly Ser Asn Pro Ala Ser Gln
35 40 45
Ile Tyr Val Ala Ser Lys Arg Lys Ala Cys Glu Glu Val Gly Phe Val
50 55 60
Ser Arg Ser Tyr Asp Leu Pro Glu Thr Thr Ser Glu Ala Glu Leu Leu
65 70 75 80
Glu Leu Ile Asp Ala Leu Asn Ala Asp Asn Thr Ile Asp Gly Ile Leu
85 90 95
Val Gln Leu Pro Leu Pro Ala Gly Ile Asp Asn Val Lys Val Leu Glu
100 105 110
Arg Ile His Pro Asp Lys Asp Val Asp Gly Phe His Pro Tyr Asn Val
115 120 125
Gly Arg Leu Cys Gln Arg Ala Pro Arg Leu Arg Pro Cys Thr Pro Arg
130 135 140
Gly Ile Val Thr Leu Leu Glu Arg Tyr Asn Ile Asp Thr Phe Gly Leu
145 150 155 160
Asn Ala Val Val Ile Gly Ala Ser Asn Ile Val Gly Arg Pro Met Ser
165 170 175
Met Glu Leu Leu Leu Ala Gly Cys Thr Thr Thr Val Thr His Arg Phe
180 185 190
Thr Lys Asn Leu Arg His His Val Glu Asn Ala Asp Leu Leu Ile Val
195 200 205
Ala Val Gly Lys Pro Gly Phe Ile Pro Gly Asp Trp Ile Lys Glu Gly
210 215 220
Ala Ile Val Ile Asp Val Gly Ile Asn Arg Leu Glu Asn Gly Lys Val
225 230 235 240
Val Gly Asp Val Val Phe Glu Asp Ala Ala Lys His Ala Ser Tyr Ile
245 250 255
Thr Pro Val Pro Gly Gly Val Gly Pro Met Thr Val Ala Thr Leu Ile
260 265 270
Glu Asn Thr Leu Gln Ala Cys Val Glu Tyr His Asp Pro Gln Asp Glu
275 280 285
<210> SEQ ID NO 35
<211> LENGTH: 760
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli (strain K12)
<400> SEQUENCE: 35
Met Ser Glu Leu Asn Glu Lys Leu Ala Thr Ala Trp Glu Gly Phe Thr
1 5 10 15
Lys Gly Asp Trp Gln Asn Glu Val Asn Val Arg Asp Phe Ile Gln Lys
20 25 30
Asn Tyr Thr Pro Tyr Glu Gly Asp Glu Ser Phe Leu Ala Gly Ala Thr
35 40 45
Glu Ala Thr Thr Thr Leu Trp Asp Lys Val Met Glu Gly Val Lys Leu
50 55 60
Glu Asn Arg Thr His Ala Pro Val Asp Phe Asp Thr Ala Val Ala Ser
65 70 75 80
Thr Ile Thr Ser His Asp Ala Gly Tyr Ile Asn Lys Gln Leu Glu Lys
85 90 95
Ile Val Gly Leu Gln Thr Glu Ala Pro Leu Lys Arg Ala Leu Ile Pro
100 105 110
Phe Gly Gly Ile Lys Met Ile Glu Gly Ser Cys Lys Ala Tyr Asn Arg
115 120 125
Glu Leu Asp Pro Met Ile Lys Lys Ile Phe Thr Glu Tyr Arg Lys Thr
130 135 140
His Asn Gln Gly Val Phe Asp Val Tyr Thr Pro Asp Ile Leu Arg Cys
145 150 155 160
Arg Lys Ser Gly Val Leu Thr Gly Leu Pro Asp Ala Tyr Gly Arg Gly
165 170 175
Arg Ile Ile Gly Asp Tyr Arg Arg Val Ala Leu Tyr Gly Ile Asp Tyr
180 185 190
Leu Met Lys Asp Lys Leu Ala Gln Phe Thr Ser Leu Gln Ala Asp Leu
195 200 205
Glu Asn Gly Val Asn Leu Glu Gln Thr Ile Arg Leu Arg Glu Glu Ile
210 215 220
Ala Glu Gln His Arg Ala Leu Gly Gln Met Lys Glu Met Ala Ala Lys
225 230 235 240
Tyr Gly Tyr Asp Ile Ser Gly Pro Ala Thr Asn Ala Gln Glu Ala Ile
245 250 255
Gln Trp Thr Tyr Phe Gly Tyr Leu Ala Ala Val Lys Ser Gln Asn Gly
260 265 270
Ala Ala Met Ser Phe Gly Arg Thr Ser Thr Phe Leu Asp Val Tyr Ile
275 280 285
Glu Arg Asp Leu Lys Ala Gly Lys Ile Thr Glu Gln Glu Ala Gln Glu
290 295 300
Met Val Asp His Leu Val Met Lys Leu Arg Met Val Arg Phe Leu Arg
305 310 315 320
Thr Pro Glu Tyr Asp Glu Leu Phe Ser Gly Asp Pro Ile Trp Ala Thr
325 330 335
Glu Ser Ile Gly Gly Met Gly Leu Asp Gly Arg Thr Leu Val Thr Lys
340 345 350
Asn Ser Phe Arg Phe Leu Asn Thr Leu Tyr Thr Met Gly Pro Ser Pro
355 360 365
Glu Pro Asn Met Thr Ile Leu Trp Ser Glu Lys Leu Pro Leu Asn Phe
370 375 380
Lys Lys Phe Ala Ala Lys Val Ser Ile Asp Thr Ser Ser Leu Gln Tyr
385 390 395 400
Glu Asn Asp Asp Leu Met Arg Pro Asp Phe Asn Asn Asp Asp Tyr Ala
405 410 415
Ile Ala Cys Cys Val Ser Pro Met Ile Val Gly Lys Gln Met Gln Phe
420 425 430
Phe Gly Ala Arg Ala Asn Leu Ala Lys Thr Met Leu Tyr Ala Ile Asn
435 440 445
Gly Gly Val Asp Glu Lys Leu Lys Met Gln Val Gly Pro Lys Ser Glu
450 455 460
Pro Ile Lys Gly Asp Val Leu Asn Tyr Asp Glu Val Met Glu Arg Met
465 470 475 480
Asp His Phe Met Asp Trp Leu Ala Lys Gln Tyr Ile Thr Ala Leu Asn
485 490 495
Ile Ile His Tyr Met His Asp Lys Tyr Ser Tyr Glu Ala Ser Leu Met
500 505 510
Ala Leu His Asp Arg Asp Val Ile Arg Thr Met Ala Cys Gly Ile Ala
515 520 525
Gly Leu Ser Val Ala Ala Asp Ser Leu Ser Ala Ile Lys Tyr Ala Lys
530 535 540
Val Lys Pro Ile Arg Asp Glu Asp Gly Leu Ala Ile Asp Phe Glu Ile
545 550 555 560
Glu Gly Glu Tyr Pro Gln Phe Gly Asn Asn Asp Pro Arg Val Asp Asp
565 570 575
Leu Ala Val Asp Leu Val Glu Arg Phe Met Lys Lys Ile Gln Lys Leu
580 585 590
His Thr Tyr Arg Asp Ala Ile Pro Thr Gln Ser Val Leu Thr Ile Thr
595 600 605
Ser Asn Val Val Tyr Gly Lys Lys Thr Gly Asn Thr Pro Asp Gly Arg
610 615 620
Arg Ala Gly Ala Pro Phe Gly Pro Gly Ala Asn Pro Met His Gly Arg
625 630 635 640
Asp Gln Lys Gly Ala Val Ala Ser Leu Thr Ser Val Ala Lys Leu Pro
645 650 655
Phe Ala Tyr Ala Lys Asp Gly Ile Ser Tyr Thr Phe Ser Ile Val Pro
660 665 670
Asn Ala Leu Gly Lys Asp Asp Glu Val Arg Lys Thr Asn Leu Ala Gly
675 680 685
Leu Met Asp Gly Tyr Phe His His Glu Ala Ser Ile Glu Gly Gly Gln
690 695 700
His Leu Asn Val Asn Val Met Asn Arg Glu Met Leu Leu Asp Ala Met
705 710 715 720
Glu Asn Pro Glu Lys Tyr Pro Gln Leu Thr Ile Arg Val Ser Gly Tyr
725 730 735
Ala Val Arg Phe Asn Ser Leu Thr Lys Glu Gln Gln Gln Asp Val Ile
740 745 750
Thr Arg Thr Phe Thr Gln Ser Met
755 760
<210> SEQ ID NO 36
<211> LENGTH: 764
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli (strain K12)
<400> SEQUENCE: 36
Met Lys Val Asp Ile Asp Thr Ser Asp Lys Leu Tyr Ala Asp Ala Trp
1 5 10 15
Leu Gly Phe Lys Gly Thr Asp Trp Lys Asn Glu Ile Asn Val Arg Asp
20 25 30
Phe Ile Gln His Asn Tyr Thr Pro Tyr Glu Gly Asp Glu Ser Phe Leu
35 40 45
Ala Glu Ala Thr Pro Ala Thr Thr Glu Leu Trp Glu Lys Val Met Glu
50 55 60
Gly Ile Arg Ile Glu Asn Ala Thr His Ala Pro Val Asp Phe Asp Thr
65 70 75 80
Asn Ile Ala Thr Thr Ile Thr Ala His Asp Ala Gly Tyr Ile Asn Gln
85 90 95
Pro Leu Glu Lys Ile Val Gly Leu Gln Thr Asp Ala Pro Leu Lys Arg
100 105 110
Ala Leu His Pro Phe Gly Gly Ile Asn Met Ile Lys Ser Ser Phe His
115 120 125
Ala Tyr Gly Arg Glu Met Asp Ser Glu Phe Glu Tyr Leu Phe Thr Asp
130 135 140
Leu Arg Lys Thr His Asn Gln Gly Val Phe Asp Val Tyr Ser Pro Asp
145 150 155 160
Met Leu Arg Cys Arg Lys Ser Gly Val Leu Thr Gly Leu Pro Asp Gly
165 170 175
Tyr Gly Arg Gly Arg Ile Ile Gly Asp Tyr Arg Arg Val Ala Leu Tyr
180 185 190
Gly Ile Ser Tyr Leu Val Arg Glu Arg Glu Leu Gln Phe Ala Asp Leu
195 200 205
Gln Ser Arg Leu Glu Lys Gly Glu Asp Leu Glu Ala Thr Ile Arg Leu
210 215 220
Arg Glu Glu Leu Ala Glu His Arg His Ala Leu Leu Gln Ile Gln Glu
225 230 235 240
Met Ala Ala Lys Tyr Gly Phe Asp Ile Ser Arg Pro Ala Gln Asn Ala
245 250 255
Gln Glu Ala Val Gln Trp Leu Tyr Phe Ala Tyr Leu Ala Ala Val Lys
260 265 270
Ser Gln Asn Gly Gly Ala Met Ser Leu Gly Arg Thr Ala Ser Phe Leu
275 280 285
Asp Ile Tyr Ile Glu Arg Asp Phe Lys Ala Gly Val Leu Asn Glu Gln
290 295 300
Gln Ala Gln Glu Leu Ile Asp His Phe Ile Met Lys Ile Arg Met Val
305 310 315 320
Arg Phe Leu Arg Thr Pro Glu Phe Asp Ser Leu Phe Ser Gly Asp Pro
325 330 335
Ile Trp Ala Thr Glu Val Ile Gly Gly Met Gly Leu Asp Gly Arg Thr
340 345 350
Leu Val Thr Lys Asn Ser Phe Arg Tyr Leu His Thr Leu His Thr Met
355 360 365
Gly Pro Ala Pro Glu Pro Asn Leu Thr Ile Leu Trp Ser Glu Glu Leu
370 375 380
Pro Ile Ala Phe Lys Lys Tyr Ala Ala Gln Val Ser Ile Val Thr Ser
385 390 395 400
Ser Leu Gln Tyr Glu Asn Asp Asp Leu Met Arg Thr Asp Phe Asn Ser
405 410 415
Asp Asp Tyr Ala Ile Ala Cys Cys Val Ser Pro Met Val Ile Gly Lys
420 425 430
Gln Met Gln Phe Phe Gly Ala Arg Ala Asn Leu Ala Lys Thr Leu Leu
435 440 445
Tyr Ala Ile Asn Gly Gly Val Asp Glu Lys Leu Lys Ile Gln Val Gly
450 455 460
Pro Lys Thr Ala Pro Leu Met Asp Asp Val Leu Asp Tyr Asp Lys Val
465 470 475 480
Met Asp Ser Leu Asp His Phe Met Asp Trp Leu Ala Val Gln Tyr Ile
485 490 495
Ser Ala Leu Asn Ile Ile His Tyr Met His Asp Lys Tyr Ser Tyr Glu
500 505 510
Ala Ser Leu Met Ala Leu His Asp Arg Asp Val Tyr Arg Thr Met Ala
515 520 525
Cys Gly Ile Ala Gly Leu Ser Val Ala Thr Asp Ser Leu Ser Ala Ile
530 535 540
Lys Tyr Ala Arg Val Lys Pro Ile Arg Asp Glu Asn Gly Leu Ala Val
545 550 555 560
Asp Phe Glu Ile Asp Gly Glu Tyr Pro Gln Tyr Gly Asn Asn Asp Glu
565 570 575
Arg Val Asp Ser Ile Ala Cys Asp Leu Val Glu Arg Phe Met Lys Lys
580 585 590
Ile Lys Ala Leu Pro Thr Tyr Arg Asn Ala Val Pro Thr Gln Ser Ile
595 600 605
Leu Thr Ile Thr Ser Asn Val Val Tyr Gly Gln Lys Thr Gly Asn Thr
610 615 620
Pro Asp Gly Arg Arg Ala Gly Thr Pro Phe Ala Pro Gly Ala Asn Pro
625 630 635 640
Met His Gly Arg Asp Arg Lys Gly Ala Val Ala Ser Leu Thr Ser Val
645 650 655
Ala Lys Leu Pro Phe Thr Tyr Ala Lys Asp Gly Ile Ser Tyr Thr Phe
660 665 670
Ser Ile Val Pro Ala Ala Leu Gly Lys Glu Asp Pro Val Arg Lys Thr
675 680 685
Asn Leu Val Gly Leu Leu Asp Gly Tyr Phe His His Glu Ala Asp Val
690 695 700
Glu Gly Gly Gln His Leu Asn Val Asn Val Met Asn Arg Glu Met Leu
705 710 715 720
Leu Asp Ala Ile Glu His Pro Glu Lys Tyr Pro Asn Leu Thr Ile Arg
725 730 735
Val Ser Gly Tyr Ala Val Arg Phe Asn Ala Leu Thr Arg Glu Gln Gln
740 745 750
Gln Asp Val Ile Ser Arg Thr Phe Thr Gln Ala Leu
755 760
<210> SEQ ID NO 37
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic
6xHis tag
<400> SEQUENCE: 37
His His His His His His
1 5
1
SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS: 37
<210> SEQ ID NO 1
<211> LENGTH: 286
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli
<400> SEQUENCE: 1
Met Ser Gln Ala Leu Lys Asn Leu Leu Thr Leu Leu Asn Leu Glu Lys
1 5 10 15
Ile Glu Glu Gly Leu Phe Arg Gly Gln Ser Glu Asp Leu Gly Leu Arg
20 25 30
Gln Val Phe Gly Gly Gln Val Val Gly Gln Ala Leu Tyr Ala Ala Lys
35 40 45
Glu Thr Val Pro Glu Glu Arg Leu Val His Ser Phe His Ser Tyr Phe
50 55 60
Leu Arg Pro Gly Asp Ser Lys Lys Pro Ile Ile Tyr Asp Val Glu Thr
65 70 75 80
Leu Arg Asp Gly Asn Ser Phe Ser Ala Arg Arg Val Ala Ala Ile Gln
85 90 95
Asn Gly Lys Pro Ile Phe Tyr Met Thr Ala Ser Phe Gln Ala Pro Glu
100 105 110
Ala Gly Phe Glu His Gln Lys Thr Met Pro Ser Ala Pro Ala Pro Asp
115 120 125
Gly Leu Pro Ser Glu Thr Gln Ile Ala Gln Ser Leu Ala His Leu Leu
130 135 140
Pro Pro Val Leu Lys Asp Lys Phe Ile Cys Asp Arg Pro Leu Glu Val
145 150 155 160
Arg Pro Val Glu Phe His Asn Pro Leu Lys Gly His Val Ala Glu Pro
165 170 175
His Arg Gln Val Trp Ile Arg Ala Asn Gly Ser Val Pro Asp Asp Leu
180 185 190
Arg Val His Gln Tyr Leu Leu Gly Tyr Ala Ser Asp Leu Asn Phe Leu
195 200 205
Pro Val Ala Leu Gln Pro His Gly Ile Gly Phe Leu Glu Pro Gly Ile
210 215 220
Gln Ile Ala Thr Ile Asp His Ser Met Trp Phe His Arg Pro Phe Asn
225 230 235 240
Leu Asn Glu Trp Leu Leu Tyr Ser Val Glu Ser Thr Ser Ala Ser Ser
245 250 255
Ala Arg Gly Phe Val Arg Gly Glu Phe Tyr Thr Gln Asp Gly Val Leu
260 265 270
Val Ala Ser Thr Val Gln Glu Gly Val Met Arg Asn His Asn
275 280 285
<210> SEQ ID NO 2
<211> LENGTH: 1174
<212> TYPE: PRT
<213> ORGANISM: Mycobacterium marinum
<400> SEQUENCE: 2
Met Ser Pro Ile Thr Arg Glu Glu Arg Leu Glu Arg Arg Ile Gln Asp
1 5 10 15
Leu Tyr Ala Asn Asp Pro Gln Phe Ala Ala Ala Lys Pro Ala Thr Ala
20 25 30
Ile Thr Ala Ala Ile Glu Arg Pro Gly Leu Pro Leu Pro Gln Ile Ile
35 40 45
Glu Thr Val Met Thr Gly Tyr Ala Asp Arg Pro Ala Leu Ala Gln Arg
50 55 60
Ser Val Glu Phe Val Thr Asp Ala Gly Thr Gly His Thr Thr Leu Arg
65 70 75 80
Leu Leu Pro His Phe Glu Thr Ile Ser Tyr Gly Glu Leu Trp Asp Arg
85 90 95
Ile Ser Ala Leu Ala Asp Val Leu Ser Thr Glu Gln Thr Val Lys Pro
100 105 110
Gly Asp Arg Val Cys Leu Leu Gly Phe Asn Ser Val Asp Tyr Ala Thr
115 120 125
Ile Asp Met Thr Leu Ala Arg Leu Gly Ala Val Ala Val Pro Leu Gln
130 135 140
Thr Ser Ala Ala Ile Thr Gln Leu Gln Pro Ile Val Ala Glu Thr Gln
145 150 155 160
Pro Thr Met Ile Ala Ala Ser Val Asp Ala Leu Ala Asp Ala Thr Glu
165 170 175
Leu Ala Leu Ser Gly Gln Thr Ala Thr Arg Val Leu Val Phe Asp His
180 185 190
His Arg Gln Val Asp Ala His Arg Ala Ala Val Glu Ser Ala Arg Glu
195 200 205
Arg Leu Ala Gly Ser Ala Val Val Glu Thr Leu Ala Glu Ala Ile Ala
210 215 220
Arg Gly Asp Val Pro Arg Gly Ala Ser Ala Gly Ser Ala Pro Gly Thr
225 230 235 240
Asp Val Ser Asp Asp Ser Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser
245 250 255
Thr Gly Ala Pro Lys Gly Ala Met Tyr Pro Arg Arg Asn Val Ala Thr
260 265 270
Phe Trp Arg Lys Arg Thr Trp Phe Glu Gly Gly Tyr Glu Pro Ser Ile
275 280 285
Thr Leu Asn Phe Met Pro Met Ser His Val Met Gly Arg Gln Ile Leu
290 295 300
Tyr Gly Thr Leu Cys Asn Gly Gly Thr Ala Tyr Phe Val Ala Lys Ser
305 310 315 320
Asp Leu Ser Thr Leu Phe Glu Asp Leu Ala Leu Val Arg Pro Thr Glu
325 330 335
Leu Thr Phe Val Pro Arg Val Trp Asp Met Val Phe Asp Glu Phe Gln
340 345 350
Ser Glu Val Asp Arg Arg Leu Val Asp Gly Ala Asp Arg Val Ala Leu
355 360 365
Glu Ala Gln Val Lys Ala Glu Ile Arg Asn Asp Val Leu Gly Gly Arg
370 375 380
Tyr Thr Ser Ala Leu Thr Gly Ser Ala Pro Ile Ser Asp Glu Met Lys
385 390 395 400
Ala Trp Val Glu Glu Leu Leu Asp Met His Leu Val Glu Gly Tyr Gly
405 410 415
Ser Thr Glu Ala Gly Met Ile Leu Ile Asp Gly Ala Ile Arg Arg Pro
420 425 430
Ala Val Leu Asp Tyr Lys Leu Val Asp Val Pro Asp Leu Gly Tyr Phe
435 440 445
Leu Thr Asp Arg Pro His Pro Arg Gly Glu Leu Leu Val Lys Thr Asp
450 455 460
Ser Leu Phe Pro Gly Tyr Tyr Gln Arg Ala Glu Val Thr Ala Asp Val
465 470 475 480
Phe Asp Ala Asp Gly Phe Tyr Arg Thr Gly Asp Ile Met Ala Glu Val
485 490 495
Gly Pro Glu Gln Phe Val Tyr Leu Asp Arg Arg Asn Asn Val Leu Lys
500 505 510
Leu Ser Gln Gly Glu Phe Val Thr Val Ser Lys Leu Glu Ala Val Phe
515 520 525
Gly Asp Ser Pro Leu Val Arg Gln Ile Tyr Ile Tyr Gly Asn Ser Ala
530 535 540
Arg Ala Tyr Leu Leu Ala Val Ile Val Pro Thr Gln Glu Ala Leu Asp
545 550 555 560
Ala Val Pro Val Glu Glu Leu Lys Ala Arg Leu Gly Asp Ser Leu Gln
565 570 575
Glu Val Ala Lys Ala Ala Gly Leu Gln Ser Tyr Glu Ile Pro Arg Asp
580 585 590
Phe Ile Ile Glu Thr Thr Pro Trp Thr Leu Glu Asn Gly Leu Leu Thr
595 600 605
Gly Ile Arg Lys Leu Ala Arg Pro Gln Leu Lys Lys His Tyr Gly Glu
610 615 620
Leu Leu Glu Gln Ile Tyr Thr Asp Leu Ala His Gly Gln Ala Asp Glu
625 630 635 640
Leu Arg Ser Leu Arg Gln Ser Gly Ala Asp Ala Pro Val Leu Val Thr
645 650 655
Val Cys Arg Ala Ala Ala Ala Leu Leu Gly Gly Ser Ala Ser Asp Val
660 665 670
Gln Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala
675 680 685
Leu Ser Phe Thr Asn Leu Leu His Glu Ile Phe Asp Ile Glu Val Pro
690 695 700
Val Gly Val Ile Val Ser Pro Ala Asn Asp Leu Gln Ala Leu Ala Asp
705 710 715 720
Tyr Val Glu Ala Ala Arg Lys Pro Gly Ser Ser Arg Pro Thr Phe Ala
725 730 735
Ser Val His Gly Ala Ser Asn Gly Gln Val Thr Glu Val His Ala Gly
740 745 750
Asp Leu Ser Leu Asp Lys Phe Ile Asp Ala Ala Thr Leu Ala Glu Ala
755 760 765
Pro Arg Leu Pro Ala Ala Asn Thr Gln Val Arg Thr Val Leu Leu Thr
770 775 780
Gly Ala Thr Gly Phe Leu Gly Arg Tyr Leu Ala Leu Glu Trp Leu Glu
785 790 795 800
Arg Met Asp Leu Val Asp Gly Lys Leu Ile Cys Leu Val Arg Ala Lys
805 810 815
Ser Asp Thr Glu Ala Arg Ala Arg Leu Asp Lys Thr Phe Asp Ser Gly
820 825 830
Asp Pro Glu Leu Leu Ala His Tyr Arg Ala Leu Ala Gly Asp His Leu
835 840 845
Glu Val Leu Ala Gly Asp Lys Gly Glu Ala Asp Leu Gly Leu Asp Arg
850 855 860
Gln Thr Trp Gln Arg Leu Ala Asp Thr Val Asp Leu Ile Val Asp Pro
865 870 875 880
Ala Ala Leu Val Asn His Val Leu Pro Tyr Ser Gln Leu Phe Gly Pro
885 890 895
Asn Ala Leu Gly Thr Ala Glu Leu Leu Arg Leu Ala Leu Thr Ser Lys
900 905 910
Ile Lys Pro Tyr Ser Tyr Thr Ser Thr Ile Gly Val Ala Asp Gln Ile
915 920 925
Pro Pro Ser Ala Phe Thr Glu Asp Ala Asp Ile Arg Val Ile Ser Ala
930 935 940
Thr Arg Ala Val Asp Asp Ser Tyr Ala Asn Gly Tyr Ser Asn Ser Lys
945 950 955 960
Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu Cys Gly Leu
965 970 975
Pro Val Ala Val Phe Arg Cys Asp Met Ile Leu Ala Asp Thr Thr Trp
980 985 990
Ala Gly Gln Leu Asn Val Pro Asp Met Phe Thr Arg Met Ile Leu Ser
995 1000 1005
Leu Ala Ala Thr Gly Ile Ala Pro Gly Ser Phe Tyr Glu Leu Ala
1010 1015 1020
Ala Asp Gly Ala Arg Gln Arg Ala His Tyr Asp Gly Leu Pro Val
1025 1030 1035
Glu Phe Ile Ala Glu Ala Ile Ser Thr Leu Gly Ala Gln Ser Gln
1040 1045 1050
Asp Gly Phe His Thr Tyr His Val Met Asn Pro Tyr Asp Asp Gly
1055 1060 1065
Ile Gly Leu Asp Glu Phe Val Asp Trp Leu Asn Glu Ser Gly Cys
1070 1075 1080
Pro Ile Gln Arg Ile Ala Asp Tyr Gly Asp Trp Leu Gln Arg Phe
1085 1090 1095
Glu Thr Ala Leu Arg Ala Leu Pro Asp Arg Gln Arg His Ser Ser
1100 1105 1110
Leu Leu Pro Leu Leu His Asn Tyr Arg Gln Pro Glu Arg Pro Val
1115 1120 1125
Arg Gly Ser Ile Ala Pro Thr Asp Arg Phe Arg Ala Ala Val Gln
1130 1135 1140
Glu Ala Lys Ile Gly Pro Asp Lys Asp Ile Pro His Val Gly Ala
1145 1150 1155
Pro Ile Ile Val Lys Tyr Val Ser Asp Leu Arg Leu Leu Gly Leu
1160 1165 1170
Leu
<210> SEQ ID NO 3
<211> LENGTH: 1173
<212> TYPE: PRT
<213> ORGANISM: Mycobacterium smegmatis
<400> SEQUENCE: 3
Met Thr Ser Asp Val His Asp Ala Thr Asp Gly Val Thr Glu Thr Ala
1 5 10 15
Leu Asp Asp Glu Gln Ser Thr Arg Arg Ile Ala Glu Leu Tyr Ala Thr
20 25 30
Asp Pro Glu Phe Ala Ala Ala Ala Pro Leu Pro Ala Val Val Asp Ala
35 40 45
Ala His Lys Pro Gly Leu Arg Leu Ala Glu Ile Leu Gln Thr Leu Phe
50 55 60
Thr Gly Tyr Gly Asp Arg Pro Ala Leu Gly Tyr Arg Ala Arg Glu Leu
65 70 75 80
Ala Thr Asp Glu Gly Gly Arg Thr Val Thr Arg Leu Leu Pro Arg Phe
85 90 95
Asp Thr Leu Thr Tyr Ala Gln Val Trp Ser Arg Val Gln Ala Val Ala
100 105 110
Ala Ala Leu Arg His Asn Phe Ala Gln Pro Ile Tyr Pro Gly Asp Ala
115 120 125
Val Ala Thr Ile Gly Phe Ala Ser Pro Asp Tyr Leu Thr Leu Asp Leu
130 135 140
Val Cys Ala Tyr Leu Gly Leu Val Ser Val Pro Leu Gln His Asn Ala
145 150 155 160
Pro Val Ser Arg Leu Ala Pro Ile Leu Ala Glu Val Glu Pro Arg Ile
165 170 175
Leu Thr Val Ser Ala Glu Tyr Leu Asp Leu Ala Val Glu Ser Val Arg
180 185 190
Asp Val Asn Ser Val Ser Gln Leu Val Val Phe Asp His His Pro Glu
195 200 205
Val Asp Asp His Arg Asp Ala Leu Ala Arg Ala Arg Glu Gln Leu Ala
210 215 220
Gly Lys Gly Ile Ala Val Thr Thr Leu Asp Ala Ile Ala Asp Glu Gly
225 230 235 240
Ala Gly Leu Pro Ala Glu Pro Ile Tyr Thr Ala Asp His Asp Gln Arg
245 250 255
Leu Ala Met Ile Leu Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly
260 265 270
Ala Met Tyr Thr Glu Ala Met Val Ala Arg Leu Trp Thr Met Ser Phe
275 280 285
Ile Thr Gly Asp Pro Thr Pro Val Ile Asn Val Asn Phe Met Pro Leu
290 295 300
Asn His Leu Gly Gly Arg Ile Pro Ile Ser Thr Ala Val Gln Asn Gly
305 310 315 320
Gly Thr Ser Tyr Phe Val Pro Glu Ser Asp Met Ser Thr Leu Phe Glu
325 330 335
Asp Leu Ala Leu Val Arg Pro Thr Glu Leu Gly Leu Val Pro Arg Val
340 345 350
Ala Asp Met Leu Tyr Gln His His Leu Ala Thr Val Asp Arg Leu Val
355 360 365
Thr Gln Gly Ala Asp Glu Leu Thr Ala Glu Lys Gln Ala Gly Ala Glu
370 375 380
Leu Arg Glu Gln Val Leu Gly Gly Arg Val Ile Thr Gly Phe Val Ser
385 390 395 400
Thr Ala Pro Leu Ala Ala Glu Met Arg Ala Phe Leu Asp Ile Thr Leu
405 410 415
Gly Ala His Ile Val Asp Gly Tyr Gly Leu Thr Glu Thr Gly Ala Val
420 425 430
Thr Arg Asp Gly Val Ile Val Arg Pro Pro Val Ile Asp Tyr Lys Leu
435 440 445
Ile Asp Val Pro Glu Leu Gly Tyr Phe Ser Thr Asp Lys Pro Tyr Pro
450 455 460
Arg Gly Glu Leu Leu Val Arg Ser Gln Thr Leu Thr Pro Gly Tyr Tyr
465 470 475 480
Lys Arg Pro Glu Val Thr Ala Ser Val Phe Asp Arg Asp Gly Tyr Tyr
485 490 495
His Thr Gly Asp Val Met Ala Glu Thr Ala Pro Asp His Leu Val Tyr
500 505 510
Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ala Gln Gly Glu Phe Val
515 520 525
Ala Val Ala Asn Leu Glu Ala Val Phe Ser Gly Ala Ala Leu Val Arg
530 535 540
Gln Ile Phe Val Tyr Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala Val
545 550 555 560
Val Val Pro Thr Pro Glu Ala Leu Glu Gln Tyr Asp Pro Ala Ala Leu
565 570 575
Lys Ala Ala Leu Ala Asp Ser Leu Gln Arg Thr Ala Arg Asp Ala Glu
580 585 590
Leu Gln Ser Tyr Glu Val Pro Ala Asp Phe Ile Val Glu Thr Glu Pro
595 600 605
Phe Ser Ala Ala Asn Gly Leu Leu Ser Gly Val Gly Lys Leu Leu Arg
610 615 620
Pro Asn Leu Lys Asp Arg Tyr Gly Gln Arg Leu Glu Gln Met Tyr Ala
625 630 635 640
Asp Ile Ala Ala Thr Gln Ala Asn Gln Leu Arg Glu Leu Arg Arg Ala
645 650 655
Ala Ala Thr Gln Pro Val Ile Asp Thr Leu Thr Gln Ala Ala Ala Thr
660 665 670
Ile Leu Gly Thr Gly Ser Glu Val Ala Ser Asp Ala His Phe Thr Asp
675 680 685
Leu Gly Gly Asp Ser Leu Ser Ala Leu Thr Leu Ser Asn Leu Leu Ser
690 695 700
Asp Phe Phe Gly Phe Glu Val Pro Val Gly Thr Ile Val Asn Pro Ala
705 710 715 720
Thr Asn Leu Ala Gln Leu Ala Gln His Ile Glu Ala Gln Arg Thr Ala
725 730 735
Gly Asp Arg Arg Pro Ser Phe Thr Thr Val His Gly Ala Asp Ala Thr
740 745 750
Glu Ile Arg Ala Ser Glu Leu Thr Leu Asp Lys Phe Ile Asp Ala Glu
755 760 765
Thr Leu Arg Ala Ala Pro Gly Leu Pro Lys Val Thr Thr Glu Pro Arg
770 775 780
Thr Val Leu Leu Ser Gly Ala Asn Gly Trp Leu Gly Arg Phe Leu Thr
785 790 795 800
Leu Gln Trp Leu Glu Arg Leu Ala Pro Val Gly Gly Thr Leu Ile Thr
805 810 815
Ile Val Arg Gly Arg Asp Asp Ala Ala Ala Arg Ala Arg Leu Thr Gln
820 825 830
Ala Tyr Asp Thr Asp Pro Glu Leu Ser Arg Arg Phe Ala Glu Leu Ala
835 840 845
Asp Arg His Leu Arg Val Val Ala Gly Asp Ile Gly Asp Pro Asn Leu
850 855 860
Gly Leu Thr Pro Glu Ile Trp His Arg Leu Ala Ala Glu Val Asp Leu
865 870 875 880
Val Val His Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr Arg Gln
885 890 895
Leu Phe Gly Pro Asn Val Val Gly Thr Ala Glu Val Ile Lys Leu Ala
900 905 910
Leu Thr Glu Arg Ile Lys Pro Val Thr Tyr Leu Ser Thr Val Ser Val
915 920 925
Ala Met Gly Ile Pro Asp Phe Glu Glu Asp Gly Asp Ile Arg Thr Val
930 935 940
Ser Pro Val Arg Pro Leu Asp Gly Gly Tyr Ala Asn Gly Tyr Gly Asn
945 950 955 960
Ser Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu Cys
965 970 975
Gly Leu Pro Val Ala Thr Phe Arg Ser Asp Met Ile Leu Ala His Pro
980 985 990
Arg Tyr Arg Gly Gln Val Asn Val Pro Asp Met Phe Thr Arg Leu Leu
995 1000 1005
Leu Ser Leu Leu Ile Thr Gly Val Ala Pro Arg Ser Phe Tyr Ile
1010 1015 1020
Gly Asp Gly Glu Arg Pro Arg Ala His Tyr Pro Gly Leu Thr Val
1025 1030 1035
Asp Phe Val Ala Glu Ala Val Thr Thr Leu Gly Ala Gln Gln Arg
1040 1045 1050
Glu Gly Tyr Val Ser Tyr Asp Val Met Asn Pro His Asp Asp Gly
1055 1060 1065
Ile Ser Leu Asp Val Phe Val Asp Trp Leu Ile Arg Ala Gly His
1070 1075 1080
Pro Ile Asp Arg Val Asp Asp Tyr Asp Asp Trp Val Arg Arg Phe
1085 1090 1095
Glu Thr Ala Leu Thr Ala Leu Pro Glu Lys Arg Arg Ala Gln Thr
1100 1105 1110
Val Leu Pro Leu Leu His Ala Phe Arg Ala Pro Gln Ala Pro Leu
1115 1120 1125
Arg Gly Ala Pro Glu Pro Thr Glu Val Phe His Ala Ala Val Arg
1130 1135 1140
Thr Ala Lys Val Gly Pro Gly Asp Ile Pro His Leu Asp Glu Ala
1145 1150 1155
Leu Ile Asp Lys Tyr Ile Arg Asp Leu Arg Glu Phe Gly Leu Ile
1160 1165 1170
<210> SEQ ID NO 4
<211> LENGTH: 1148
<212> TYPE: PRT
<213> ORGANISM: Segniliparus rugosus
<400> SEQUENCE: 4
Met Gly Asp Gly Glu Glu Arg Ala Lys Arg Phe Phe Gln Arg Ile Gly
1 5 10 15
Glu Leu Ser Ala Thr Asp Pro Gln Phe Ala Ala Ala Ala Pro Asp Pro
20 25 30
Ala Val Val Glu Ala Val Ser Asp Pro Ser Leu Ser Phe Thr Arg Tyr
35 40 45
Leu Asp Thr Leu Met Arg Gly Tyr Ala Glu Arg Pro Ala Leu Ala His
50 55 60
Arg Val Gly Ala Gly Tyr Glu Thr Ile Ser Tyr Gly Glu Leu Trp Ala
65 70 75 80
Arg Val Gly Ala Ile Ala Ala Ala Trp Gln Ala Asp Gly Leu Ala Pro
85 90 95
Gly Asp Phe Val Ala Thr Val Gly Phe Thr Ser Pro Asp Tyr Val Ala
100 105 110
Val Asp Leu Ala Ala Ala Arg Ser Gly Leu Val Ser Val Pro Leu Gln
115 120 125
Ala Gly Ala Ser Leu Ala Gln Leu Val Gly Ile Leu Glu Glu Thr Glu
130 135 140
Pro Lys Val Leu Ala Ala Ser Ala Ser Ser Leu Glu Gly Ala Val Ala
145 150 155 160
Cys Ala Leu Ala Ala Pro Ser Val Gln Arg Leu Val Val Phe Asp Leu
165 170 175
Arg Gly Pro Asp Ala Ser Glu Ser Ala Ala Asp Glu Arg Arg Gly Ala
180 185 190
Leu Ala Asp Ala Glu Glu Gln Leu Ala Arg Ala Gly Arg Ala Val Val
195 200 205
Val Glu Thr Leu Ala Asp Leu Ala Ala Arg Gly Glu Ala Leu Pro Glu
210 215 220
Ala Pro Leu Phe Glu Pro Ala Glu Gly Glu Asp Pro Leu Ala Leu Leu
225 230 235 240
Ile Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly Ala Met Tyr Ser
245 250 255
Gln Arg Leu Val Ser Gln Leu Trp Gly Arg Thr Pro Val Val Pro Gly
260 265 270
Met Pro Asn Ile Ser Leu His Tyr Met Pro Leu Ser His Ser Tyr Gly
275 280 285
Arg Ala Val Leu Ala Gly Ala Leu Ser Ala Gly Gly Thr Ala His Phe
290 295 300
Thr Ala Asn Ser Asp Leu Ser Thr Leu Phe Glu Asp Ile Ala Leu Ala
305 310 315 320
Arg Pro Thr Phe Leu Ala Leu Val Pro Arg Val Cys Glu Met Leu Phe
325 330 335
Gln Glu Ser Gln Arg Gly Gln Asp Val Ala Glu Leu Arg Glu Arg Val
340 345 350
Leu Gly Gly Arg Leu Leu Val Ala Val Cys Gly Ser Ala Pro Leu Ser
355 360 365
Pro Glu Met Arg Ala Phe Met Glu Glu Val Leu Gly Phe Pro Leu Leu
370 375 380
Asp Gly Tyr Gly Ser Thr Glu Ala Leu Gly Val Met Arg Asn Gly Ile
385 390 395 400
Ile Gln Arg Pro Pro Val Ile Asp Tyr Lys Leu Val Asp Val Pro Glu
405 410 415
Leu Gly Tyr Arg Thr Thr Asp Lys Pro Tyr Pro Arg Gly Glu Leu Cys
420 425 430
Ile Arg Ser Thr Ser Leu Ile Ser Gly Tyr Tyr Lys Arg Pro Glu Ile
435 440 445
Thr Ala Glu Val Phe Asp Ala Gln Gly Tyr Tyr Lys Thr Gly Asp Val
450 455 460
Met Ala Glu Ile Ala Pro Asp His Leu Val Tyr Val Asp Arg Ser Lys
465 470 475 480
Asn Val Leu Lys Leu Ser Gln Gly Glu Phe Val Ala Val Ala Lys Leu
485 490 495
Glu Ala Ala Tyr Gly Thr Ser Pro Tyr Val Lys Gln Ile Phe Val Tyr
500 505 510
Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala Val Val Val Pro Asn Ala
515 520 525
Glu Val Leu Gly Ala Arg Asp Gln Glu Glu Ala Lys Pro Leu Ile Ala
530 535 540
Ala Ser Leu Gln Lys Ile Ala Lys Glu Ala Gly Leu Gln Ser Tyr Glu
545 550 555 560
Val Pro Arg Asp Phe Leu Ile Glu Thr Glu Pro Phe Thr Thr Gln Asn
565 570 575
Gly Leu Leu Ser Glu Val Gly Lys Leu Leu Arg Pro Lys Leu Lys Ala
580 585 590
Arg Tyr Gly Glu Ala Leu Glu Ala Arg Tyr Asp Glu Ile Ala His Gly
595 600 605
Gln Ala Asp Glu Leu Arg Ala Leu Arg Asp Gly Ala Gly Gln Arg Pro
610 615 620
Val Val Glu Thr Val Val Arg Ala Ala Val Ala Ile Ser Gly Ser Glu
625 630 635 640
Gly Ala Glu Val Gly Pro Glu Ala Asn Phe Ala Asp Leu Gly Gly Asp
645 650 655
Ser Leu Ser Ala Leu Ser Leu Ala Asn Leu Leu His Asp Val Phe Glu
660 665 670
Val Glu Val Pro Val Arg Ile Ile Ile Gly Pro Thr Ala Ser Leu Ala
675 680 685
Gly Ile Ala Lys His Ile Glu Ala Glu Arg Ala Gly Ala Ser Ala Pro
690 695 700
Thr Ala Ala Ser Val His Gly Ala Gly Ala Thr Arg Ile Arg Ala Ser
705 710 715 720
Glu Leu Thr Leu Glu Lys Phe Leu Pro Glu Asp Leu Leu Ala Ala Ala
725 730 735
Lys Gly Leu Pro Ala Ala Asp Gln Val Arg Thr Val Leu Leu Thr Gly
740 745 750
Ala Asn Gly Trp Leu Gly Arg Phe Leu Ala Leu Glu Gln Leu Glu Arg
755 760 765
Leu Ala Arg Ser Gly Gln Asp Gly Gly Lys Leu Ile Cys Leu Val Arg
770 775 780
Gly Lys Asp Ala Ala Ala Ala Arg Arg Arg Ile Glu Glu Thr Leu Gly
785 790 795 800
Thr Asp Pro Ala Leu Ala Ala Arg Phe Ala Glu Leu Ala Glu Gly Arg
805 810 815
Leu Glu Val Val Pro Gly Asp Val Gly Glu Pro Lys Phe Gly Leu Asp
820 825 830
Asp Ala Ala Trp Asp Arg Leu Ala Glu Glu Val Asp Val Ile Val His
835 840 845
Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr His Gln Leu Phe Gly
850 855 860
Pro Asn Val Val Gly Thr Ala Glu Ile Ile Arg Leu Ala Ile Thr Ala
865 870 875 880
Lys Arg Lys Pro Val Thr Tyr Leu Ser Thr Val Ala Val Ala Ala Gly
885 890 895
Val Glu Pro Ser Ser Phe Glu Glu Asp Gly Asp Ile Arg Ala Val Val
900 905 910
Pro Glu Arg Pro Leu Gly Asp Gly Tyr Ala Asn Gly Tyr Gly Asn Ser
915 920 925
Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Glu Leu Val Gly
930 935 940
Leu Pro Val Ala Val Phe Arg Ser Asp Met Ile Leu Ala His Thr Arg
945 950 955 960
Tyr Thr Gly Gln Leu Asn Val Pro Asp Gln Phe Thr Arg Leu Val Leu
965 970 975
Ser Leu Leu Ala Thr Gly Ile Ala Pro Lys Ser Phe Tyr Gln Gln Gly
980 985 990
Ala Ala Gly Glu Arg Gln Arg Ala His Tyr Asp Gly Ile Pro Val Asp
995 1000 1005
Phe Thr Ala Glu Ala Ile Thr Thr Leu Gly Ala Glu Pro Ser Trp
1010 1015 1020
Phe Asp Gly Gly Ala Gly Phe Arg Ser Phe Asp Val Phe Asn Pro
1025 1030 1035
His His Asp Gly Val Gly Leu Asp Glu Phe Val Asp Trp Leu Ile
1040 1045 1050
Glu Ala Gly His Pro Ile Ser Arg Ile Asp Asp His Lys Glu Trp
1055 1060 1065
Phe Ala Arg Phe Glu Thr Ala Val Arg Gly Leu Pro Glu Ala Gln
1070 1075 1080
Arg Gln His Ser Leu Leu Pro Leu Leu Arg Ala Tyr Ser Phe Pro
1085 1090 1095
His Pro Pro Val Asp Gly Ser Val Tyr Pro Thr Gly Lys Phe Gln
1100 1105 1110
Gly Ala Val Lys Ala Ala Gln Val Gly Ser Asp His Asp Val Pro
1115 1120 1125
His Leu Gly Lys Ala Leu Ile Val Lys Tyr Ala Asp Asp Leu Lys
1130 1135 1140
Ala Leu Gly Leu Leu
1145
<210> SEQ ID NO 5
<211> LENGTH: 1168
<212> TYPE: PRT
<213> ORGANISM: Mycobacterium smegmatis
<400> SEQUENCE: 5
Met Thr Ile Glu Thr Arg Glu Asp Arg Phe Asn Arg Arg Ile Asp His
1 5 10 15
Leu Phe Glu Thr Asp Pro Gln Phe Ala Ala Ala Arg Pro Asp Glu Ala
20 25 30
Ile Ser Ala Ala Ala Ala Asp Pro Glu Leu Arg Leu Pro Ala Ala Val
35 40 45
Lys Gln Ile Leu Ala Gly Tyr Ala Asp Arg Pro Ala Leu Gly Lys Arg
50 55 60
Ala Val Glu Phe Val Thr Asp Glu Glu Gly Arg Thr Thr Ala Lys Leu
65 70 75 80
Leu Pro Arg Phe Asp Thr Ile Thr Tyr Arg Gln Leu Ala Gly Arg Ile
85 90 95
Gln Ala Val Thr Asn Ala Trp His Asn His Pro Val Asn Ala Gly Asp
100 105 110
Arg Val Ala Ile Leu Gly Phe Thr Ser Val Asp Tyr Thr Thr Ile Asp
115 120 125
Ile Ala Leu Leu Glu Leu Gly Ala Val Ser Val Pro Leu Gln Thr Ser
130 135 140
Ala Pro Val Ala Gln Leu Gln Pro Ile Val Ala Glu Thr Glu Pro Lys
145 150 155 160
Val Ile Ala Ser Ser Val Asp Phe Leu Ala Asp Ala Val Ala Leu Val
165 170 175
Glu Ser Gly Pro Ala Pro Ser Arg Leu Val Val Phe Asp Tyr Ser His
180 185 190
Glu Val Asp Asp Gln Arg Glu Ala Phe Glu Ala Ala Lys Gly Lys Leu
195 200 205
Ala Gly Thr Gly Val Val Val Glu Thr Ile Thr Asp Ala Leu Asp Arg
210 215 220
Gly Arg Ser Leu Ala Asp Ala Pro Leu Tyr Val Pro Asp Glu Ala Asp
225 230 235 240
Pro Leu Thr Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro Lys
245 250 255
Gly Ala Met Tyr Pro Glu Ser Lys Thr Ala Thr Met Trp Gln Ala Gly
260 265 270
Ser Lys Ala Arg Trp Asp Glu Thr Leu Gly Val Met Pro Ser Ile Thr
275 280 285
Leu Asn Phe Met Pro Met Ser His Val Met Gly Arg Gly Ile Leu Cys
290 295 300
Ser Thr Leu Ala Ser Gly Gly Thr Ala Tyr Phe Ala Ala Arg Ser Asp
305 310 315 320
Leu Ser Thr Phe Leu Glu Asp Leu Ala Leu Val Arg Pro Thr Gln Leu
325 330 335
Asn Phe Val Pro Arg Ile Trp Asp Met Leu Phe Gln Glu Tyr Gln Ser
340 345 350
Arg Leu Asp Asn Arg Arg Ala Glu Gly Ser Glu Asp Arg Ala Glu Ala
355 360 365
Ala Val Leu Glu Glu Val Arg Thr Gln Leu Leu Gly Gly Arg Phe Val
370 375 380
Ser Ala Leu Thr Gly Ser Ala Pro Ile Ser Ala Glu Met Lys Ser Trp
385 390 395 400
Val Glu Asp Leu Leu Asp Met His Leu Leu Glu Gly Tyr Gly Ser Thr
405 410 415
Glu Ala Gly Ala Val Phe Ile Asp Gly Gln Ile Gln Arg Pro Pro Val
420 425 430
Ile Asp Tyr Lys Leu Val Asp Val Pro Asp Leu Gly Tyr Phe Ala Thr
435 440 445
Asp Arg Pro Tyr Pro Arg Gly Glu Leu Leu Val Lys Ser Glu Gln Met
450 455 460
Phe Pro Gly Tyr Tyr Lys Arg Pro Glu Ile Thr Ala Glu Met Phe Asp
465 470 475 480
Glu Asp Gly Tyr Tyr Arg Thr Gly Asp Ile Val Ala Glu Leu Gly Pro
485 490 495
Asp His Leu Glu Tyr Leu Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
500 505 510
Gln Gly Glu Phe Val Thr Val Ser Lys Leu Glu Ala Val Phe Gly Asp
515 520 525
Ser Pro Leu Val Arg Gln Ile Tyr Val Tyr Gly Asn Ser Ala Arg Ser
530 535 540
Tyr Leu Leu Ala Val Val Val Pro Thr Glu Glu Ala Leu Ser Arg Trp
545 550 555 560
Asp Gly Asp Glu Leu Lys Ser Arg Ile Ser Asp Ser Leu Gln Asp Ala
565 570 575
Ala Arg Ala Ala Gly Leu Gln Ser Tyr Glu Ile Pro Arg Asp Phe Leu
580 585 590
Val Glu Thr Thr Pro Phe Thr Leu Glu Asn Gly Leu Leu Thr Gly Ile
595 600 605
Arg Lys Leu Ala Arg Pro Lys Leu Lys Ala His Tyr Gly Glu Arg Leu
610 615 620
Glu Gln Leu Tyr Thr Asp Leu Ala Glu Gly Gln Ala Asn Glu Leu Arg
625 630 635 640
Glu Leu Arg Arg Asn Gly Ala Asp Arg Pro Val Val Glu Thr Val Ser
645 650 655
Arg Ala Ala Val Ala Leu Leu Gly Ala Ser Val Thr Asp Leu Arg Ser
660 665 670
Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser
675 680 685
Phe Ser Asn Leu Leu His Glu Ile Phe Asp Val Asp Val Pro Val Gly
690 695 700
Val Ile Val Ser Pro Ala Thr Asp Leu Ala Gly Val Ala Ala Tyr Ile
705 710 715 720
Glu Gly Glu Leu Arg Gly Ser Lys Arg Pro Thr Tyr Ala Ser Val His
725 730 735
Gly Arg Asp Ala Thr Glu Val Arg Ala Arg Asp Leu Ala Leu Gly Lys
740 745 750
Phe Ile Asp Ala Lys Thr Leu Ser Ala Ala Pro Gly Leu Pro Arg Ser
755 760 765
Gly Thr Glu Ile Arg Thr Val Leu Leu Thr Gly Ala Thr Gly Phe Leu
770 775 780
Gly Arg Tyr Leu Ala Leu Glu Trp Leu Glu Arg Met Asp Leu Val Asp
785 790 795 800
Gly Lys Val Ile Cys Leu Val Arg Ala Arg Ser Asp Asp Glu Ala Arg
805 810 815
Ala Arg Leu Asp Ala Thr Phe Asp Thr Gly Asp Ala Thr Leu Leu Glu
820 825 830
His Tyr Arg Ala Leu Ala Ala Asp His Leu Glu Val Ile Ala Gly Asp
835 840 845
Lys Gly Glu Ala Asp Leu Gly Leu Asp His Asp Thr Trp Gln Arg Leu
850 855 860
Ala Asp Thr Val Asp Leu Ile Val Asp Pro Ala Ala Leu Val Asn His
865 870 875 880
Val Leu Pro Tyr Ser Gln Met Phe Gly Pro Asn Ala Leu Gly Thr Ala
885 890 895
Glu Leu Ile Arg Ile Ala Leu Thr Thr Thr Ile Lys Pro Tyr Val Tyr
900 905 910
Val Ser Thr Ile Gly Val Gly Gln Gly Ile Ser Pro Glu Ala Phe Val
915 920 925
Glu Asp Ala Asp Ile Arg Glu Ile Ser Ala Thr Arg Arg Val Asp Asp
930 935 940
Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu
945 950 955 960
Leu Arg Glu Ala His Asp Trp Cys Gly Leu Pro Val Ser Val Phe Arg
965 970 975
Cys Asp Met Ile Leu Ala Asp Thr Thr Tyr Ser Gly Gln Leu Asn Leu
980 985 990
Pro Asp Met Phe Thr Arg Leu Met Leu Ser Leu Val Ala Thr Gly Ile
995 1000 1005
Ala Pro Gly Ser Phe Tyr Glu Leu Asp Ala Asp Gly Asn Arg Gln
1010 1015 1020
Arg Ala His Tyr Asp Gly Leu Pro Val Glu Phe Ile Ala Glu Ala
1025 1030 1035
Ile Ser Thr Ile Gly Ser Gln Val Thr Asp Gly Phe Glu Thr Phe
1040 1045 1050
His Val Met Asn Pro Tyr Asp Asp Gly Ile Gly Leu Asp Glu Tyr
1055 1060 1065
Val Asp Trp Leu Ile Glu Ala Gly Tyr Pro Val His Arg Val Asp
1070 1075 1080
Asp Tyr Ala Thr Trp Leu Ser Arg Phe Glu Thr Ala Leu Arg Ala
1085 1090 1095
Leu Pro Glu Arg Gln Arg Gln Ala Ser Leu Leu Pro Leu Leu His
1100 1105 1110
Asn Tyr Gln Gln Pro Ser Pro Pro Val Cys Gly Ala Met Ala Pro
1115 1120 1125
Thr Asp Arg Phe Arg Ala Ala Val Gln Asp Ala Lys Ile Gly Pro
1130 1135 1140
Asp Lys Asp Ile Pro His Val Thr Ala Asp Val Ile Val Lys Tyr
1145 1150 1155
Ile Ser Asn Leu Gln Met Leu Gly Leu Leu
1160 1165
<210> SEQ ID NO 6
<211> LENGTH: 1185
<212> TYPE: PRT
<213> ORGANISM: Mycobacterium massiliense
<400> SEQUENCE: 6
Met Thr Asn Glu Thr Asn Pro Gln Gln Glu Gln Leu Ser Arg Arg Ile
1 5 10 15
Glu Ser Leu Arg Glu Ser Asp Pro Gln Phe Arg Ala Ala Gln Pro Asp
20 25 30
Pro Ala Val Ala Glu Gln Val Leu Arg Pro Gly Leu His Leu Ser Glu
35 40 45
Ala Ile Ala Ala Leu Met Thr Gly Tyr Ala Glu Arg Pro Ala Leu Gly
50 55 60
Glu Arg Ala Arg Glu Leu Val Ile Asp Gln Asp Gly Arg Thr Thr Leu
65 70 75 80
Arg Leu Leu Pro Arg Phe Asp Thr Thr Thr Tyr Gly Glu Leu Trp Ser
85 90 95
Arg Thr Thr Ser Val Ala Ala Ala Trp His His Asp Ala Thr His Pro
100 105 110
Val Lys Ala Gly Asp Leu Val Ala Thr Leu Gly Phe Thr Ser Ile Asp
115 120 125
Tyr Thr Val Leu Asp Leu Ala Ile Met Ile Leu Gly Gly Val Ala Val
130 135 140
Pro Leu Gln Thr Ser Ala Pro Ala Ser Gln Trp Thr Thr Ile Leu Ala
145 150 155 160
Glu Ala Glu Pro Asn Thr Leu Ala Val Ser Ile Glu Leu Ile Gly Ala
165 170 175
Ala Met Glu Ser Val Arg Ala Thr Pro Ser Ile Lys Gln Val Val Val
180 185 190
Phe Asp Tyr Thr Pro Glu Val Asp Asp Gln Arg Glu Ala Phe Glu Ala
195 200 205
Ala Ser Thr Gln Leu Ala Gly Thr Gly Ile Ala Leu Glu Thr Leu Asp
210 215 220
Ala Val Ile Ala Arg Gly Ala Ala Leu Pro Ala Ala Pro Leu Tyr Ala
225 230 235 240
Pro Ser Ala Gly Asp Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly
245 250 255
Ser Thr Gly Ala Pro Lys Gly Ala Met His Ser Glu Asn Ile Val Arg
260 265 270
Arg Trp Trp Ile Arg Glu Asp Val Met Ala Gly Thr Glu Asn Leu Pro
275 280 285
Met Ile Gly Leu Asn Phe Met Pro Met Ser His Ile Met Gly Arg Gly
290 295 300
Thr Leu Thr Ser Thr Leu Ser Thr Gly Gly Thr Gly Tyr Phe Ala Ala
305 310 315 320
Ser Ser Asp Met Ser Thr Leu Phe Glu Asp Met Glu Leu Ile Arg Pro
325 330 335
Thr Ala Leu Ala Leu Val Pro Arg Val Cys Asp Met Val Phe Gln Arg
340 345 350
Phe Gln Thr Glu Val Asp Arg Arg Leu Ala Ser Gly Asp Thr Ala Ser
355 360 365
Ala Glu Ala Val Ala Ala Glu Val Lys Ala Asp Ile Arg Asp Asn Leu
370 375 380
Phe Gly Gly Arg Val Ser Ala Val Met Val Gly Ser Ala Pro Leu Ser
385 390 395 400
Glu Glu Leu Gly Glu Phe Ile Glu Ser Cys Phe Glu Leu Asn Leu Thr
405 410 415
Asp Gly Tyr Gly Ser Thr Glu Ala Gly Met Val Phe Arg Asp Gly Ile
420 425 430
Val Gln Arg Pro Pro Val Ile Asp Tyr Lys Leu Val Asp Val Pro Glu
435 440 445
Leu Gly Tyr Phe Ser Thr Asp Lys Pro His Pro Arg Gly Glu Leu Leu
450 455 460
Leu Lys Thr Asp Gly Met Phe Leu Gly Tyr Tyr Lys Arg Pro Glu Val
465 470 475 480
Thr Ala Ser Val Phe Asp Ala Asp Gly Phe Tyr Met Thr Gly Asp Ile
485 490 495
Val Ala Glu Leu Ala His Asp Asn Ile Glu Ile Ile Asp Arg Arg Asn
500 505 510
Asn Val Leu Lys Leu Ser Gln Gly Glu Phe Val Ala Val Ala Thr Leu
515 520 525
Glu Ala Glu Tyr Ala Asn Ser Pro Val Val His Gln Ile Tyr Val Tyr
530 535 540
Gly Ser Ser Glu Arg Ser Tyr Leu Leu Ala Val Val Val Pro Thr Pro
545 550 555 560
Glu Ala Val Ala Ala Ala Lys Gly Asp Ala Ala Ala Leu Lys Thr Thr
565 570 575
Ile Ala Asp Ser Leu Gln Asp Ile Ala Lys Glu Ile Gln Leu Gln Ser
580 585 590
Tyr Glu Val Pro Arg Asp Phe Ile Ile Glu Pro Gln Pro Phe Thr Gln
595 600 605
Gly Asn Gly Leu Leu Thr Gly Ile Ala Lys Leu Ala Arg Pro Asn Leu
610 615 620
Lys Ala His Tyr Gly Pro Arg Leu Glu Gln Met Tyr Ala Glu Ile Ala
625 630 635 640
Glu Gln Gln Ala Ala Glu Leu Arg Ala Leu His Gly Val Asp Pro Asp
645 650 655
Lys Pro Ala Leu Glu Thr Val Leu Lys Ala Ala Gln Ala Leu Leu Gly
660 665 670
Val Ser Ser Ala Glu Leu Ala Ala Asp Ala His Phe Thr Asp Leu Gly
675 680 685
Gly Asp Ser Leu Ser Ala Leu Ser Phe Ser Asp Leu Leu Arg Asp Ile
690 695 700
Phe Ala Val Glu Val Pro Val Gly Val Ile Val Ser Ala Ala Asn Asp
705 710 715 720
Leu Gly Gly Val Ala Lys Phe Val Asp Glu Gln Arg His Ser Gly Gly
725 730 735
Thr Arg Pro Thr Ala Glu Thr Val His Gly Ala Gly His Thr Glu Ile
740 745 750
Arg Ala Ala Asp Leu Thr Leu Asp Lys Phe Ile Asp Glu Ala Thr Leu
755 760 765
His Ala Ala Pro Ser Leu Pro Lys Ala Ala Gly Ile Pro His Thr Val
770 775 780
Leu Leu Thr Gly Ser Asn Gly Tyr Leu Gly His Tyr Leu Ala Leu Glu
785 790 795 800
Trp Leu Glu Arg Leu Asp Lys Thr Asp Gly Lys Leu Ile Val Ile Val
805 810 815
Arg Gly Lys Asn Ala Glu Ala Ala Tyr Gly Arg Leu Glu Glu Ala Phe
820 825 830
Asp Thr Gly Asp Thr Glu Leu Leu Ala His Phe Arg Ser Leu Ala Asp
835 840 845
Lys His Leu Glu Val Leu Ala Gly Asp Ile Gly Asp Pro Asn Leu Gly
850 855 860
Leu Asp Ala Asp Thr Trp Gln Arg Leu Ala Asp Thr Val Asp Val Ile
865 870 875 880
Val His Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr Asn Gln Leu
885 890 895
Phe Gly Pro Asn Val Val Gly Thr Ala Glu Ile Ile Lys Leu Ala Ile
900 905 910
Thr Thr Lys Ile Lys Pro Val Thr Tyr Leu Ser Thr Val Ala Val Ala
915 920 925
Ala Tyr Val Asp Pro Thr Thr Phe Asp Glu Glu Ser Asp Ile Arg Leu
930 935 940
Ile Ser Ala Val Arg Pro Ile Asp Asp Gly Tyr Ala Asn Gly Tyr Gly
945 950 955 960
Asn Ala Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu
965 970 975
Cys Gly Leu Pro Val Ala Val Phe Arg Ser Asp Met Ile Leu Ala His
980 985 990
Ser Arg Tyr Thr Gly Gln Leu Asn Val Pro Asp Gln Phe Thr Arg Leu
995 1000 1005
Ile Leu Ser Leu Ile Ala Thr Gly Ile Ala Pro Gly Ser Phe Tyr
1010 1015 1020
Gln Ala Gln Thr Thr Gly Glu Arg Pro Leu Ala His Tyr Asp Gly
1025 1030 1035
Leu Pro Gly Asp Phe Thr Ala Glu Ala Ile Thr Thr Leu Gly Thr
1040 1045 1050
Gln Val Pro Glu Gly Ser Glu Gly Phe Val Thr Tyr Asp Cys Val
1055 1060 1065
Asn Pro His Ala Asp Gly Ile Ser Leu Asp Asn Phe Val Asp Trp
1070 1075 1080
Leu Ile Glu Ala Gly Tyr Pro Ile Ala Arg Ile Asp Asn Tyr Thr
1085 1090 1095
Glu Trp Phe Thr Arg Phe Asp Thr Ala Ile Arg Gly Leu Ser Glu
1100 1105 1110
Lys Gln Lys Gln His Ser Leu Leu Pro Leu Leu His Ala Phe Glu
1115 1120 1125
Gln Pro Ser Ala Ala Glu Asn His Gly Val Val Pro Ala Lys Arg
1130 1135 1140
Phe Gln His Ala Val Gln Ala Ala Gly Ile Gly Pro Val Gly Gln
1145 1150 1155
Asp Gly Thr Thr Asp Ile Pro His Leu Ser Arg Arg Leu Ile Val
1160 1165 1170
Lys Tyr Ala Lys Asp Leu Glu Gln Leu Gly Leu Leu
1175 1180 1185
<210> SEQ ID NO 7
<211> LENGTH: 1186
<212> TYPE: PRT
<213> ORGANISM: Segniliparus rotundus
<400> SEQUENCE: 7
Met Thr Gln Ser His Thr Gln Gly Pro Gln Ala Ser Ala Ala His Ser
1 5 10 15
Arg Leu Ala Arg Arg Ala Ala Glu Leu Leu Ala Thr Asp Pro Gln Ala
20 25 30
Ala Ala Thr Leu Pro Asp Pro Glu Val Val Arg Gln Ala Thr Arg Pro
35 40 45
Gly Leu Arg Leu Ala Glu Arg Val Asp Ala Ile Leu Ser Gly Tyr Ala
50 55 60
Asp Arg Pro Ala Leu Gly Gln Arg Ser Phe Gln Thr Val Lys Asp Pro
65 70 75 80
Ile Thr Gly Arg Ser Ser Val Glu Leu Leu Pro Thr Phe Asp Thr Ile
85 90 95
Thr Tyr Arg Glu Leu Arg Glu Arg Ala Thr Ala Ile Ala Ser Asp Leu
100 105 110
Ala His His Pro Gln Ala Pro Ala Lys Pro Gly Asp Phe Leu Ala Ser
115 120 125
Ile Gly Phe Ile Ser Val Asp Tyr Val Ala Ile Asp Ile Ala Gly Val
130 135 140
Phe Ala Gly Leu Thr Ala Val Pro Leu Gln Thr Gly Ala Thr Leu Ala
145 150 155 160
Thr Leu Thr Ala Ile Thr Ala Glu Thr Ala Pro Thr Leu Phe Ala Ala
165 170 175
Ser Ile Glu His Leu Pro Thr Ala Val Asp Ala Val Leu Ala Thr Pro
180 185 190
Ser Val Arg Arg Leu Leu Val Phe Asp Tyr Arg Ala Gly Ser Asp Glu
195 200 205
Asp Arg Glu Ala Val Glu Ala Ala Lys Arg Lys Ile Ala Asp Ala Gly
210 215 220
Ser Ser Val Leu Val Asp Val Leu Asp Glu Val Ile Ala Arg Gly Lys
225 230 235 240
Ser Ala Pro Lys Ala Pro Leu Pro Pro Ala Thr Asp Ala Gly Asp Asp
245 250 255
Ser Leu Ser Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro Lys
260 265 270
Gly Ala Met Tyr Pro Glu Arg Asn Val Ala His Phe Trp Gly Gly Val
275 280 285
Trp Ala Ala Ala Phe Asp Glu Asp Ala Ala Pro Pro Val Pro Ala Ile
290 295 300
Asn Ile Thr Phe Leu Pro Leu Ser His Val Ala Ser Arg Leu Ser Leu
305 310 315 320
Met Pro Thr Leu Ala Arg Gly Gly Leu Met His Phe Val Ala Lys Ser
325 330 335
Asp Leu Ser Thr Leu Phe Glu Asp Leu Lys Leu Ala Arg Pro Thr Asn
340 345 350
Leu Phe Leu Val Pro Arg Val Val Glu Met Leu Tyr Gln His Tyr Gln
355 360 365
Ser Glu Leu Asp Arg Arg Gly Val Gln Asp Gly Thr Arg Glu Ala Glu
370 375 380
Ala Val Lys Asp Asp Leu Arg Thr Gly Leu Leu Gly Gly Arg Ile Leu
385 390 395 400
Thr Ala Gly Phe Gly Ser Ala Pro Leu Ser Ala Glu Leu Ala Gly Phe
405 410 415
Ile Glu Ser Leu Leu Gln Ile His Leu Val Asp Gly Tyr Gly Ser Thr
420 425 430
Glu Ala Gly Pro Val Trp Arg Asp Gly Tyr Leu Val Lys Pro Pro Val
435 440 445
Thr Asp Tyr Lys Leu Ile Asp Val Pro Glu Leu Gly Tyr Phe Ser Thr
450 455 460
Asp Ser Pro His Pro Arg Gly Glu Leu Ala Ile Lys Thr Gln Thr Ile
465 470 475 480
Leu Pro Gly Tyr Tyr Lys Arg Pro Glu Thr Thr Ala Glu Val Phe Asp
485 490 495
Glu Asp Gly Phe Tyr Leu Thr Gly Asp Val Val Ala Gln Ile Gly Pro
500 505 510
Glu Gln Phe Ala Tyr Val Asp Arg Arg Lys Asn Val Leu Lys Leu Ser
515 520 525
Gln Gly Glu Phe Val Thr Leu Ala Lys Leu Glu Ala Ala Tyr Ser Ser
530 535 540
Ser Pro Leu Val Arg Gln Leu Phe Val Tyr Gly Ser Ser Glu Arg Ser
545 550 555 560
Tyr Leu Leu Ala Val Ile Val Pro Thr Pro Asp Ala Leu Lys Lys Phe
565 570 575
Gly Val Gly Glu Ala Ala Lys Ala Ala Leu Gly Glu Ser Leu Gln Lys
580 585 590
Ile Ala Arg Asp Glu Gly Leu Gln Ser Tyr Glu Val Pro Arg Asp Phe
595 600 605
Ile Ile Glu Thr Asp Pro Phe Thr Val Glu Asn Gly Leu Leu Ser Asp
610 615 620
Ala Arg Lys Ser Leu Arg Pro Lys Leu Lys Glu His Tyr Gly Glu Arg
625 630 635 640
Leu Glu Ala Met Tyr Lys Glu Leu Ala Asp Gly Gln Ala Asn Glu Leu
645 650 655
Arg Asp Ile Arg Arg Gly Val Gln Gln Arg Pro Thr Leu Glu Thr Val
660 665 670
Arg Arg Ala Ala Ala Ala Met Leu Gly Ala Ser Ala Ala Glu Ile Lys
675 680 685
Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu
690 695 700
Thr Phe Ser Asn Phe Leu His Asp Leu Phe Glu Val Asp Val Pro Val
705 710 715 720
Gly Val Ile Val Ser Ala Ala Asn Thr Leu Gly Ser Val Ala Glu His
725 730 735
Ile Asp Ala Gln Leu Ala Gly Gly Arg Ala Arg Pro Thr Phe Ala Thr
740 745 750
Val His Gly Lys Gly Ser Thr Thr Ile Lys Ala Ser Asp Leu Thr Leu
755 760 765
Asp Lys Phe Ile Asp Glu Gln Thr Leu Glu Ala Ala Lys His Leu Pro
770 775 780
Lys Pro Ala Asp Pro Pro Arg Thr Val Leu Leu Thr Gly Ala Asn Gly
785 790 795 800
Trp Leu Gly Arg Phe Leu Ala Leu Glu Trp Leu Glu Arg Leu Ala Pro
805 810 815
Ala Gly Gly Lys Leu Ile Thr Ile Val Arg Gly Lys Asp Ala Ala Gln
820 825 830
Ala Lys Ala Arg Leu Asp Ala Ala Tyr Glu Ser Gly Asp Pro Lys Leu
835 840 845
Ala Gly His Tyr Gln Asp Leu Ala Ala Thr Thr Leu Glu Val Leu Ala
850 855 860
Gly Asp Phe Ser Glu Pro Arg Leu Gly Leu Asp Glu Ala Thr Trp Asn
865 870 875 880
Arg Leu Ala Asp Glu Val Asp Phe Ile Ser His Pro Gly Ala Leu Val
885 890 895
Asn His Val Leu Pro Tyr Asn Gln Leu Phe Gly Pro Asn Val Ala Gly
900 905 910
Val Ala Glu Ile Ile Lys Leu Ala Ile Thr Thr Arg Ile Lys Pro Val
915 920 925
Thr Tyr Leu Ser Thr Val Ala Val Ala Ala Gly Val Glu Pro Ser Ala
930 935 940
Leu Asp Glu Asp Gly Asp Ile Arg Thr Val Ser Ala Glu Arg Ser Val
945 950 955 960
Asp Glu Gly Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Gly Gly Glu
965 970 975
Val Leu Leu Arg Glu Ala His Asp Arg Thr Gly Leu Pro Val Arg Val
980 985 990
Phe Arg Ser Asp Met Ile Leu Ala His Gln Lys Tyr Thr Gly Gln Val
995 1000 1005
Asn Ala Thr Asp Gln Phe Thr Arg Leu Val Gln Ser Leu Leu Ala
1010 1015 1020
Thr Gly Leu Ala Pro Lys Ser Phe Tyr Glu Leu Asp Ala Gln Gly
1025 1030 1035
Asn Arg Gln Arg Ala His Tyr Asp Gly Ile Pro Val Asp Phe Thr
1040 1045 1050
Ala Glu Ser Ile Thr Thr Leu Gly Gly Asp Gly Leu Glu Gly Tyr
1055 1060 1065
Arg Ser Tyr Asn Val Phe Asn Pro His Arg Asp Gly Val Gly Leu
1070 1075 1080
Asp Glu Phe Val Asp Trp Leu Ile Glu Ala Gly His Pro Ile Thr
1085 1090 1095
Arg Ile Asp Asp Tyr Asp Gln Trp Leu Ser Arg Phe Glu Thr Ser
1100 1105 1110
Leu Arg Gly Leu Pro Glu Ser Lys Arg Gln Ala Ser Val Leu Pro
1115 1120 1125
Leu Leu His Ala Phe Ala Arg Pro Gly Pro Ala Val Asp Gly Ser
1130 1135 1140
Pro Phe Arg Asn Thr Val Phe Arg Thr Asp Val Gln Lys Ala Lys
1145 1150 1155
Ile Gly Ala Glu His Asp Ile Pro His Leu Gly Lys Ala Leu Val
1160 1165 1170
Leu Lys Tyr Ala Asp Asp Ile Lys Gln Leu Gly Leu Leu
1175 1180 1185
<210> SEQ ID NO 8
<211> LENGTH: 459
<212> TYPE: PRT
<213> ORGANISM: Chromobacterium violaceum
<400> SEQUENCE: 8
Met Gln Lys Gln Arg Thr Thr Ser Gln Trp Arg Glu Leu Asp Ala Ala
1 5 10 15
His His Leu His Pro Phe Thr Asp Thr Ala Ser Leu Asn Gln Ala Gly
20 25 30
Ala Arg Val Met Thr Arg Gly Glu Gly Val Tyr Leu Trp Asp Ser Glu
35 40 45
Gly Asn Lys Ile Ile Asp Gly Met Ala Gly Leu Trp Cys Val Asn Val
50 55 60
Gly Tyr Gly Arg Lys Asp Phe Ala Glu Ala Ala Arg Arg Gln Met Glu
65 70 75 80
Glu Leu Pro Phe Tyr Asn Thr Phe Phe Lys Thr Thr His Pro Ala Val
85 90 95
Val Glu Leu Ser Ser Leu Leu Ala Glu Val Thr Pro Ala Gly Phe Asp
100 105 110
Arg Val Phe Tyr Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Met Ile
115 120 125
Arg Met Val Arg Arg Tyr Trp Asp Val Gln Gly Lys Pro Glu Lys Lys
130 135 140
Thr Leu Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr Ile Gly Gly
145 150 155 160
Ala Ser Leu Gly Gly Met Lys Tyr Met His Glu Gln Gly Asp Leu Pro
165 170 175
Ile Pro Gly Met Ala His Ile Glu Gln Pro Trp Trp Tyr Lys His Gly
180 185 190
Lys Asp Met Thr Pro Asp Glu Phe Gly Val Val Ala Ala Arg Trp Leu
195 200 205
Glu Glu Lys Ile Leu Glu Ile Gly Ala Asp Lys Val Ala Ala Phe Val
210 215 220
Gly Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro Pro Ala Thr
225 230 235 240
Tyr Trp Pro Glu Ile Glu Arg Ile Cys Arg Lys Tyr Asp Val Leu Leu
245 250 255
Val Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Trp Phe
260 265 270
Gly His Gln His Phe Gly Phe Gln Pro Asp Leu Phe Thr Ala Ala Lys
275 280 285
Gly Leu Ser Ser Gly Tyr Leu Pro Ile Gly Ala Val Phe Val Gly Lys
290 295 300
Arg Val Ala Glu Gly Leu Ile Ala Gly Gly Asp Phe Asn His Gly Phe
305 310 315 320
Thr Tyr Ser Gly His Pro Val Cys Ala Ala Val Ala His Ala Asn Val
325 330 335
Ala Ala Leu Arg Asp Glu Gly Ile Val Gln Arg Val Lys Asp Asp Ile
340 345 350
Gly Pro Tyr Met Gln Lys Arg Trp Arg Glu Thr Phe Ser Arg Phe Glu
355 360 365
His Val Asp Asp Val Arg Gly Val Gly Met Val Gln Ala Phe Thr Leu
370 375 380
Val Lys Asn Lys Ala Lys Arg Glu Leu Phe Pro Asp Phe Gly Glu Ile
385 390 395 400
Gly Thr Leu Cys Arg Asp Ile Phe Phe Arg Asn Asn Leu Ile Met Arg
405 410 415
Ala Cys Gly Asp His Ile Val Ser Ala Pro Pro Leu Val Met Thr Arg
420 425 430
Ala Glu Val Asp Glu Met Leu Ala Val Ala Glu Arg Cys Leu Glu Glu
435 440 445
Phe Glu Gln Thr Leu Lys Ala Arg Gly Leu Ala
450 455
<210> SEQ ID NO 9
<211> LENGTH: 468
<212> TYPE: PRT
<213> ORGANISM: Pseudomonas aeruginosa
<400> SEQUENCE: 9
Met Asn Ala Arg Leu His Ala Thr Ser Pro Leu Gly Asp Ala Asp Leu
1 5 10 15
Val Arg Ala Asp Gln Ala His Tyr Met His Gly Tyr His Val Phe Asp
20 25 30
Asp His Arg Val Asn Gly Ser Leu Asn Ile Ala Ala Gly Asp Gly Ala
35 40 45
Tyr Ile Tyr Asp Thr Ala Gly Asn Arg Tyr Leu Asp Ala Val Gly Gly
50 55 60
Met Trp Cys Thr Asn Ile Gly Leu Gly Arg Glu Glu Met Ala Arg Thr
65 70 75 80
Val Ala Glu Gln Thr Arg Leu Leu Ala Tyr Ser Asn Pro Phe Cys Asp
85 90 95
Met Ala Asn Pro Arg Ala Ile Glu Leu Cys Arg Lys Leu Ala Glu Leu
100 105 110
Ala Pro Gly Asp Leu Asp His Val Phe Leu Thr Thr Gly Gly Ser Thr
115 120 125
Ala Val Asp Thr Ala Ile Arg Leu Met His Tyr Tyr Gln Asn Cys Arg
130 135 140
Gly Lys Arg Ala Lys Lys His Val Ile Thr Arg Ile Asn Ala Tyr His
145 150 155 160
Gly Ser Thr Phe Leu Gly Met Ser Leu Gly Gly Lys Ser Ala Asp Arg
165 170 175
Pro Ala Glu Phe Asp Phe Leu Asp Glu Arg Ile His His Leu Ala Cys
180 185 190
Pro Tyr Tyr Tyr Arg Ala Pro Glu Gly Leu Gly Glu Ala Glu Phe Leu
195 200 205
Asp Gly Leu Val Asp Glu Phe Glu Arg Lys Ile Leu Glu Leu Gly Ala
210 215 220
Asp Arg Val Gly Ala Phe Ile Ser Glu Pro Val Phe Gly Ser Gly Gly
225 230 235 240
Val Ile Val Pro Pro Ala Gly Tyr His Arg Arg Met Trp Glu Leu Cys
245 250 255
Gln Arg Tyr Asp Val Leu Tyr Ile Ser Asp Glu Val Val Thr Ser Phe
260 265 270
Gly Arg Leu Gly His Phe Phe Ala Ser Gln Ala Val Phe Gly Val Gln
275 280 285
Pro Asp Ile Ile Leu Thr Ala Lys Gly Leu Thr Ser Gly Tyr Gln Pro
290 295 300
Leu Gly Ala Cys Ile Phe Ser Arg Arg Ile Trp Glu Val Ile Ala Glu
305 310 315 320
Pro Asp Lys Gly Arg Cys Phe Ser His Gly Phe Thr Tyr Ser Gly His
325 330 335
Pro Val Ala Cys Ala Ala Ala Leu Lys Asn Ile Glu Ile Ile Glu Arg
340 345 350
Glu Gly Leu Leu Ala His Ala Asp Glu Val Gly Arg Tyr Phe Glu Glu
355 360 365
Arg Leu Gln Ser Leu Arg Asp Leu Pro Ile Val Gly Asp Val Arg Gly
370 375 380
Met Arg Phe Met Ala Cys Val Glu Phe Val Ala Asp Lys Ala Ser Lys
385 390 395 400
Ala Leu Phe Pro Glu Ser Leu Asn Ile Gly Glu Trp Val His Leu Arg
405 410 415
Ala Gln Lys Arg Gly Leu Leu Val Arg Pro Ile Val His Leu Asn Val
420 425 430
Met Ser Pro Pro Leu Ile Leu Thr Arg Glu Gln Val Asp Thr Val Val
435 440 445
Arg Val Leu Arg Glu Ser Ile Glu Glu Thr Val Glu Asp Leu Val Arg
450 455 460
Ala Gly His Arg
465
<210> SEQ ID NO 10
<211> LENGTH: 454
<212> TYPE: PRT
<213> ORGANISM: Pseudomonas syringae
<400> SEQUENCE: 10
Met Ser Ala Asn Asn Pro Gln Thr Leu Glu Trp Gln Ala Leu Ser Ser
1 5 10 15
Glu His His Leu Ala Pro Phe Ser Asp Tyr Lys Gln Leu Lys Glu Lys
20 25 30
Gly Pro Arg Ile Ile Thr Arg Ala Glu Gly Val Tyr Leu Trp Asp Ser
35 40 45
Glu Gly Asn Lys Ile Leu Asp Gly Met Ser Gly Leu Trp Cys Val Ala
50 55 60
Ile Gly Tyr Gly Arg Glu Glu Leu Ala Asp Ala Ala Ser Lys Gln Met
65 70 75 80
Arg Glu Leu Pro Tyr Tyr Asn Leu Phe Phe Gln Thr Ala His Pro Pro
85 90 95
Val Leu Glu Leu Ala Lys Ala Ile Ser Asp Ile Ala Pro Glu Gly Met
100 105 110
Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Gly Asn Asp Thr Met
115 120 125
Leu Arg Met Val Arg His Tyr Trp Ala Leu Lys Gly Gln Pro Asn Lys
130 135 140
Lys Thr Ile Ile Ser Arg Val Asn Gly Tyr His Gly Ser Thr Val Ala
145 150 155 160
Gly Ala Ser Leu Gly Gly Met Thr Tyr Met His Glu Gln Gly Asp Leu
165 170 175
Pro Ile Pro Gly Val Val His Ile Pro Gln Pro Tyr Trp Phe Gly Glu
180 185 190
Gly Gly Asp Met Thr Pro Asp Glu Phe Gly Ile Trp Ala Ala Glu Gln
195 200 205
Leu Glu Lys Lys Ile Leu Glu Leu Gly Val Glu Asn Val Gly Ala Phe
210 215 220
Ile Ala Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro Pro Asp
225 230 235 240
Ser Tyr Trp Pro Lys Ile Lys Glu Ile Leu Ser Arg Tyr Asp Ile Leu
245 250 255
Phe Ala Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Ser Glu Trp
260 265 270
Phe Gly Ser Asp Phe Tyr Gly Leu Arg Pro Asp Met Met Thr Ile Ala
275 280 285
Lys Gly Leu Thr Ser Gly Tyr Val Pro Met Gly Gly Leu Ile Val Arg
290 295 300
Asp Glu Ile Val Ala Val Leu Asn Glu Gly Gly Asp Phe Asn His Gly
305 310 315 320
Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu Glu Asn
325 330 335
Ile Arg Ile Leu Arg Glu Glu Lys Ile Val Glu Arg Val Arg Ser Glu
340 345 350
Thr Ala Pro Tyr Leu Gln Lys Arg Leu Arg Glu Leu Ser Asp His Pro
355 360 365
Leu Val Gly Glu Val Arg Gly Val Gly Leu Leu Gly Ala Ile Glu Leu
370 375 380
Val Lys Asp Lys Thr Thr Arg Glu Arg Tyr Thr Asp Lys Gly Ala Gly
385 390 395 400
Met Ile Cys Arg Thr Phe Cys Phe Asp Asn Gly Leu Ile Met Arg Ala
405 410 415
Val Gly Asp Thr Met Ile Ile Ala Pro Pro Leu Val Ile Ser Phe Ala
420 425 430
Gln Ile Asp Glu Leu Val Glu Lys Ala Arg Thr Cys Leu Asp Leu Thr
435 440 445
Leu Ala Val Leu Gln Gly
450
<210> SEQ ID NO 11
<211> LENGTH: 467
<212> TYPE: PRT
<213> ORGANISM: Rhodobacter sphaeroides
<400> SEQUENCE: 11
Met Thr Arg Asn Asp Ala Thr Asn Ala Ala Gly Ala Val Gly Ala Ala
1 5 10 15
Met Arg Asp His Ile Leu Leu Pro Ala Gln Glu Met Ala Lys Leu Gly
20 25 30
Lys Ser Ala Gln Pro Val Leu Thr His Ala Glu Gly Ile Tyr Val His
35 40 45
Thr Glu Asp Gly Arg Arg Leu Ile Asp Gly Pro Ala Gly Met Trp Cys
50 55 60
Ala Gln Val Gly Tyr Gly Arg Arg Glu Ile Val Asp Ala Met Ala His
65 70 75 80
Gln Ala Met Val Leu Pro Tyr Ala Ser Pro Trp Tyr Met Ala Thr Ser
85 90 95
Pro Ala Ala Arg Leu Ala Glu Lys Ile Ala Thr Leu Thr Pro Gly Asp
100 105 110
Leu Asn Arg Ile Phe Phe Thr Thr Gly Gly Ser Thr Ala Val Asp Ser
115 120 125
Ala Leu Arg Phe Ser Glu Phe Tyr Asn Asn Val Leu Gly Arg Pro Gln
130 135 140
Lys Lys Arg Ile Ile Val Arg Tyr Asp Gly Tyr His Gly Ser Thr Ala
145 150 155 160
Leu Thr Ala Ala Cys Thr Gly Arg Thr Gly Asn Trp Pro Asn Phe Asp
165 170 175
Ile Ala Gln Asp Arg Ile Ser Phe Leu Ser Ser Pro Asn Pro Arg His
180 185 190
Ala Gly Asn Arg Ser Gln Glu Ala Phe Leu Asp Asp Leu Val Gln Glu
195 200 205
Phe Glu Asp Arg Ile Glu Ser Leu Gly Pro Asp Thr Ile Ala Ala Phe
210 215 220
Leu Ala Glu Pro Ile Leu Ala Ser Gly Gly Val Ile Ile Pro Pro Ala
225 230 235 240
Gly Tyr His Ala Arg Phe Lys Ala Ile Cys Glu Lys His Asp Ile Leu
245 250 255
Tyr Ile Ser Asp Glu Val Val Thr Gly Phe Gly Arg Cys Gly Glu Trp
260 265 270
Phe Ala Ser Glu Lys Val Phe Gly Val Val Pro Asp Ile Ile Thr Phe
275 280 285
Ala Lys Gly Val Thr Ser Gly Tyr Val Pro Leu Gly Gly Leu Ala Ile
290 295 300
Ser Glu Ala Val Leu Ala Arg Ile Ser Gly Glu Asn Ala Lys Gly Ser
305 310 315 320
Trp Phe Thr Asn Gly Tyr Thr Tyr Ser Asn Gln Pro Val Ala Cys Ala
325 330 335
Ala Ala Leu Ala Asn Ile Glu Leu Met Glu Arg Glu Gly Ile Val Asp
340 345 350
Gln Ala Arg Glu Met Ala Asp Tyr Phe Ala Ala Ala Leu Ala Ser Leu
355 360 365
Arg Asp Leu Pro Gly Val Ala Glu Thr Arg Ser Val Gly Leu Val Gly
370 375 380
Cys Val Gln Cys Leu Leu Asp Pro Thr Arg Ala Asp Gly Thr Ala Glu
385 390 395 400
Asp Lys Ala Phe Thr Leu Lys Ile Asp Glu Arg Cys Phe Glu Leu Gly
405 410 415
Leu Ile Val Arg Pro Leu Gly Asp Leu Cys Val Ile Ser Pro Pro Leu
420 425 430
Ile Ile Ser Arg Ala Gln Ile Asp Glu Met Val Ala Ile Met Arg Gln
435 440 445
Ala Ile Thr Glu Val Ser Ala Ala His Gly Leu Thr Ala Lys Glu Pro
450 455 460
Ala Ala Val
465
<210> SEQ ID NO 12
<211> LENGTH: 459
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli
<400> SEQUENCE: 12
Met Asn Arg Leu Pro Ser Ser Ala Ser Ala Leu Ala Cys Ser Ala His
1 5 10 15
Ala Leu Asn Leu Ile Glu Lys Arg Thr Leu Asp His Glu Glu Met Lys
20 25 30
Ala Leu Asn Arg Glu Val Ile Glu Tyr Phe Lys Glu His Val Asn Pro
35 40 45
Gly Phe Leu Glu Tyr Arg Lys Ser Val Thr Ala Gly Gly Asp Tyr Gly
50 55 60
Ala Val Glu Trp Gln Ala Gly Ser Leu Asn Thr Leu Val Asp Thr Gln
65 70 75 80
Gly Gln Glu Phe Ile Asp Cys Leu Gly Gly Phe Gly Ile Phe Asn Val
85 90 95
Gly His Arg Asn Pro Val Val Val Ser Ala Val Gln Asn Gln Leu Ala
100 105 110
Lys Gln Pro Leu His Ser Gln Glu Leu Leu Asp Pro Leu Arg Ala Met
115 120 125
Leu Ala Lys Thr Leu Ala Ala Leu Thr Pro Gly Lys Leu Lys Tyr Ser
130 135 140
Phe Phe Cys Asn Ser Gly Thr Glu Ser Val Glu Ala Ala Leu Lys Leu
145 150 155 160
Ala Lys Ala Tyr Gln Ser Pro Arg Gly Lys Phe Thr Phe Ile Ala Thr
165 170 175
Ser Gly Ala Phe His Gly Lys Ser Leu Gly Ala Leu Ser Ala Thr Ala
180 185 190
Lys Ser Thr Phe Arg Lys Pro Phe Met Pro Leu Leu Pro Gly Phe Arg
195 200 205
His Val Pro Phe Gly Asn Ile Glu Ala Met Arg Thr Ala Leu Asn Glu
210 215 220
Cys Lys Lys Thr Gly Asp Asp Val Ala Ala Val Ile Leu Glu Pro Ile
225 230 235 240
Gln Gly Glu Gly Gly Val Ile Leu Pro Pro Pro Gly Tyr Leu Thr Ala
245 250 255
Val Arg Lys Leu Cys Asp Glu Phe Gly Ala Leu Met Ile Leu Asp Glu
260 265 270
Val Gln Thr Gly Met Gly Arg Thr Gly Lys Met Phe Ala Cys Glu His
275 280 285
Glu Asn Val Gln Pro Asp Ile Leu Cys Leu Ala Lys Ala Leu Gly Gly
290 295 300
Gly Val Met Pro Ile Gly Ala Thr Ile Ala Thr Glu Glu Val Phe Ser
305 310 315 320
Val Leu Phe Asp Asn Pro Phe Leu His Thr Thr Thr Phe Gly Gly Asn
325 330 335
Pro Leu Ala Cys Ala Ala Ala Leu Ala Thr Ile Asn Val Leu Leu Glu
340 345 350
Gln Asn Leu Pro Ala Gln Ala Glu Gln Lys Gly Asp Met Leu Leu Asp
355 360 365
Gly Phe Arg Gln Leu Ala Arg Glu Tyr Pro Asp Leu Val Gln Glu Ala
370 375 380
Arg Gly Lys Gly Met Leu Met Ala Ile Glu Phe Val Asp Asn Glu Ile
385 390 395 400
Gly Tyr Asn Phe Ala Ser Glu Met Phe Arg Gln Arg Val Leu Val Ala
405 410 415
Gly Thr Leu Asn Asn Ala Lys Thr Ile Arg Ile Glu Pro Pro Leu Thr
420 425 430
Leu Thr Ile Glu Gln Cys Glu Leu Val Ile Lys Ala Ala Arg Lys Ala
435 440 445
Leu Ala Ala Met Arg Val Ser Val Glu Glu Ala
450 455
<210> SEQ ID NO 13
<211> LENGTH: 453
<212> TYPE: PRT
<213> ORGANISM: Vibrio fluvialis
<400> SEQUENCE: 13
Met Asn Lys Pro Gln Ser Trp Glu Ala Arg Ala Glu Thr Tyr Ser Leu
1 5 10 15
Tyr Gly Phe Thr Asp Met Pro Ser Leu His Gln Arg Gly Thr Val Val
20 25 30
Val Thr His Gly Glu Gly Pro Tyr Ile Val Asp Val Asn Gly Arg Arg
35 40 45
Tyr Leu Asp Ala Asn Ser Gly Leu Trp Asn Met Val Ala Gly Phe Asp
50 55 60
His Lys Gly Leu Ile Asp Ala Ala Lys Ala Gln Tyr Glu Arg Phe Pro
65 70 75 80
Gly Tyr His Ala Phe Phe Gly Arg Met Ser Asp Gln Thr Val Met Leu
85 90 95
Ser Glu Lys Leu Val Glu Val Ser Pro Phe Asp Ser Gly Arg Val Phe
100 105 110
Tyr Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr Met Val Lys Met Leu
115 120 125
Trp Phe Leu His Ala Ala Glu Gly Lys Pro Gln Lys Arg Lys Ile Leu
130 135 140
Thr Arg Trp Asn Ala Tyr His Gly Val Thr Ala Val Ser Ala Ser Met
145 150 155 160
Thr Gly Lys Pro Tyr Asn Ser Val Phe Gly Leu Pro Leu Pro Gly Phe
165 170 175
Val His Leu Thr Cys Pro His Tyr Trp Arg Tyr Gly Glu Glu Gly Glu
180 185 190
Thr Glu Glu Gln Phe Val Ala Arg Leu Ala Arg Glu Leu Glu Glu Thr
195 200 205
Ile Gln Arg Glu Gly Ala Asp Thr Ile Ala Gly Phe Phe Ala Glu Pro
210 215 220
Val Met Gly Ala Gly Gly Val Ile Pro Pro Ala Lys Gly Tyr Phe Gln
225 230 235 240
Ala Ile Leu Pro Ile Leu Arg Lys Tyr Asp Ile Pro Val Ile Ser Asp
245 250 255
Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Asn Thr Trp Gly Cys Val
260 265 270
Thr Tyr Asp Phe Thr Pro Asp Ala Ile Ile Ser Ser Lys Asn Leu Thr
275 280 285
Ala Gly Phe Phe Pro Met Gly Ala Val Ile Leu Gly Pro Glu Leu Ser
290 295 300
Lys Arg Leu Glu Thr Ala Ile Glu Ala Ile Glu Glu Phe Pro His Gly
305 310 315 320
Phe Thr Ala Ser Gly His Pro Val Gly Cys Ala Ile Ala Leu Lys Ala
325 330 335
Ile Asp Val Val Met Asn Glu Gly Leu Ala Glu Asn Val Arg Arg Leu
340 345 350
Ala Pro Arg Phe Glu Glu Arg Leu Lys His Ile Ala Glu Arg Pro Asn
355 360 365
Ile Gly Glu Tyr Arg Gly Ile Gly Phe Met Trp Ala Leu Glu Ala Val
370 375 380
Lys Asp Lys Ala Ser Lys Thr Pro Phe Asp Gly Asn Leu Ser Val Ser
385 390 395 400
Glu Arg Ile Ala Asn Thr Cys Thr Asp Leu Gly Leu Ile Cys Arg Pro
405 410 415
Leu Gly Gln Ser Val Val Leu Cys Pro Pro Phe Ile Leu Thr Glu Ala
420 425 430
Gln Met Asp Glu Met Phe Asp Lys Leu Glu Lys Ala Leu Asp Lys Val
435 440 445
Phe Ala Glu Val Ala
450
<210> SEQ ID NO 14
<211> LENGTH: 224
<212> TYPE: PRT
<213> ORGANISM: Bacillus subtilis
<400> SEQUENCE: 14
Met Lys Ile Tyr Gly Ile Tyr Met Asp Arg Pro Leu Ser Gln Glu Glu
1 5 10 15
Asn Glu Arg Phe Met Ser Phe Ile Ser Pro Glu Lys Arg Glu Lys Cys
20 25 30
Arg Arg Phe Tyr His Lys Glu Asp Ala His Arg Thr Leu Leu Gly Asp
35 40 45
Val Leu Val Arg Ser Val Ile Ser Arg Gln Tyr Gln Leu Asp Lys Ser
50 55 60
Asp Ile Arg Phe Ser Thr Gln Glu Tyr Gly Lys Pro Cys Ile Pro Asp
65 70 75 80
Leu Pro Asp Ala His Phe Asn Ile Ser His Ser Gly Arg Trp Val Ile
85 90 95
Cys Ala Phe Asp Ser Gln Pro Ile Gly Ile Asp Ile Glu Lys Thr Lys
100 105 110
Pro Ile Ser Leu Glu Ile Ala Lys Arg Phe Phe Ser Lys Thr Glu Tyr
115 120 125
Ser Asp Leu Leu Ala Lys Asp Lys Asp Glu Gln Thr Asp Tyr Phe Tyr
130 135 140
His Leu Trp Ser Met Lys Glu Ser Phe Ile Lys Gln Glu Gly Lys Gly
145 150 155 160
Leu Ser Leu Pro Leu Asp Ser Phe Ser Val Arg Leu His Gln Asp Gly
165 170 175
Gln Val Ser Ile Glu Leu Pro Asp Ser His Ser Pro Cys Tyr Ile Lys
180 185 190
Thr Tyr Glu Val Asp Pro Gly Tyr Lys Met Ala Val Cys Ala Ala His
195 200 205
Pro Asp Phe Pro Glu Asp Ile Thr Met Val Ser Tyr Glu Glu Leu Leu
210 215 220
<210> SEQ ID NO 15
<211> LENGTH: 222
<212> TYPE: PRT
<213> ORGANISM: Nocardia sp. NRRL 5646
<400> SEQUENCE: 15
Met Ile Glu Thr Ile Leu Pro Ala Gly Val Glu Ser Ala Glu Leu Leu
1 5 10 15
Glu Tyr Pro Glu Asp Leu Lys Ala His Pro Ala Glu Glu His Leu Ile
20 25 30
Ala Lys Ser Val Glu Lys Arg Arg Arg Asp Phe Ile Gly Ala Arg His
35 40 45
Cys Ala Arg Leu Ala Leu Ala Glu Leu Gly Glu Pro Pro Val Ala Ile
50 55 60
Gly Lys Gly Glu Arg Gly Ala Pro Ile Trp Pro Arg Gly Val Val Gly
65 70 75 80
Ser Leu Thr His Cys Asp Gly Tyr Arg Ala Ala Ala Val Ala His Lys
85 90 95
Met Arg Phe Arg Ser Ile Gly Ile Asp Ala Glu Pro His Ala Thr Leu
100 105 110
Pro Glu Gly Val Leu Asp Ser Val Ser Leu Pro Pro Glu Arg Glu Trp
115 120 125
Leu Lys Thr Thr Asp Ser Ala Leu His Leu Asp Arg Leu Leu Phe Cys
130 135 140
Ala Lys Glu Ala Thr Tyr Lys Ala Trp Trp Pro Leu Thr Ala Arg Trp
145 150 155 160
Leu Gly Phe Glu Glu Ala His Ile Thr Phe Glu Ile Glu Asp Gly Ser
165 170 175
Ala Asp Ser Gly Asn Gly Thr Phe His Ser Glu Leu Leu Val Pro Gly
180 185 190
Gln Thr Asn Asp Gly Gly Thr Pro Leu Leu Ser Phe Asp Gly Arg Trp
195 200 205
Leu Ile Ala Asp Gly Phe Ile Leu Thr Ala Ile Ala Tyr Ala
210 215 220
<210> SEQ ID NO 16
<211> LENGTH: 269
<212> TYPE: PRT
<213> ORGANISM: Bacillus cereus
<400> SEQUENCE: 16
Met Ile Asn Lys Thr Leu Leu Gln Lys Arg Phe Asn Val Ala Ala Val
1 5 10 15
Ser Tyr Asp Gln Tyr Ala Asn Val Gln Lys Lys Met Ala His Ser Leu
20 25 30
Leu Ser Thr Leu Asn Arg Arg Tyr Ser Thr Asn Ser Ser Ile Arg Ile
35 40 45
Leu Glu Leu Gly Cys Gly Thr Gly Tyr Val Thr Glu Gln Leu Ser Asn
50 55 60
Leu Phe Pro Lys Ala Gln Ile Thr Ala Ile Asp Phe Ala Glu Ser Met
65 70 75 80
Ile Ala Val Ala Lys Thr Arg Gln Asn Val Asn Asn Val Thr Phe Tyr
85 90 95
Cys Glu Asp Ile Glu Arg Leu Arg Leu Glu Glu Thr Tyr Asp Val Ile
100 105 110
Ile Ser Asn Ala Thr Phe Gln Trp Leu Asn Asp Leu Lys Gln Val Ile
115 120 125
Thr Asn Leu Phe Arg His Leu Ser Ile Glu Gly Ile Leu Leu Phe Ser
130 135 140
Thr Phe Gly Gln Glu Thr Phe Gln Glu Leu His Ala Ser Phe Gln Arg
145 150 155 160
Ala Lys Glu Glu Lys Asn Ile Gln Asn Glu Thr Ser Ile Gly Gln Arg
165 170 175
Phe Tyr Ser Lys Asn Gln Leu Arg His Ile Cys Glu Ile Glu Thr Gly
180 185 190
Asp Val His Val Ser Glu Thr Cys Tyr Ile Glu Arg Phe Thr Glu Val
195 200 205
Arg Glu Phe Leu His Ser Ile Arg Lys Val Gly Ala Thr Asn Ser Asn
210 215 220
Glu Glu Ser Tyr Cys Gln Ser Pro Ser Leu Phe Arg Ala Met Leu Arg
225 230 235 240
Ile Tyr Glu Arg Asp Phe Thr Gly Asn Glu Gly Ile Met Ala Thr Tyr
245 250 255
His Ala Leu Phe Val His Ile Thr Lys Glu Gly Lys Arg
260 265
<210> SEQ ID NO 17
<211> LENGTH: 256
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli
<400> SEQUENCE: 17
Met Asn Asn Ile Trp Trp Gln Thr Lys Gly Gln Gly Asn Val His Leu
1 5 10 15
Val Leu Leu His Gly Trp Gly Leu Asn Ala Glu Val Trp Arg Cys Ile
20 25 30
Asp Glu Glu Leu Ser Ser His Phe Thr Leu His Leu Val Asp Leu Pro
35 40 45
Gly Phe Gly Arg Ser Arg Gly Phe Gly Ala Leu Ser Leu Ala Asp Met
50 55 60
Ala Glu Ala Val Leu Gln Gln Ala Pro Asp Lys Ala Ile Trp Leu Gly
65 70 75 80
Trp Ser Leu Gly Gly Leu Val Ala Ser Gln Ile Ala Leu Thr His Pro
85 90 95
Glu Arg Val Gln Ala Leu Val Thr Val Ala Ser Ser Pro Cys Phe Ser
100 105 110
Ala Arg Asp Glu Trp Pro Gly Ile Lys Pro Asp Val Leu Ala Gly Phe
115 120 125
Gln Gln Gln Leu Ser Asp Asp Phe Gln Arg Thr Val Glu Arg Phe Leu
130 135 140
Ala Leu Gln Thr Met Gly Thr Glu Thr Ala Arg Gln Asp Ala Arg Ala
145 150 155 160
Leu Lys Lys Thr Val Leu Ala Leu Pro Met Pro Glu Val Asp Val Leu
165 170 175
Asn Gly Gly Leu Glu Ile Leu Lys Thr Val Asp Leu Arg Gln Pro Leu
180 185 190
Gln Asn Val Ser Met Pro Phe Leu Arg Leu Tyr Gly Tyr Leu Asp Gly
195 200 205
Leu Val Pro Arg Lys Val Val Pro Met Leu Asp Lys Leu Trp Pro His
210 215 220
Ser Glu Ser Tyr Ile Phe Ala Lys Ala Ala His Ala Pro Phe Ile Ser
225 230 235 240
His Pro Ala Glu Phe Cys His Leu Leu Val Ala Leu Lys Gln Arg Val
245 250 255
<210> SEQ ID NO 18
<211> LENGTH: 556
<212> TYPE: PRT
<213> ORGANISM: Clostridium cylindrosporum
<400> SEQUENCE: 18
Met Lys Thr Asp Val Gln Ile Ala Gln Glu Ala Gln Met Lys Pro Ile
1 5 10 15
Thr Glu Val Ala Asn Tyr Leu Gly Ile Gln Asp Asp Glu Leu Glu Leu
20 25 30
Tyr Gly Lys Tyr Lys Ala Lys Val Ser Leu Asp Val Leu Glu Arg Gln
35 40 45
Lys Asp Lys Glu Asp Ala Lys Leu Val Leu Val Thr Ala Ile Asn Pro
50 55 60
Thr Pro Ala Gly Glu Gly Lys Thr Thr Thr Asn Val Gly Leu Ser Met
65 70 75 80
Gly Leu Asn Lys Ile Gly Lys Arg Thr Ile Thr Ala Leu Arg Glu Pro
85 90 95
Ser Leu Gly Pro Cys Phe Gly Val Lys Gly Gly Ala Ala Gly Gly Gly
100 105 110
Tyr Ala Gln Val Val Pro Met Asp Asp Ile Asn Leu His Phe Thr Gly
115 120 125
Asp Phe His Ala Ile Thr Ser Ala His Asn Leu Leu Ala Ala Leu Leu
130 135 140
Asp Asn His Leu His Gln Gly Asn Ala Leu Asn Ile Asn Pro Lys Lys
145 150 155 160
Ile Val Trp Lys Arg Val Ile Asp Met Asn Asp Arg Ser Leu Arg Asn
165 170 175
Val Ile Ile Gly Leu Gly Gly Asn Gly Asp Gly Phe Val Arg Gln Ala
180 185 190
Gln Phe Asp Ile Thr Val Ala Ser Glu Ile Met Ala Ile Leu Cys Leu
195 200 205
Ala Thr Ser Met Ser Asp Leu Lys Glu Arg Leu Ser Lys Met Ile Val
210 215 220
Ala Tyr Ala Lys Asp Gly Ser Ala Val Thr Ala Gly Gln Leu Glu Ala
225 230 235 240
Thr Gly Ala Met Ala Leu Leu Leu Lys Asp Ala Val Lys Pro Asn Leu
245 250 255
Val Gln Thr Leu Glu Asn Thr Pro Ala Phe Ile His Gly Gly Pro Phe
260 265 270
Ala Asn Ile Ala His Gly Cys Asn Ser Val Leu Ala Thr Lys Val Ala
275 280 285
Leu Lys Leu Ala Asp Tyr Val Val Thr Glu Gly Gly Phe Gly Ala Asp
290 295 300
Leu Gly Ala Glu Lys Phe Phe Asp Ile Lys Ser Arg Phe Ala Gly Leu
305 310 315 320
Lys Pro Asn Cys Asp Val Ser Val Ala Thr Val Arg Ala Leu Lys Met
325 330 335
Asn Gly Gly Val Pro Lys Thr Glu Leu Ala Ala Glu Asn Val Glu Ala
340 345 350
Val Lys Lys Gly Val Ala Asn Leu Glu Arg His Ile Glu Asn Val Ala
355 360 365
Lys Phe Gly Val Pro Ala Val Val Ala Ile Asn Lys Phe Pro Leu Asp
370 375 380
Thr Glu Ala Glu Leu Lys Ala Val Glu Asp Ala Cys Asn Ala Lys Gly
385 390 395 400
Ala Asp Val Val Leu Ser Asp Val Trp Ala Asn Gly Gly Glu Gly Gly
405 410 415
Val Glu Met Ala Lys Lys Val Val Glu Ile Cys Glu Lys Asn Glu Ala
420 425 430
Asn Phe Ala Pro Leu Tyr Asp Val Asn Leu Ser Ile Pro Glu Lys Ile
435 440 445
Glu Lys Ile Ala Thr Thr Ile Tyr Arg Ala Asp Gly Val Asp Phe Thr
450 455 460
Ser Asp Cys Lys Lys Gln Ile Ala Glu Leu Glu Lys Leu Gly Leu Asp
465 470 475 480
Lys Met Pro Ile Cys Met Ala Lys Thr Gln Tyr Ser Phe Ser Asp Asp
485 490 495
Pro Thr Leu Leu Gly Ala Pro Thr Gly Phe Arg Ile Thr Val Arg Glu
500 505 510
Val Arg Val Ser Ala Gly Ala Gly Phe Ile Val Ala Leu Thr Gly Asn
515 520 525
Met Met Thr Met Pro Gly Leu Pro Lys Val Pro Ala Ala Asn Gly Met
530 535 540
Asp Ile Leu Glu Ser Gly Glu Ile Ile Gly Leu Ser
545 550 555
<210> SEQ ID NO 19
<211> LENGTH: 556
<212> TYPE: PRT
<213> ORGANISM: Alkaliphilus oremlandii (strain OhILAs)
<400> SEQUENCE: 19
Met Lys Thr Asp Val Gln Ile Ala Gln Glu Ala Lys Met Leu Pro Ile
1 5 10 15
Ala Asp Ile Ala Ala Gly Leu Gly Ile Gln Asp Asp Glu Leu Glu Leu
20 25 30
Tyr Gly Lys Tyr Lys Ala Lys Val Ser Leu Asp Val Phe Asp Arg Leu
35 40 45
Lys Asp Lys Pro Asp Gly Lys Leu Ile Leu Val Thr Ala Ile Asn Pro
50 55 60
Thr Pro Ala Gly Glu Gly Lys Thr Thr Thr Asn Val Gly Leu Ser Met
65 70 75 80
Gly Leu Asn Lys Ile Gly Lys Lys Thr Ile Thr Ala Leu Arg Glu Pro
85 90 95
Ser Leu Gly Pro Asn Phe Gly Val Lys Gly Gly Ala Ala Gly Gly Gly
100 105 110
Tyr Ala Gln Val Val Pro Met Glu Asp Ile Asn Leu His Phe Thr Gly
115 120 125
Asp Ile His Ala Ile Thr Thr Ala His Asn Leu Leu Ala Ala Leu Leu
130 135 140
Asp Asn His Leu His Gln Gly Asn Lys Leu Asn Ile Asp Ser Arg Arg
145 150 155 160
Ile Val Trp Arg Arg Val Leu Asp Met Asn Asp Arg Ala Leu Arg Asn
165 170 175
Thr Val Ile Gly Leu Gly Ser Arg Gly Asp Gly Val Pro Arg Gln Asp
180 185 190
Gly Phe Asp Ile Thr Val Ala Ser Glu Ile Met Ala Ile Leu Cys Leu
195 200 205
Ser Asn Ser Leu Glu Asp Leu Lys Asp Arg Ile Ser Arg Met Val Val
210 215 220
Ala Tyr Asn Leu Asp Asn Gln Pro Ile Thr Val Asn Asp Leu Glu Ala
225 230 235 240
Thr Gly Ala Leu Ser Leu Leu Leu Lys Asp Ala Ile Lys Pro Asn Leu
245 250 255
Val Gln Thr Leu Glu Asn Thr Pro Ala Phe Ile His Gly Gly Pro Phe
260 265 270
Ala Asn Ile Ala His Gly Cys Asn Ser Val Leu Ala Thr Lys Leu Gly
275 280 285
Leu Lys Leu Ala Asp Tyr Val Val Thr Glu Ala Gly Phe Gly Ala Asp
290 295 300
Leu Gly Ala Glu Lys Phe Phe Asp Ile Lys Cys Arg Phe Ala Gly Leu
305 310 315 320
Lys Pro Asp Cys Ala Val Ile Val Ala Thr Val Arg Ala Leu Lys Asn
325 330 335
His Gly Gly Val Pro Lys Ala Glu Leu Asn Asn Glu Asn Leu Glu Ala
340 345 350
Leu Glu Lys Gly Tyr Arg Asn Leu Glu Lys His Ile Glu Asn Val Gln
355 360 365
Lys Phe Gly Val Pro Ala Val Val Ala Ile Asn Lys Phe Pro Thr Asp
370 375 380
Thr Glu Ala Glu Leu Asn Phe Leu Arg Lys His Cys Ala Glu Met Gly
385 390 395 400
Ala Glu Val Val Leu Ser Asp Val Trp Ala Asn Gly Gly Asp Gly Gly
405 410 415
Ile Glu Met Ala Lys Lys Val Val Glu Val Val Glu Ser Lys Glu Ser
420 425 430
Asn Phe Lys Pro Leu Tyr Asp Val Asn Ala Ser Ile Val Glu Lys Ile
435 440 445
Asn Thr Ile Ala Lys Glu Val Tyr Gly Ala Asp Gly Val Asp Phe Thr
450 455 460
Lys Ser Ala Gln Thr Gln Ile Lys Lys Tyr Glu Asp Leu Gly Leu Asp
465 470 475 480
Lys Met Pro Ile Cys Met Ala Lys Thr Gln Tyr Ser Leu Ser Asp Asp
485 490 495
Pro Ser Leu Ile Gly Arg Pro Ser Gly Phe Arg Ile Thr Val Lys Glu
500 505 510
Ile Arg Leu Ser Ala Gly Ala Gly Phe Leu Val Ala Leu Thr Gly Asp
515 520 525
Ile Met Val Met Pro Gly Leu Pro Lys Val Pro Ala Ala Asn His Met
530 535 540
Asp Ile Leu Glu Ser Gly Glu Ile Ile Gly Leu Phe
545 550 555
<210> SEQ ID NO 20
<211> LENGTH: 556
<212> TYPE: PRT
<213> ORGANISM: Clostridium acidurici
<400> SEQUENCE: 20
Met Lys Thr Asp Ile Gln Ile Ala Gln Glu Ala Gln Met Lys His Ile
1 5 10 15
Lys Asp Val Ala Glu Leu Ile Asp Ile His Glu Asp Asp Leu Glu Leu
20 25 30
Tyr Gly Lys Tyr Lys Ala Lys Val Ser Leu Asp Val Leu Asp Gln Leu
35 40 45
Lys Asp Lys Pro Asp Gly Lys Leu Val Leu Val Thr Ala Ile Asn Pro
50 55 60
Thr Pro Ala Gly Glu Gly Lys Thr Thr Thr Asn Ile Gly Leu Ser Met
65 70 75 80
Gly Leu Asn Lys Leu Gly Lys Lys Thr Ser Thr Ala Leu Arg Glu Pro
85 90 95
Ser Leu Gly Pro Ser Phe Gly Val Lys Gly Gly Ala Ala Gly Gly Gly
100 105 110
Tyr Ala Gln Val Val Pro Met Ala Asp Ile Asn Leu His Phe Thr Gly
115 120 125
Asp Phe His Ala Ile Thr Ser Ala His Ser Leu Leu Ala Ala Leu Val
130 135 140
Asp Asn His Leu His His Gly Asn Ala Leu Arg Ile Asp Thr Asn Arg
145 150 155 160
Ile Val Trp Lys Arg Val Val Asp Met Asn Asp Arg Ala Leu Arg Lys
165 170 175
Ile Val Val Gly Leu Gly Gly Lys Ala Gln Gly Ile Thr Arg Glu Asp
180 185 190
Gly Phe Asp Ile Thr Val Ala Ser Glu Ile Met Ala Ile Leu Cys Leu
195 200 205
Ala Asn Asp Arg Glu Asp Leu Lys Glu Arg Leu Gly Asn Met Val Val
210 215 220
Ala Tyr Asn Val Asp Gly Asp Ala Val Arg Ala Lys Asp Leu Glu Ala
225 230 235 240
Gln Gly Ala Leu Thr Leu Ile Leu Lys Asp Ala Ile Asn Pro Asn Ile
245 250 255
Val Gln Thr Leu Glu Asn Thr Pro Ala Phe Ile His Gly Gly Pro Phe
260 265 270
Ala Asn Ile Ala His Gly Cys Asn Ser Val Leu Ala Thr Lys Leu Ala
275 280 285
Leu Lys Thr Gly Asp Tyr Ala Val Thr Glu Ala Gly Phe Gly Ala Asp
290 295 300
Leu Gly Ala Glu Lys Phe Phe Asp Ile Lys Cys Arg Tyr Ala Gly Leu
305 310 315 320
Asn Pro Asp Val Ala Val Ile Val Ala Thr Val Arg Ala Leu Lys Met
325 330 335
His Gly Gly Val Ala Lys Glu Asp Leu Gly Thr Glu Asn Leu Asp Ala
340 345 350
Leu Ala Lys Gly Met Thr Asn Leu Glu Arg His Ile Glu Asn Val Ala
355 360 365
Lys Phe Gly Val Pro Ser Val Val Ala Ile Asn Ala Phe Pro Thr Asp
370 375 380
Thr Glu Ala Glu Lys Gln Leu Val Phe Asp Lys Cys Lys Glu Met Gly
385 390 395 400
Val Asp Val Ala Ile Ser Asp Val Phe Ala Lys Gly Gly Asp Gly Gly
405 410 415
Val Glu Leu Ala Gln Lys Val Ile Asp Val Cys Glu Asn Lys Lys Ser
420 425 430
Asp Phe Lys Val Leu Tyr Asp Val Glu Glu Ser Ile Pro Glu Lys Ile
435 440 445
Thr Lys Ile Ala Lys Glu Ile Tyr Arg Ala Asp Lys Val Asn Phe Ser
450 455 460
Lys Ala Ala Lys Lys Gln Ile Ala Glu Leu Glu Lys Leu Gly Leu Asp
465 470 475 480
Lys Leu Pro Ile Cys Met Ala Lys Thr Gln Tyr Ser Phe Ser Asp Asp
485 490 495
Pro Ala Leu Leu Gly Ala Pro Glu Gly Phe Glu Leu Thr Ile Arg Asp
500 505 510
Leu Glu Leu Ala Ala Gly Ala Gly Phe Ile Val Ala Leu Thr Gly Asp
515 520 525
Ile Met Arg Met Pro Gly Leu Pro Lys Val Pro Ala Ala Asn Arg Met
530 535 540
Asp Val Leu Pro Asn Gly Glu Ile Ile Gly Leu Phe
545 550 555
<210> SEQ ID NO 21
<211> LENGTH: 556
<212> TYPE: PRT
<213> ORGANISM: Eubacterium acidaminophilum
<400> SEQUENCE: 21
Met Lys Thr Asp Val Gln Ile Ala Gln Glu Ala Lys Met Leu Pro Ile
1 5 10 15
Met Glu Val Ala Lys Gln Ile Gly Leu Gly Glu Asp Asp Ile Glu Leu
20 25 30
Tyr Gly Lys Tyr Lys Ala Lys Ile Ser Leu Asp Val Tyr Lys Arg Leu
35 40 45
Ala Asp Lys Pro Asp Gly Lys Leu Val Leu Val Thr Ala Ile Asn Pro
50 55 60
Thr Pro Ala Gly Glu Gly Lys Thr Thr Thr Asn Val Gly Leu Ser Met
65 70 75 80
Gly Leu Asn Lys Ile Gly Lys Lys Thr Ile Thr Ala Leu Asn Glu Pro
85 90 95
Ser Leu Gly Pro Cys Phe Gly Val Lys Gly Gly Ala Ala Gly Gly Gly
100 105 110
Tyr Ala Gln Val Val Pro Met Asp Asp Ile Asn Leu His Phe Thr Gly
115 120 125
Asp Ile His Ala Ile Thr Thr Ala His Asn Leu Leu Ala Ala Leu Met
130 135 140
Asp Asn His Ile Lys Gln Gly Asn Ala Leu Gly Ile Asp Ile Asn Lys
145 150 155 160
Ile Thr Trp Lys Arg Val Leu Asp Met Asn Asp Arg Ala Leu Arg Asp
165 170 175
Ile Val Ile Gly Leu Gly Gly Thr Ala Asn Gly Ile Pro Arg Gln Asp
180 185 190
Gly Phe Asp Ile Thr Val Ala Ser Glu Ile Met Ala Ile Met Cys Leu
195 200 205
Ala Thr Ser Leu Ser Asp Leu Lys Asp Arg Leu Ser Arg Met Ile Val
210 215 220
Gly Tyr Thr Ser Arg Arg Leu Ala Val Thr Ala Asp Ser Leu Thr Leu
225 230 235 240
Arg Gly Ala Leu Ala Leu Leu Leu Lys Asp Ala Leu Lys Pro Asn Leu
245 250 255
Val Gln Thr Leu Glu Asn Thr Pro Ala Ile Ile His Gly Gly Pro Phe
260 265 270
Ala Asn Ile Ala His Gly Cys Asn Ser Val Thr Thr Thr Lys Thr Ala
275 280 285
Leu Lys Ile Ala Asp Tyr Val Val Thr Glu Ala Gly Phe Gly Ala Asp
290 295 300
Leu Gly Ala Glu Lys Phe Phe Asp Ile Lys Cys Arg Phe Ala Asp Leu
305 310 315 320
Lys Pro Asp Val Ala Val Ile Val Ala Thr Val Arg Ala Leu Lys Asn
325 330 335
His Gly Gly Val Ala Lys Ala Asn Leu Gly Ala Glu Asn Met Lys Ala
340 345 350
Leu Glu Asp Gly Phe Gly Asn Leu Glu Arg His Ile Glu Asn Val His
355 360 365
Lys Phe Gly Val Pro Ala Val Val Ala Ile Asn Ala Phe Pro Thr Asp
370 375 380
Thr Glu Lys Glu Leu Lys Phe Val Glu Asp Ala Cys Arg Lys Leu Gly
385 390 395 400
Ala Asp Val Val Leu Ser Glu Val Trp Ala Lys Gly Gly Glu Gly Gly
405 410 415
Val Glu Leu Ala Lys Lys Val Val Glu Val Thr Glu Lys Gly Ala Ala
420 425 430
Lys Phe Lys Pro Leu Tyr Pro Ala Glu Met Pro Leu Lys Gln Lys Ile
435 440 445
Glu Thr Ile Ala Lys Glu Ile Tyr Arg Ala Asp Gly Val Glu Phe Ser
450 455 460
Ala Lys Ala Ser Lys Glu Leu Asp Lys Phe Glu Lys Leu Gly Phe Gly
465 470 475 480
Asn Leu Pro Ile Cys Val Ala Lys Thr Gln Tyr Ser Phe Ser Asp Asn
485 490 495
Pro Asn Leu Lys Gly Ala Pro Lys Gly Phe Thr Val Ser Val Ser Asn
500 505 510
Ala Arg Ile Ser Ala Gly Ala Gly Phe Ile Val Val Leu Thr Gly Asp
515 520 525
Ile Met Thr Met Pro Gly Leu Pro Lys Val Pro Ala Ala Asn His Met
530 535 540
Asp Val Leu Glu Ser Gly Glu Ile Val Gly Leu Phe
545 550 555
<210> SEQ ID NO 22
<211> LENGTH: 556
<212> TYPE: PRT
<213> ORGANISM: Desulfitobacterium hafniense (strain Y51)
<400> SEQUENCE: 22
Met Lys Thr Asp Ile Glu Ile Ala Gln Glu Ala Thr Met Lys Pro Ile
1 5 10 15
Thr Glu Ile Ala Gln Gly Leu Asp Leu Leu Glu Asp Glu Ile Glu Leu
20 25 30
Tyr Gly Lys Tyr Lys Ala Lys Val Asn Phe Ser Ala Trp Glu Arg Leu
35 40 45
Lys Asp Lys Pro Asp Ala Lys Leu Ile Leu Val Thr Ala Ile Asn Pro
50 55 60
Thr Pro Ala Gly Glu Gly Lys Thr Thr Thr Thr Val Gly Leu Gly Gln
65 70 75 80
Ala Met Ser Lys Ile Gly Lys Asn Ala Met Ile Ala Leu Arg Glu Pro
85 90 95
Ser Leu Gly Pro Cys Phe Gly Val Lys Gly Gly Ala Ala Gly Gly Gly
100 105 110
Tyr Ala Gln Val Val Pro Met Glu Asp Ile Asn Leu His Phe Thr Gly
115 120 125
Asp Phe His Ala Ile Thr Ser Thr His Asn Leu Leu Ala Ala Leu Leu
130 135 140
Asp Asn His Ile Gln Gln Gly Asn Leu Leu Asn Ile Asp Pro Arg Gln
145 150 155 160
Ile Val Phe Arg Arg Val Met Asp Met Asn Asp Arg Ala Leu Arg Lys
165 170 175
Ile Val Ile Gly Leu Gly Gly Arg Thr Glu Gly Ile Pro Arg Glu Asn
180 185 190
Gly Phe Asp Ile Thr Val Ala Ser Glu Ile Met Ala Ile Leu Cys Leu
195 200 205
Ala Lys Asp Leu Met Asp Leu Lys Glu Arg Phe Gly Arg Ile Val Val
210 215 220
Ala Tyr Thr Tyr Asp Gly Lys Ala Ile Thr Ala His Asp Leu Glu Ala
225 230 235 240
Glu Gly Ala Met Ala Leu Leu Met Lys Asp Ala Ile Lys Pro Asn Leu
245 250 255
Val Gln Thr Leu Glu Asn Thr Pro Val Phe Ile His Gly Gly Pro Phe
260 265 270
Ala Asn Ile Ala His Gly Cys Asn Ser Val Val Ala Thr Arg Met Ala
275 280 285
Met Lys Leu Ala Asp Tyr Val Ile Thr Glu Ala Gly Phe Gly Ala Asp
290 295 300
Leu Gly Ala Glu Lys Phe Tyr Asp Leu Lys Cys Arg Phe Ala Glu Leu
305 310 315 320
Lys Pro Ala Ala Thr Val Ile Val Ala Thr Val Arg Ala Leu Lys Met
325 330 335
Asn Gly Gly Val Ala Lys Glu Asp Leu Gly Pro Glu Asn Leu Glu Ala
340 345 350
Leu Ala Lys Gly Ile Val Asn Leu Glu Lys His Ile Glu Asn Ile Gly
355 360 365
Lys Phe Gly Val Pro Ala Val Val Ala Ile Asn Arg Phe Pro Thr Asp
370 375 380
Thr Asp Ala Glu Leu Glu Phe Val Ala Glu Arg Cys Arg Gln Leu Gly
385 390 395 400
Ala Glu Phe Ala Leu Ser Glu Val Phe Thr Lys Gly Gly Glu Gly Gly
405 410 415
Ile Glu Leu Ala Lys Ala Val Leu Asn Ile Val Asp Asn Lys Glu Ser
420 425 430
Asn Phe His Val Leu Tyr Glu Leu Asp Leu Pro Ile Ala Lys Lys Ile
435 440 445
Glu Thr Ile Cys Lys Glu Val Tyr Gly Ala Asp Gly Val Asn Phe Thr
450 455 460
Lys Glu Ala Leu Thr Ser Met Lys Lys Tyr Glu Glu Leu Gly Tyr Gly
465 470 475 480
Gln Leu Pro Ile Cys Met Ala Lys Thr Gln Tyr Ser Leu Thr Asp Asp
485 490 495
Gln Asn Val Leu Gly Arg Pro Ser Gly Phe Thr Ile Thr Val Arg Glu
500 505 510
Leu Arg Leu Ser Ala Gly Ala Gly Phe Leu Val Ala Ile Thr Gly Ala
515 520 525
Ile Met Thr Met Pro Gly Leu Pro Lys Arg Pro Ala Ala Leu Arg Met
530 535 540
Asp Ile Asp Ala Ala Gly Arg Ile Thr Gly Leu Phe
545 550 555
<210> SEQ ID NO 23
<211> LENGTH: 369
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli (strain K12)
<400> SEQUENCE: 23
Met Lys Ser Arg Ala Ala Val Ala Phe Ala Pro Gly Lys Pro Leu Glu
1 5 10 15
Ile Val Glu Ile Asp Val Ala Pro Pro Lys Lys Gly Glu Val Leu Ile
20 25 30
Lys Val Thr His Thr Gly Val Cys His Thr Asp Ala Phe Thr Leu Ser
35 40 45
Gly Asp Asp Pro Glu Gly Val Phe Pro Val Val Leu Gly His Glu Gly
50 55 60
Ala Gly Val Val Val Glu Val Gly Glu Gly Val Thr Ser Val Lys Pro
65 70 75 80
Gly Asp His Val Ile Pro Leu Tyr Thr Ala Glu Cys Gly Glu Cys Glu
85 90 95
Phe Cys Arg Ser Gly Lys Thr Asn Leu Cys Val Ala Val Arg Glu Thr
100 105 110
Gln Gly Lys Gly Leu Met Pro Asp Gly Thr Thr Arg Phe Ser Tyr Asn
115 120 125
Gly Gln Pro Leu Tyr His Tyr Met Gly Cys Ser Thr Phe Ser Glu Tyr
130 135 140
Thr Val Val Ala Glu Val Ser Leu Ala Lys Ile Asn Pro Glu Ala Asn
145 150 155 160
His Glu His Val Cys Leu Leu Gly Cys Gly Val Thr Thr Gly Ile Gly
165 170 175
Ala Val His Asn Thr Ala Lys Val Gln Pro Gly Asp Ser Val Ala Val
180 185 190
Phe Gly Leu Gly Ala Ile Gly Leu Ala Val Val Gln Gly Ala Arg Gln
195 200 205
Ala Lys Ala Gly Arg Ile Ile Ala Ile Asp Thr Asn Pro Lys Lys Phe
210 215 220
Asp Leu Ala Arg Arg Phe Gly Ala Thr Asp Cys Ile Asn Pro Asn Asp
225 230 235 240
Tyr Asp Lys Pro Ile Lys Asp Val Leu Leu Asp Ile Asn Lys Trp Gly
245 250 255
Ile Asp His Thr Phe Glu Cys Ile Gly Asn Val Asn Val Met Arg Ala
260 265 270
Ala Leu Glu Ser Ala His Arg Gly Trp Gly Gln Ser Val Ile Ile Gly
275 280 285
Val Ala Val Ala Gly Gln Glu Ile Ser Thr Arg Pro Phe Gln Leu Val
290 295 300
Thr Gly Arg Val Trp Lys Gly Ser Ala Phe Gly Gly Val Lys Gly Arg
305 310 315 320
Ser Gln Leu Pro Gly Met Val Glu Asp Ala Met Lys Gly Asp Ile Asp
325 330 335
Leu Glu Pro Phe Val Thr His Thr Met Ser Leu Asp Glu Ile Asn Asp
340 345 350
Ala Phe Asp Leu Met His Glu Gly Lys Ser Ile Arg Thr Val Ile Arg
355 360 365
Tyr
<210> SEQ ID NO 24
<211> LENGTH: 369
<212> TYPE: PRT
<213> ORGANISM: Shigella sonnei (strain Ss046)
<400> SEQUENCE: 24
Met Lys Ser Arg Ala Ala Val Ala Phe Ala Pro Gly Lys Pro Leu Glu
1 5 10 15
Ile Val Glu Ile Asp Val Ala Pro Pro Lys Lys Gly Glu Val Leu Ile
20 25 30
Lys Val Thr His Thr Gly Val Cys His Thr Asp Ala Phe Thr Leu Ser
35 40 45
Gly Asp Asp Pro Glu Gly Val Phe Pro Val Val Leu Gly His Glu Gly
50 55 60
Ala Gly Val Val Val Glu Val Gly Glu Gly Val Thr Ser Val Lys Pro
65 70 75 80
Gly Asp His Val Ile Pro Leu Tyr Thr Ala Glu Cys Gly Glu Cys Glu
85 90 95
Phe Cys Arg Ser Gly Lys Thr Asn Leu Cys Val Ala Val Arg Glu Thr
100 105 110
Gln Gly Lys Gly Leu Met Pro Asp Gly Thr Thr Arg Phe Ser Tyr Asn
115 120 125
Gly Gln Pro Leu Tyr His Tyr Met Gly Cys Ser Thr Phe Ser Glu Tyr
130 135 140
Thr Val Val Ala Glu Val Ser Leu Ala Lys Ile Asn Pro Glu Ala Asn
145 150 155 160
His Glu His Val Cys Leu Leu Gly Cys Gly Val Thr Thr Gly Ile Gly
165 170 175
Ala Val His Asn Thr Ala Lys Val Gln Pro Gly Asp Ser Val Ala Val
180 185 190
Phe Gly Leu Gly Ala Ile Gly Leu Ala Val Val Gln Gly Ala Arg Gln
195 200 205
Ala Lys Ala Gly Arg Ile Ile Ala Ile Asp Thr Asn Pro Lys Lys Phe
210 215 220
Asp Leu Ala Arg Arg Phe Gly Ala Thr Asp Cys Ile Asn Pro Asn Asp
225 230 235 240
Tyr Asp Lys Pro Ile Lys Asp Val Leu Leu Asp Ile Asn Lys Trp Gly
245 250 255
Ile Asp His Thr Phe Glu Cys Ile Gly Asn Val Asn Val Met Arg Ala
260 265 270
Ala Leu Glu Ser Ala His Arg Gly Trp Gly Gln Ser Val Ile Ile Gly
275 280 285
Val Ala Gly Ser Gly Gln Glu Ile Ser Thr Arg Pro Phe Gln Leu Val
290 295 300
Thr Gly Arg Val Trp Lys Gly Ser Ala Phe Gly Gly Val Lys Gly Arg
305 310 315 320
Ser Gln Leu Pro Gly Met Val Glu Asp Ala Met Lys Gly Asp Ile Asp
325 330 335
Leu Glu Pro Phe Val Thr His Thr Met Ser Leu Asp Glu Ile Asn Asp
340 345 350
Ala Phe Asp Leu Met His Glu Gly Lys Ser Ile Arg Thr Val Ile Arg
355 360 365
Tyr
<210> SEQ ID NO 25
<211> LENGTH: 369
<212> TYPE: PRT
<213> ORGANISM: Achromobacter sp.
<400> SEQUENCE: 25
Met Lys Ser Arg Ala Ala Val Ala Phe Ala Pro Gly Lys Pro Leu Glu
1 5 10 15
Ile Val Glu Ile Asp Val Ala Pro Pro Lys Lys Gly Glu Val Leu Ile
20 25 30
Lys Val Thr His Thr Gly Val Cys His Thr Asp Ala Phe Thr Leu Ser
35 40 45
Gly Asp Asp Pro Glu Gly Val Phe Pro Val Val Leu Gly His Glu Gly
50 55 60
Ala Gly Val Val Val Glu Val Gly Glu Gly Val Thr Ser Val Lys Pro
65 70 75 80
Gly Asp His Val Ile Pro Leu Tyr Thr Ala Glu Cys Gly Glu Cys Glu
85 90 95
Phe Cys Arg Ser Gly Lys Thr Asn Leu Cys Val Ala Val Arg Glu Thr
100 105 110
Gln Gly Lys Gly Leu Met Pro Asp Gly Thr Thr Arg Phe Ser Tyr Asn
115 120 125
Gly Gln Pro Leu Tyr His Tyr Met Gly Cys Ser Thr Phe Ser Glu Tyr
130 135 140
Thr Val Val Ala Glu Val Ser Leu Ala Lys Ile Asn Pro Glu Ala Asn
145 150 155 160
His Glu His Val Cys Leu Leu Gly Cys Gly Val Thr Thr Gly Ile Gly
165 170 175
Ala Val His Asn Thr Ala Lys Val Gln Pro Gly Asp Ser Val Ala Val
180 185 190
Phe Gly Leu Gly Ala Ile Gly Leu Ala Val Val Gln Gly Ala Arg Gln
195 200 205
Ala Lys Ala Gly Arg Ile Ile Ala Ile Asp Thr Asn Pro Lys Lys Phe
210 215 220
Asp Leu Ala Arg Arg Phe Gly Ala Thr Asp Cys Ile Asn Pro Asn Asp
225 230 235 240
Tyr Asp Lys Pro Ile Lys Asp Val Leu Leu Asp Ile Asn Lys Trp Gly
245 250 255
Ile Asp His Thr Phe Glu Cys Ile Gly Asn Val Asn Val Met Arg Ala
260 265 270
Ala Leu Glu Ser Ala His Arg Gly Trp Gly Gln Ser Val Ile Ile Gly
275 280 285
Val Ala Gly Ala Gly Gln Glu Ile Ser Thr Arg Pro Phe Gln Leu Val
290 295 300
Thr Gly Arg Val Trp Lys Gly Ser Ala Phe Gly Gly Val Lys Gly Arg
305 310 315 320
Ser Gln Leu Pro Gly Met Val Glu Asp Ala Met Lys Gly Asp Ile Asp
325 330 335
Leu Glu Pro Phe Val Thr His Thr Met Ser Leu Asp Glu Ile Asn Asp
340 345 350
Ala Phe Asp Leu Met His Glu Gly Lys Ser Ile Arg Thr Val Ile Arg
355 360 365
Tyr
<210> SEQ ID NO 26
<211> LENGTH: 369
<212> TYPE: PRT
<213> ORGANISM: Klebsiella pneumoniae IS22
<400> SEQUENCE: 26
Met Lys Ser Arg Ala Ala Val Ala Phe Ala Pro Gly Lys Pro Leu Glu
1 5 10 15
Ile Val Glu Ile Asp Val Ala Pro Pro Lys Lys Gly Glu Val Leu Ile
20 25 30
Lys Val Thr His Thr Gly Val Cys His Thr Asp Ala Phe Thr Leu Ser
35 40 45
Gly Asp Asp Pro Glu Gly Val Phe Pro Val Val Leu Gly His Glu Gly
50 55 60
Ala Gly Val Val Val Glu Val Gly Glu Gly Val Thr Ser Val Lys Pro
65 70 75 80
Gly Asp His Val Ile Pro Leu Tyr Thr Ala Glu Cys Gly Glu Cys Glu
85 90 95
Phe Cys Arg Ser Gly Lys Thr Asn Leu Cys Val Ala Val Arg Glu Thr
100 105 110
Gln Gly Lys Gly Leu Met Pro Asp Gly Thr Thr Arg Phe Ser Tyr Asn
115 120 125
Gly Gln Pro Leu Tyr His Tyr Met Gly Cys Ser Thr Phe Ser Glu Tyr
130 135 140
Thr Val Val Ala Glu Val Ser Leu Ala Lys Ile Asn Pro Glu Ala Asn
145 150 155 160
His Glu His Val Cys Leu Leu Gly Cys Gly Val Thr Thr Gly Ile Gly
165 170 175
Ala Val His Asn Thr Ala Lys Val Gln Pro Gly Asp Ser Val Ala Val
180 185 190
Phe Gly Leu Gly Ala Ile Gly Leu Ala Val Val Gln Gly Ala Arg Gln
195 200 205
Ala Lys Ala Gly Arg Ile Ile Ala Ile Asp Thr Asn Pro Lys Lys Phe
210 215 220
Asp Leu Ala Arg Arg Phe Gly Ala Thr Asp Cys Ile Asn Pro Asn Asp
225 230 235 240
Tyr Asp Lys Pro Ile Lys Asp Val Leu Leu Asp Ile Asn Lys Trp Gly
245 250 255
Ile Asp His Thr Phe Glu Cys Ile Gly Asn Val Asn Val Met Arg Ala
260 265 270
Ala Leu Glu Ser Ala His Arg Gly Trp Gly Gln Ser Val Ile Ile Gly
275 280 285
Val Ala Gly Ala Gly Gln Glu Ile Ser Thr Arg Pro Phe Gln Leu Val
290 295 300
Thr Gly Arg Val Trp Lys Gly Ser Ala Phe Gly Gly Val Lys Gly Arg
305 310 315 320
Ser Gln Leu Pro Gly Met Val Glu Asp Ala Met Lys Gly Asp Ile Asp
325 330 335
Leu Glu Pro Phe Val Thr His Thr Met Ser Leu Asp Glu Ile Asn Asp
340 345 350
Ala Phe Asp Leu Met His Glu Gly Lys Ser Ile Arg Thr Val Ile Arg
355 360 365
Tyr
<210> SEQ ID NO 27
<211> LENGTH: 277
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli (strain K12)
<400> SEQUENCE: 27
Met Glu Leu Ile Glu Lys His Val Ser Phe Gly Gly Trp Gln Asn Met
1 5 10 15
Tyr Arg His Tyr Ser Gln Ser Leu Lys Cys Glu Met Asn Val Gly Val
20 25 30
Tyr Leu Pro Pro Lys Ala Ala Asn Glu Lys Leu Pro Val Leu Tyr Trp
35 40 45
Leu Ser Gly Leu Thr Cys Asn Glu Gln Asn Phe Ile Thr Lys Ser Gly
50 55 60
Met Gln Arg Tyr Ala Ala Glu His Asn Ile Ile Val Val Ala Pro Asp
65 70 75 80
Thr Ser Pro Arg Gly Ser His Val Ala Asp Ala Asp Arg Tyr Asp Leu
85 90 95
Gly Gln Gly Ala Gly Phe Tyr Leu Asn Ala Thr Gln Ala Pro Trp Asn
100 105 110
Glu His Tyr Lys Met Tyr Asp Tyr Ile Arg Asn Glu Leu Pro Asp Leu
115 120 125
Val Met His His Phe Pro Ala Thr Ala Lys Lys Ser Ile Ser Gly His
130 135 140
Ser Met Gly Gly Leu Gly Ala Leu Val Leu Ala Leu Arg Asn Pro Asp
145 150 155 160
Glu Tyr Val Ser Val Ser Ala Phe Ser Pro Ile Val Ser Pro Ser Gln
165 170 175
Val Pro Trp Gly Gln Gln Ala Phe Ala Ala Tyr Leu Ala Glu Asn Lys
180 185 190
Asp Ala Trp Leu Asp Tyr Asp Pro Val Ser Leu Ile Ser Gln Gly Gln
195 200 205
Arg Val Ala Glu Ile Met Val Asp Gln Gly Leu Ser Asp Asp Phe Tyr
210 215 220
Ala Glu Gln Leu Arg Thr Pro Asn Leu Glu Lys Ile Cys Gln Glu Met
225 230 235 240
Asn Ile Lys Thr Leu Ile Arg Tyr His Glu Gly Tyr Asp His Ser Tyr
245 250 255
Tyr Phe Val Ser Ser Phe Ile Gly Glu His Ile Ala Tyr His Ala Asn
260 265 270
Lys Leu Asn Met Arg
275
<210> SEQ ID NO 28
<211> LENGTH: 277
<212> TYPE: PRT
<213> ORGANISM: Shigella sonnei (strain Ss046)
<400> SEQUENCE: 28
Met Glu Leu Ile Glu Lys His Ala Ser Phe Gly Gly Trp Gln Asn Val
1 5 10 15
Tyr Arg His Tyr Ser Gln Ser Leu Lys Cys Glu Met Asn Val Gly Val
20 25 30
Tyr Leu Pro Pro Lys Ala Ala Asn Glu Lys Leu Pro Val Leu Tyr Trp
35 40 45
Leu Ser Gly Leu Thr Cys Asn Glu Gln Asn Phe Ile Thr Lys Ser Gly
50 55 60
Met Gln Arg Tyr Ala Ala Glu His Asn Ile Ile Val Val Ala Pro Asp
65 70 75 80
Thr Ser Pro Arg Gly Ser His Val Ala Asp Ala Asp Arg Tyr Asp Leu
85 90 95
Gly Gln Gly Ala Gly Phe Tyr Leu Asn Ala Thr Gln Ala Pro Trp Asn
100 105 110
Glu His Tyr Lys Met Tyr Asp Tyr Ile Arg Asn Glu Leu Pro Asp Leu
115 120 125
Val Met Gln His Phe Pro Ala Thr Thr Arg Lys Ser Ile Ser Gly His
130 135 140
Ser Met Gly Gly Leu Gly Ala Leu Val Leu Ala Leu Arg Asn Pro Asp
145 150 155 160
Glu Tyr Val Ser Val Ser Ala Phe Ser Pro Ile Val Ser Pro Ser Gln
165 170 175
Val Pro Trp Gly Gln Gln Ala Phe Ala Ala Tyr Leu Gly Glu Asn Lys
180 185 190
Asp Ala Trp Leu Asp Tyr Asp Pro Val Ser Leu Ile Ser Gln Gly Gln
195 200 205
Arg Val Ala Glu Ile Met Val Asp Gln Gly Leu Ser Asp Asp Phe Tyr
210 215 220
Ala Glu Gln Leu Arg Thr Pro Asn Leu Glu Lys Ile Cys Gln Glu Met
225 230 235 240
Asn Ile Lys Thr Leu Ile Arg Tyr His Glu Gly Tyr Asp His Ser Tyr
245 250 255
Tyr Phe Val Ser Ser Phe Ile Gly Glu His Ile Ala Tyr His Ala Asn
260 265 270
Lys Leu Asn Met Arg
275
<210> SEQ ID NO 29
<211> LENGTH: 277
<212> TYPE: PRT
<213> ORGANISM: Achromobacter sp.
<400> SEQUENCE: 29
Met Glu Leu Ile Glu Lys His Val Ser Phe Gly Gly Trp Gln Asn Val
1 5 10 15
Tyr Arg His Tyr Ser Gln Ser Leu Lys Cys Glu Met Asn Val Gly Val
20 25 30
Tyr Leu Pro Pro Lys Ala Ala Asn Glu Lys Leu Pro Val Leu Tyr Trp
35 40 45
Leu Ser Gly Leu Thr Cys Asn Glu Gln Asn Phe Ile Thr Lys Ser Gly
50 55 60
Met Gln Arg Tyr Ala Ala Glu His Asn Ile Ile Val Val Ala Pro Asp
65 70 75 80
Thr Ser Pro Arg Gly Ser His Val Ala Asp Ala Asp Arg Tyr Asp Leu
85 90 95
Gly Gln Gly Ala Gly Phe Tyr Leu Asn Ala Thr Gln Ala Pro Trp Asn
100 105 110
Glu His Tyr Lys Met Tyr Asp Tyr Ile Arg Asn Glu Leu Pro Asp Leu
115 120 125
Val Met His His Phe Pro Ala Thr Ala Lys Lys Ser Ile Ser Gly His
130 135 140
Ser Met Gly Gly Leu Gly Ala Leu Val Leu Ala Leu Arg Asn Pro Asp
145 150 155 160
Glu Tyr Val Ser Val Ser Ala Phe Ser Pro Ile Val Ser Pro Ser Gln
165 170 175
Val Pro Trp Gly Gln Gln Ala Phe Ala Ala Tyr Leu Ala Glu Asn Lys
180 185 190
Asp Ala Trp Leu Asp Tyr Asp Pro Val Ser Leu Ile Ser Gln Gly Gln
195 200 205
Arg Val Ala Glu Ile Met Val Asp Gln Gly Leu Ser Asp Asp Phe Tyr
210 215 220
Ala Glu Gln Leu Arg Thr Pro Asn Leu Glu Lys Ile Cys Gln Glu Met
225 230 235 240
Asn Ile Lys Thr Leu Ile Arg Tyr His Glu Gly Tyr Asp His Ser Tyr
245 250 255
Tyr Phe Val Ser Ser Phe Ile Gly Glu His Ile Ala Tyr His Ala Asn
260 265 270
Lys Leu Asn Met Arg
275
<210> SEQ ID NO 30
<211> LENGTH: 277
<212> TYPE: PRT
<213> ORGANISM: Klebsiella pneumoniae IS22
<400> SEQUENCE: 30
Met Glu Leu Ile Glu Lys His Ala Ser Phe Gly Gly Trp Gln Asn Val
1 5 10 15
Tyr Arg His Tyr Ser Gln Ser Leu Lys Cys Glu Met Asn Val Gly Val
20 25 30
Tyr Leu Pro Pro Lys Ala Ala Asn Glu Lys Leu Pro Val Leu Tyr Trp
35 40 45
Leu Ser Gly Leu Thr Cys Asn Glu Gln Asn Phe Ile Thr Lys Ser Gly
50 55 60
Met Gln Arg Tyr Ala Ala Glu His Asn Ile Ile Val Val Ala Pro Asp
65 70 75 80
Thr Ser Pro Arg Gly Ser His Val Ala Asp Ala Asp Arg Tyr Asp Leu
85 90 95
Gly Gln Gly Ala Gly Phe Tyr Leu Asn Ala Thr Gln Ala Pro Trp Asn
100 105 110
Glu His Tyr Lys Met Tyr Asp Tyr Ile Arg Asn Glu Leu Pro Asp Leu
115 120 125
Val Met His His Phe Pro Ala Thr Ala Lys Lys Ser Ile Ser Gly His
130 135 140
Ser Met Gly Gly Leu Gly Ala Leu Val Leu Ala Leu Arg Asn Pro Asp
145 150 155 160
Glu Tyr Val Ser Val Ser Ala Phe Ser Pro Ile Val Ser Pro Ser Gln
165 170 175
Val Pro Trp Gly Gln Gln Ala Phe Ala Ala Tyr Leu Ala Glu Asn Lys
180 185 190
Asp Ala Trp Leu Asp Tyr Asp Pro Val Ser Leu Ile Ser Gln Gly Gln
195 200 205
Arg Val Ala Glu Ile Met Val Asp Gln Gly Leu Ser Asp Asp Phe Tyr
210 215 220
Ala Glu Gln Leu Arg Thr Pro Asn Leu Glu Lys Ile Cys Gln Glu Met
225 230 235 240
Asn Ile Lys Thr Leu Ile Arg Tyr His Glu Gly Tyr Asp His Ser Tyr
245 250 255
Tyr Phe Val Ser Ser Phe Ile Gly Glu His Ile Ala Tyr His Ala Asn
260 265 270
Lys Leu Asn Met Arg
275
<210> SEQ ID NO 31
<211> LENGTH: 387
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli (strain K12)
<400> SEQUENCE: 31
Met Asn Asn Phe Asn Leu His Thr Pro Thr Arg Ile Leu Phe Gly Lys
1 5 10 15
Gly Ala Ile Ala Gly Leu Arg Glu Gln Ile Pro His Asp Ala Arg Val
20 25 30
Leu Ile Thr Tyr Gly Gly Gly Ser Val Lys Lys Thr Gly Val Leu Asp
35 40 45
Gln Val Leu Asp Ala Leu Lys Gly Met Asp Val Leu Glu Phe Gly Gly
50 55 60
Ile Glu Pro Asn Pro Ala Tyr Glu Thr Leu Met Asn Ala Val Lys Leu
65 70 75 80
Val Arg Glu Gln Lys Val Thr Phe Leu Leu Ala Val Gly Gly Gly Ser
85 90 95
Val Leu Asp Gly Thr Lys Phe Ile Ala Ala Ala Ala Asn Tyr Pro Glu
100 105 110
Asn Ile Asp Pro Trp His Ile Leu Gln Thr Gly Gly Lys Glu Ile Lys
115 120 125
Ser Ala Ile Pro Met Gly Cys Val Leu Thr Leu Pro Ala Thr Gly Ser
130 135 140
Glu Ser Asn Ala Gly Ala Val Ile Ser Arg Lys Thr Thr Gly Asp Lys
145 150 155 160
Gln Ala Phe His Ser Ala His Val Gln Pro Val Phe Ala Val Leu Asp
165 170 175
Pro Val Tyr Thr Tyr Thr Leu Pro Pro Arg Gln Val Ala Asn Gly Val
180 185 190
Val Asp Ala Phe Val His Thr Val Glu Gln Tyr Val Thr Lys Pro Val
195 200 205
Asp Ala Lys Ile Gln Asp Arg Phe Ala Glu Gly Ile Leu Leu Thr Leu
210 215 220
Ile Glu Asp Gly Pro Lys Ala Leu Lys Glu Pro Glu Asn Tyr Asp Val
225 230 235 240
Arg Ala Asn Val Met Trp Ala Ala Thr Gln Ala Leu Asn Gly Leu Ile
245 250 255
Gly Ala Gly Val Pro Gln Asp Trp Ala Thr His Met Leu Gly His Glu
260 265 270
Leu Thr Ala Met His Gly Leu Asp His Ala Gln Thr Leu Ala Ile Val
275 280 285
Leu Pro Ala Leu Trp Asn Glu Lys Arg Asp Thr Lys Arg Ala Lys Leu
290 295 300
Leu Gln Tyr Ala Glu Arg Val Trp Asn Ile Thr Glu Gly Ser Asp Asp
305 310 315 320
Glu Arg Ile Asp Ala Ala Ile Ala Ala Thr Arg Asn Phe Phe Glu Gln
325 330 335
Leu Gly Val Pro Thr His Leu Ser Asp Tyr Gly Leu Asp Gly Ser Ser
340 345 350
Ile Pro Ala Leu Leu Lys Lys Leu Glu Glu His Gly Met Thr Gln Leu
355 360 365
Gly Glu Asn His Asp Ile Thr Leu Asp Val Ser Arg Arg Ile Tyr Glu
370 375 380
Ala Ala Arg
385
<210> SEQ ID NO 32
<211> LENGTH: 288
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli (strain K12)
<400> SEQUENCE: 32
Met Ala Ala Lys Ile Ile Asp Gly Lys Thr Ile Ala Gln Gln Val Arg
1 5 10 15
Ser Glu Val Ala Gln Lys Val Gln Ala Arg Ile Ala Ala Gly Leu Arg
20 25 30
Ala Pro Gly Leu Ala Val Val Leu Val Gly Ser Asn Pro Ala Ser Gln
35 40 45
Ile Tyr Val Ala Ser Lys Arg Lys Ala Cys Glu Glu Val Gly Phe Val
50 55 60
Ser Arg Ser Tyr Asp Leu Pro Glu Thr Thr Ser Glu Ala Glu Leu Leu
65 70 75 80
Glu Leu Ile Asp Thr Leu Asn Ala Asp Asn Thr Ile Asp Gly Ile Leu
85 90 95
Val Gln Leu Pro Leu Pro Ala Gly Ile Asp Asn Val Lys Val Leu Glu
100 105 110
Arg Ile His Pro Asp Lys Asp Val Asp Gly Phe His Pro Tyr Asn Val
115 120 125
Gly Arg Leu Cys Gln Arg Ala Pro Arg Leu Arg Pro Cys Thr Pro Arg
130 135 140
Gly Ile Val Thr Leu Leu Glu Arg Tyr Asn Ile Asp Thr Phe Gly Leu
145 150 155 160
Asn Ala Val Val Ile Gly Ala Ser Asn Ile Val Gly Arg Pro Met Ser
165 170 175
Met Glu Leu Leu Leu Ala Gly Cys Thr Thr Thr Val Thr His Arg Phe
180 185 190
Thr Lys Asn Leu Arg His His Val Glu Asn Ala Asp Leu Leu Ile Val
195 200 205
Ala Val Gly Lys Pro Gly Phe Ile Pro Gly Asp Trp Ile Lys Glu Gly
210 215 220
Ala Ile Val Ile Asp Val Gly Ile Asn Arg Leu Glu Asn Gly Lys Val
225 230 235 240
Val Gly Asp Val Val Phe Glu Asp Ala Ala Lys Arg Ala Ser Tyr Ile
245 250 255
Thr Pro Val Pro Gly Gly Val Gly Pro Met Thr Val Ala Thr Leu Ile
260 265 270
Glu Asn Thr Leu Gln Ala Cys Val Glu Tyr His Asp Pro Gln Asp Glu
275 280 285
<210> SEQ ID NO 33
<211> LENGTH: 288
<212> TYPE: PRT
<213> ORGANISM: Escherichia fergusonii (strain ATCC
35469/DSM 13698/CDC 0568-73)
<400> SEQUENCE: 33
Met Ala Ala Lys Ile Ile Asp Gly Lys Thr Ile Ala Gln Gln Val Arg
1 5 10 15
Ser Glu Val Ala Gln Lys Val Gln Ala Arg Val Ala Ala Gly Leu Arg
20 25 30
Ala Pro Gly Leu Ala Val Val Leu Val Gly Ser Asn Pro Ala Ser Gln
35 40 45
Ile Tyr Val Ala Ser Lys Arg Lys Ala Cys Glu Glu Val Gly Phe Val
50 55 60
Ser Arg Ser Tyr Asp Leu Pro Glu Thr Thr Ser Glu Ala Glu Leu Leu
65 70 75 80
Glu Leu Ile Asp Val Leu Asn Ala Asp Asn Thr Ile Asp Gly Ile Leu
85 90 95
Val Gln Leu Pro Leu Pro Ala Gly Ile Asp Asn Val Lys Val Leu Glu
100 105 110
Arg Ile His Pro Asp Lys Asp Val Asp Gly Phe His Pro Tyr Asn Val
115 120 125
Gly Arg Leu Cys Gln Arg Ala Pro Arg Leu Arg Pro Cys Thr Pro Arg
130 135 140
Gly Ile Val Thr Leu Leu Glu Arg Tyr Asn Ile Asp Thr Phe Gly Leu
145 150 155 160
Asn Ala Val Val Ile Gly Ala Ser Asn Ile Val Gly Arg Pro Met Ser
165 170 175
Met Glu Leu Leu Leu Ala Gly Cys Thr Thr Thr Val Thr His Arg Phe
180 185 190
Thr Lys Asn Leu Arg His His Val Glu Asn Ala Asp Leu Leu Ile Val
195 200 205
Ala Val Gly Lys Pro Gly Phe Ile Pro Gly Asp Trp Ile Lys Glu Gly
210 215 220
Ala Ile Val Ile Asp Val Gly Ile Asn Arg Leu Glu Asn Gly Lys Val
225 230 235 240
Val Gly Asp Val Val Phe Glu Asp Ala Ala Lys Arg Ala Ser Tyr Ile
245 250 255
Thr Pro Val Pro Gly Gly Val Gly Pro Met Thr Val Ala Thr Leu Ile
260 265 270
Glu Asn Thr Leu Gln Ala Cys Val Glu Tyr His Asp Pro Gln Asp Glu
275 280 285
<210> SEQ ID NO 34
<211> LENGTH: 288
<212> TYPE: PRT
<213> ORGANISM: Shigella dysenteriae serotype 1 (strain Sd197)
<400> SEQUENCE: 34
Met Ala Ala Lys Ile Ile Asp Gly Lys Thr Ile Ala Gln Gln Val Arg
1 5 10 15
Ser Glu Val Ala Gln Lys Val Gln Ala Arg Ile Ala Ala Gly Leu Arg
20 25 30
Ala Pro Gly Leu Ala Val Val Leu Val Gly Ser Asn Pro Ala Ser Gln
35 40 45
Ile Tyr Val Ala Ser Lys Arg Lys Ala Cys Glu Glu Val Gly Phe Val
50 55 60
Ser Arg Ser Tyr Asp Leu Pro Glu Thr Thr Ser Glu Ala Glu Leu Leu
65 70 75 80
Glu Leu Ile Asp Ala Leu Asn Ala Asp Asn Thr Ile Asp Gly Ile Leu
85 90 95
Val Gln Leu Pro Leu Pro Ala Gly Ile Asp Asn Val Lys Val Leu Glu
100 105 110
Arg Ile His Pro Asp Lys Asp Val Asp Gly Phe His Pro Tyr Asn Val
115 120 125
Gly Arg Leu Cys Gln Arg Ala Pro Arg Leu Arg Pro Cys Thr Pro Arg
130 135 140
Gly Ile Val Thr Leu Leu Glu Arg Tyr Asn Ile Asp Thr Phe Gly Leu
145 150 155 160
Asn Ala Val Val Ile Gly Ala Ser Asn Ile Val Gly Arg Pro Met Ser
165 170 175
Met Glu Leu Leu Leu Ala Gly Cys Thr Thr Thr Val Thr His Arg Phe
180 185 190
Thr Lys Asn Leu Arg His His Val Glu Asn Ala Asp Leu Leu Ile Val
195 200 205
Ala Val Gly Lys Pro Gly Phe Ile Pro Gly Asp Trp Ile Lys Glu Gly
210 215 220
Ala Ile Val Ile Asp Val Gly Ile Asn Arg Leu Glu Asn Gly Lys Val
225 230 235 240
Val Gly Asp Val Val Phe Glu Asp Ala Ala Lys His Ala Ser Tyr Ile
245 250 255
Thr Pro Val Pro Gly Gly Val Gly Pro Met Thr Val Ala Thr Leu Ile
260 265 270
Glu Asn Thr Leu Gln Ala Cys Val Glu Tyr His Asp Pro Gln Asp Glu
275 280 285
<210> SEQ ID NO 35
<211> LENGTH: 760
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli (strain K12)
<400> SEQUENCE: 35
Met Ser Glu Leu Asn Glu Lys Leu Ala Thr Ala Trp Glu Gly Phe Thr
1 5 10 15
Lys Gly Asp Trp Gln Asn Glu Val Asn Val Arg Asp Phe Ile Gln Lys
20 25 30
Asn Tyr Thr Pro Tyr Glu Gly Asp Glu Ser Phe Leu Ala Gly Ala Thr
35 40 45
Glu Ala Thr Thr Thr Leu Trp Asp Lys Val Met Glu Gly Val Lys Leu
50 55 60
Glu Asn Arg Thr His Ala Pro Val Asp Phe Asp Thr Ala Val Ala Ser
65 70 75 80
Thr Ile Thr Ser His Asp Ala Gly Tyr Ile Asn Lys Gln Leu Glu Lys
85 90 95
Ile Val Gly Leu Gln Thr Glu Ala Pro Leu Lys Arg Ala Leu Ile Pro
100 105 110
Phe Gly Gly Ile Lys Met Ile Glu Gly Ser Cys Lys Ala Tyr Asn Arg
115 120 125
Glu Leu Asp Pro Met Ile Lys Lys Ile Phe Thr Glu Tyr Arg Lys Thr
130 135 140
His Asn Gln Gly Val Phe Asp Val Tyr Thr Pro Asp Ile Leu Arg Cys
145 150 155 160
Arg Lys Ser Gly Val Leu Thr Gly Leu Pro Asp Ala Tyr Gly Arg Gly
165 170 175
Arg Ile Ile Gly Asp Tyr Arg Arg Val Ala Leu Tyr Gly Ile Asp Tyr
180 185 190
Leu Met Lys Asp Lys Leu Ala Gln Phe Thr Ser Leu Gln Ala Asp Leu
195 200 205
Glu Asn Gly Val Asn Leu Glu Gln Thr Ile Arg Leu Arg Glu Glu Ile
210 215 220
Ala Glu Gln His Arg Ala Leu Gly Gln Met Lys Glu Met Ala Ala Lys
225 230 235 240
Tyr Gly Tyr Asp Ile Ser Gly Pro Ala Thr Asn Ala Gln Glu Ala Ile
245 250 255
Gln Trp Thr Tyr Phe Gly Tyr Leu Ala Ala Val Lys Ser Gln Asn Gly
260 265 270
Ala Ala Met Ser Phe Gly Arg Thr Ser Thr Phe Leu Asp Val Tyr Ile
275 280 285
Glu Arg Asp Leu Lys Ala Gly Lys Ile Thr Glu Gln Glu Ala Gln Glu
290 295 300
Met Val Asp His Leu Val Met Lys Leu Arg Met Val Arg Phe Leu Arg
305 310 315 320
Thr Pro Glu Tyr Asp Glu Leu Phe Ser Gly Asp Pro Ile Trp Ala Thr
325 330 335
Glu Ser Ile Gly Gly Met Gly Leu Asp Gly Arg Thr Leu Val Thr Lys
340 345 350
Asn Ser Phe Arg Phe Leu Asn Thr Leu Tyr Thr Met Gly Pro Ser Pro
355 360 365
Glu Pro Asn Met Thr Ile Leu Trp Ser Glu Lys Leu Pro Leu Asn Phe
370 375 380
Lys Lys Phe Ala Ala Lys Val Ser Ile Asp Thr Ser Ser Leu Gln Tyr
385 390 395 400
Glu Asn Asp Asp Leu Met Arg Pro Asp Phe Asn Asn Asp Asp Tyr Ala
405 410 415
Ile Ala Cys Cys Val Ser Pro Met Ile Val Gly Lys Gln Met Gln Phe
420 425 430
Phe Gly Ala Arg Ala Asn Leu Ala Lys Thr Met Leu Tyr Ala Ile Asn
435 440 445
Gly Gly Val Asp Glu Lys Leu Lys Met Gln Val Gly Pro Lys Ser Glu
450 455 460
Pro Ile Lys Gly Asp Val Leu Asn Tyr Asp Glu Val Met Glu Arg Met
465 470 475 480
Asp His Phe Met Asp Trp Leu Ala Lys Gln Tyr Ile Thr Ala Leu Asn
485 490 495
Ile Ile His Tyr Met His Asp Lys Tyr Ser Tyr Glu Ala Ser Leu Met
500 505 510
Ala Leu His Asp Arg Asp Val Ile Arg Thr Met Ala Cys Gly Ile Ala
515 520 525
Gly Leu Ser Val Ala Ala Asp Ser Leu Ser Ala Ile Lys Tyr Ala Lys
530 535 540
Val Lys Pro Ile Arg Asp Glu Asp Gly Leu Ala Ile Asp Phe Glu Ile
545 550 555 560
Glu Gly Glu Tyr Pro Gln Phe Gly Asn Asn Asp Pro Arg Val Asp Asp
565 570 575
Leu Ala Val Asp Leu Val Glu Arg Phe Met Lys Lys Ile Gln Lys Leu
580 585 590
His Thr Tyr Arg Asp Ala Ile Pro Thr Gln Ser Val Leu Thr Ile Thr
595 600 605
Ser Asn Val Val Tyr Gly Lys Lys Thr Gly Asn Thr Pro Asp Gly Arg
610 615 620
Arg Ala Gly Ala Pro Phe Gly Pro Gly Ala Asn Pro Met His Gly Arg
625 630 635 640
Asp Gln Lys Gly Ala Val Ala Ser Leu Thr Ser Val Ala Lys Leu Pro
645 650 655
Phe Ala Tyr Ala Lys Asp Gly Ile Ser Tyr Thr Phe Ser Ile Val Pro
660 665 670
Asn Ala Leu Gly Lys Asp Asp Glu Val Arg Lys Thr Asn Leu Ala Gly
675 680 685
Leu Met Asp Gly Tyr Phe His His Glu Ala Ser Ile Glu Gly Gly Gln
690 695 700
His Leu Asn Val Asn Val Met Asn Arg Glu Met Leu Leu Asp Ala Met
705 710 715 720
Glu Asn Pro Glu Lys Tyr Pro Gln Leu Thr Ile Arg Val Ser Gly Tyr
725 730 735
Ala Val Arg Phe Asn Ser Leu Thr Lys Glu Gln Gln Gln Asp Val Ile
740 745 750
Thr Arg Thr Phe Thr Gln Ser Met
755 760
<210> SEQ ID NO 36
<211> LENGTH: 764
<212> TYPE: PRT
<213> ORGANISM: Escherichia coli (strain K12)
<400> SEQUENCE: 36
Met Lys Val Asp Ile Asp Thr Ser Asp Lys Leu Tyr Ala Asp Ala Trp
1 5 10 15
Leu Gly Phe Lys Gly Thr Asp Trp Lys Asn Glu Ile Asn Val Arg Asp
20 25 30
Phe Ile Gln His Asn Tyr Thr Pro Tyr Glu Gly Asp Glu Ser Phe Leu
35 40 45
Ala Glu Ala Thr Pro Ala Thr Thr Glu Leu Trp Glu Lys Val Met Glu
50 55 60
Gly Ile Arg Ile Glu Asn Ala Thr His Ala Pro Val Asp Phe Asp Thr
65 70 75 80
Asn Ile Ala Thr Thr Ile Thr Ala His Asp Ala Gly Tyr Ile Asn Gln
85 90 95
Pro Leu Glu Lys Ile Val Gly Leu Gln Thr Asp Ala Pro Leu Lys Arg
100 105 110
Ala Leu His Pro Phe Gly Gly Ile Asn Met Ile Lys Ser Ser Phe His
115 120 125
Ala Tyr Gly Arg Glu Met Asp Ser Glu Phe Glu Tyr Leu Phe Thr Asp
130 135 140
Leu Arg Lys Thr His Asn Gln Gly Val Phe Asp Val Tyr Ser Pro Asp
145 150 155 160
Met Leu Arg Cys Arg Lys Ser Gly Val Leu Thr Gly Leu Pro Asp Gly
165 170 175
Tyr Gly Arg Gly Arg Ile Ile Gly Asp Tyr Arg Arg Val Ala Leu Tyr
180 185 190
Gly Ile Ser Tyr Leu Val Arg Glu Arg Glu Leu Gln Phe Ala Asp Leu
195 200 205
Gln Ser Arg Leu Glu Lys Gly Glu Asp Leu Glu Ala Thr Ile Arg Leu
210 215 220
Arg Glu Glu Leu Ala Glu His Arg His Ala Leu Leu Gln Ile Gln Glu
225 230 235 240
Met Ala Ala Lys Tyr Gly Phe Asp Ile Ser Arg Pro Ala Gln Asn Ala
245 250 255
Gln Glu Ala Val Gln Trp Leu Tyr Phe Ala Tyr Leu Ala Ala Val Lys
260 265 270
Ser Gln Asn Gly Gly Ala Met Ser Leu Gly Arg Thr Ala Ser Phe Leu
275 280 285
Asp Ile Tyr Ile Glu Arg Asp Phe Lys Ala Gly Val Leu Asn Glu Gln
290 295 300
Gln Ala Gln Glu Leu Ile Asp His Phe Ile Met Lys Ile Arg Met Val
305 310 315 320
Arg Phe Leu Arg Thr Pro Glu Phe Asp Ser Leu Phe Ser Gly Asp Pro
325 330 335
Ile Trp Ala Thr Glu Val Ile Gly Gly Met Gly Leu Asp Gly Arg Thr
340 345 350
Leu Val Thr Lys Asn Ser Phe Arg Tyr Leu His Thr Leu His Thr Met
355 360 365
Gly Pro Ala Pro Glu Pro Asn Leu Thr Ile Leu Trp Ser Glu Glu Leu
370 375 380
Pro Ile Ala Phe Lys Lys Tyr Ala Ala Gln Val Ser Ile Val Thr Ser
385 390 395 400
Ser Leu Gln Tyr Glu Asn Asp Asp Leu Met Arg Thr Asp Phe Asn Ser
405 410 415
Asp Asp Tyr Ala Ile Ala Cys Cys Val Ser Pro Met Val Ile Gly Lys
420 425 430
Gln Met Gln Phe Phe Gly Ala Arg Ala Asn Leu Ala Lys Thr Leu Leu
435 440 445
Tyr Ala Ile Asn Gly Gly Val Asp Glu Lys Leu Lys Ile Gln Val Gly
450 455 460
Pro Lys Thr Ala Pro Leu Met Asp Asp Val Leu Asp Tyr Asp Lys Val
465 470 475 480
Met Asp Ser Leu Asp His Phe Met Asp Trp Leu Ala Val Gln Tyr Ile
485 490 495
Ser Ala Leu Asn Ile Ile His Tyr Met His Asp Lys Tyr Ser Tyr Glu
500 505 510
Ala Ser Leu Met Ala Leu His Asp Arg Asp Val Tyr Arg Thr Met Ala
515 520 525
Cys Gly Ile Ala Gly Leu Ser Val Ala Thr Asp Ser Leu Ser Ala Ile
530 535 540
Lys Tyr Ala Arg Val Lys Pro Ile Arg Asp Glu Asn Gly Leu Ala Val
545 550 555 560
Asp Phe Glu Ile Asp Gly Glu Tyr Pro Gln Tyr Gly Asn Asn Asp Glu
565 570 575
Arg Val Asp Ser Ile Ala Cys Asp Leu Val Glu Arg Phe Met Lys Lys
580 585 590
Ile Lys Ala Leu Pro Thr Tyr Arg Asn Ala Val Pro Thr Gln Ser Ile
595 600 605
Leu Thr Ile Thr Ser Asn Val Val Tyr Gly Gln Lys Thr Gly Asn Thr
610 615 620
Pro Asp Gly Arg Arg Ala Gly Thr Pro Phe Ala Pro Gly Ala Asn Pro
625 630 635 640
Met His Gly Arg Asp Arg Lys Gly Ala Val Ala Ser Leu Thr Ser Val
645 650 655
Ala Lys Leu Pro Phe Thr Tyr Ala Lys Asp Gly Ile Ser Tyr Thr Phe
660 665 670
Ser Ile Val Pro Ala Ala Leu Gly Lys Glu Asp Pro Val Arg Lys Thr
675 680 685
Asn Leu Val Gly Leu Leu Asp Gly Tyr Phe His His Glu Ala Asp Val
690 695 700
Glu Gly Gly Gln His Leu Asn Val Asn Val Met Asn Arg Glu Met Leu
705 710 715 720
Leu Asp Ala Ile Glu His Pro Glu Lys Tyr Pro Asn Leu Thr Ile Arg
725 730 735
Val Ser Gly Tyr Ala Val Arg Phe Asn Ala Leu Thr Arg Glu Gln Gln
740 745 750
Gln Asp Val Ile Ser Arg Thr Phe Thr Gln Ala Leu
755 760
<210> SEQ ID NO 37
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic
6xHis tag
<400> SEQUENCE: 37
His His His His His His
1 5
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