Patent application title: METHODS AND MATERIALS FOR PRODUCING 5 AND 7-CARBON MONOMERS
Inventors:
IPC8 Class: AC12P1300FI
USPC Class:
1 1
Class name:
Publication date: 2017-06-08
Patent application number: 20170159086
Abstract:
This document describes biochemical pathways for biosynthesizing a
3-oxo-7-hydroxyheptanoyl-CoA intermediate using a .beta.-ketothiolase,
and enzymatically converting 3-oxo-7-hydroxyheptanoyl-CoA to
7-hydroxyheptanoic acid. -7-hydroxyheptanoic acid can be further
enzymatically converted to pimelic acid, 7-aminoheptanoic acid,
heptamethylenediamine or 1,7-heptanediol. This document also describes
recombinant hosts producing 7-hydroxyheptanoic acid as well as pimelic
acid, 7-aminoheptanoic acid, heptamethylenediamine and 1,7-heptanediol.Claims:
1. A method of producing 3-oxo-7-hydroxyheptanoyl-CoA or a salt thereof,
said method comprising enzymatically converting 5-hydroxypentanoyl-CoA to
3-oxo-7-hydroxyheptanoyl-CoA using a polypeptide having the activity of a
.beta.-ketothiolase classified under EC. 2.3.1.-.
2. The method of claim 1, wherein said .beta.-ketothiolase is classified under EC 2.3.1.16.
3. The method of claim 1, wherein said .beta.-ketothiolase is classified under EC 2.3.1.174.
4. The method of claim 1, wherein said .beta.-ketothiolase (a) has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO: 13 or (b) has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO: 13 and is capable of converting 5-hydroxypentanoyl-CoA to 3-oxo-7-hydroxyheptanoyl-CoA.
5. The method of claim 1, further comprising enzymatically converting 3-oxo-7-hydroxyheptanoyl-CoA to 7-hydroxyheptanoate using a polypeptide having the activity of a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoA reductase, an enoyl-CoA hydratase, a trans-2-enoyl-CoA reductase, and a thioesterase or a CoA transferase.
6. The method of claim 5, wherein said 3-hydroxyacyl-CoA dehydrogenase or said 3-oxoacyl-CoA reductase is classified under EC 1.1.1.35, EC 1.1.1.36, EC 1.1.1.100, or EC 1.1.1.157.
7. The method of claim 5, wherein said enoyl-CoA hydratase is classified under EC 4.2.1.17 or EC 4.2.1.119.
8. The method of claim 5, wherein said trans-2-enoyl-CoA reductase is classified under EC 1.3.1.38, EC 1.3.1.44, or EC 1.3.1.8.
9. (canceled)
10. (canceled)
11. The method of claim 5, said method further comprising enzymatically converting 7-hydroxyheptanoate to pimelic acid, 7-aminoheptanoate, heptamethylenediamine, or 1,7-heptanediol in one or more steps.
12. The method of claim 11, wherein 7-hydroxyheptanoate is converted to pimelic acid using one or more of a monooxygenase, an alcohol dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 5-hydroxyvalerate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 5-oxovalerate dehydrogenase, or an aldehyde dehydrogenase.
13. The method of claim 11, wherein 7-hydroxyheptanoate is converted to 7-aminoheptanoate using one or more of a polypeptide having the activity of an alcohol dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase, and a .omega.-transaminase.
14. The method of claim 11, wherein 7-hydroxyheptanoate is converted to heptamethylenediamine using one or more of a carboxylate reductase, a .omega.-transaminase, an alcohol dehydrogenase, an N-acetyltransferase, and an acetylputrescine deacylase.
15. The method of claim 13, wherein said .omega.-transaminase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs.: 7-12.
16. The method of claim 11, wherein 7-hydroxyheptanoate is converted to 1,7-heptanediol using a carboxylate reductase and an alcohol dehydrogenase.
17. The method of claim 16, wherein said carboxylate reductase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs.: 2-6.
18. The method of claim 1, wherein said 5-hydroxypentanoyl-CoA is enzymatically produced from 2-oxoadipate or malonyl-CoA.
19. The method of claim 18, wherein 5-hydroxypentanoyl-CoA is enzymatically produced from 2-oxoadipate--using one or more of an alpha-aminotransaminase; a 2-oxoadipate decarboxylase; a branch chain decarboxylase; a glutamate decarboxylase; a .omega.-transaminase; a CoA transferase, a CoA ligase, and an alcohol dehydrogenase.
20. The method of claim 18, wherein 5-hydroxypentanoyl-CoA is enzymatically produced from malonyl-CoA using one or more of a malonyl-CoA reductase; a 3-hydroxypropionate dehydrogenase; a 3-hydroxypropionyl-CoA synthase; a CoA-transferase; a .beta.-ketothiolase; a 3-hydroxyacyl-CoA dehydrogenase; a 3-oxoacyl-CoA reductase, an enoyl-CoA hydratase, and a trans-2-enoyl-CoA reductase.
21. The method of claim 1, wherein said method is performed in a recombinant organism.
22. The method of claim 21, wherein said organism is subjected to a cultivation strategy under aerobic, anaerobic or, micro-aerobic cultivation conditions.
23. The method of claim 21, wherein said organism is cultured under conditions of nutrient limitation.
24. (canceled)
25. The method of claim 21, wherein the principal carbon source fed to the fermentation derives from a biological feedstock.
26. (canceled)
27. The method of claim 21, wherein the principal carbon source fed to the fermentation derives from a non-biological feedstock.
28. (canceled)
29. The method of claim 21, wherein the organism is a prokaryote.
30. (canceled)
31. (canceled)
32. The method of claim 21, wherein the organism is a eukaryote.
33. (canceled)
34. (canceled)
35. The method of claim 21, wherein the organism's tolerance to high concentrations of a C7 building block is improved through continuous cultivation in a selective environment.
36. The method of claim 21, wherein said host comprises an attenuation to one or more of the following enzymes: polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, a phosphotransacetylase forming acetate, an acetate kinase, a lactate dehydrogenase, a menaquinol-fumarate oxidoreductase, an alcohol dehydrogenase forming ethanol, a triose phosphate isomerase, a pyruvate decarboxylase, a glucose-6-phosphate isomerase, NADH-consuming transhydrogenase, an NADH-specific glutamate dehydrogenase, a NADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoA dehydrogenase, an acyl-CoA dehydrogenase accepting C7 building blocks and central precursors as substrates, a butaryl-CoA dehydrogenase, or an adipyl-CoA synthetase.
37. The method of claim 21, wherein said organism overexpresses one or more genes encoding: an acetyl-CoA synthetase; a 6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase; a glucose dehydrogenase; a fructose 1,6 diphosphatase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase; a formate dehydrogenase; a L-glutamine synthetase; a diamine transporter, a dicarboxylate transporter, and/or a multidrug transporter.
38. A recombinant organism comprising at least one exogenous nucleic acid encoding (i) a .beta.-ketothiolase, (ii) a thioesterase or a CoA transferase, and one or more of (iii) a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoA reductase, (iv) an enoyl-CoA hydratase, and (v) a trans-2-enoyl-CoA reductase, said organism producing 7-hydroxyheptanoate.
39. The recombinant organism of claim 38, said host further comprising one or more of the following exogenous enzymes: a monooxygenase, an alcohol dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 5-hydroxyvalerate dehydrogenase, a 6-hydroxyhextanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 5-oxovalerate dehydrogenase, or an aldehyde dehydrogenase, said host further producing pimelic acid.
40. The recombinant organism of claim 38, said host further comprising one or more of the following exogenous enzymes: a monooxygenase, a transaminase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase, and an alcohol dehydrogenase, said host further producing 7-aminoheptanoate.
41. The recombinant organism of claim 38, said host further comprising one or more of the following exogenous enzymes: a carboxylate reductase, a .omega.-transaminase, a deacylase, an N-acetyl transferase, or an alcohol dehydrogenase, said host further producing heptamethylenediamine.
42. The recombinant organism of claim 38, said host further comprising an exogenous carboxylate reductase and an exogenous alcohol dehydrogenase, said host further producing 1,7-heptanediol.
43. The recombinant organism of claim 38, said host further comprising one or more of the following exogenous enzymes: an alpha-aminotransaminase; a 2-oxoadipate decarboxylase; a branch-chain decarboxylase; a glutamate decarboxylase; a .omega.-transaminase; a CoA-ligase; a CoA-transferase; and an alcohol dehydrogenase.
44. (canceled)
45. A nucleic acid construct or expression vector comprising (a) a polynucleotide encoding a polypeptide having .beta.-ketothiolase 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 .beta.-ketothiolase activity is selected from the group consisting of: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NOs: 1 or 13; (b) a polynucleotide encoding a polypeptide having .omega.-transaminase 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 .omega.-transaminase activity is selected from the group consisting of: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NOs: 7-12.
46. A composition comprising the nucleic acid construct or expression vector of claim 45.
47. A non-naturally occurring biochemical network comprising at least one substrate of FIG. 1, at least one exogenous nucleic acid encoding a polypeptide having the activity of at least one enzyme of FIG. 1 and at least one product of FIG. 1.
48. A bio-derived product, bio-based product or fermentation-derived product, wherein said product comprises: i. a composition comprising at least one bio-derived, bio-based or fermentation-derived compound according to claim 1, or of FIG. 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.
49. The method of claim 14, wherein said .omega.-transaminase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs.: 7-12.
Description:
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No. 62/255,303, filed Nov. 13, 2015, the entire contents of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] This invention provides materials and methods for biosynthesizing 5 carbon and 7 carbon monomers. For example, the invention provides 3-oxo-7-hydroxyheptanoyl-CoA using a .beta.-ketothiolase, and enzymatically converting 3-oxo-7-hydroxyheptanoyl-CoA to 7-hydroxyheptanoic acid using one or more of an isolated 3-hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl-CoA reductase, an enoyl-CoA hydratase, a trans-2-enoyl-CoA reductase, and a thioesterase, or using host having at least one exogenous nucleic acid capable of making one or more of such enzymes. This invention also provides methods for converting 7-hydroxyheptanoic acid to one or more of pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine, and 1,7-heptanediol using one or more isolated enzymes such as dehydrogenases, reductases, hydratases, thioesterases, monooxygenases, and transaminases or using host cells expressing one or more such enzymes.
BACKGROUND
[0003] Nylons are polyamides which are generally synthesized by the condensation polymerization of a diamine with a dicarboxylic acid. Similarly, Nylons also may be produced by the condensation polymerization of lactams. Nylon 7 is produced by polymerisation of 7-aminoheptanoic acid, whereas Nylon 7,7 is produced by condensation polymerisation of pimelic acid and heptamethylenediamine. No economically viable petrochemical routes exist for producing the monomers for Nylon 7 and Nylon 7,7.
[0004] Given that there are no economically viable 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 chemical compounds.
[0005] Both bioderived feedstocks and petrochemical feedstocks can be viable starting materials for the biocatalysis processes.
SUMMARY
[0006] Accordingly, against this background, it is clear that there is a need for sustainable methods for producing one or more of pimelic acid, 7-hydroxyheptanoic acid, 7-aminoheptanoic acid, heptamethylenediamine, and 1,7-heptanediol or derivatives thereof, wherein the methods are biocatalyst based. This document is based at least in part on the discovery that it is possible to construct biochemical pathways for using, inter alia, a .beta.-ketothiolase to produce, for example, 7-hydroxyheptanoate, which can be converted in one or more enzymatic steps to pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine, or 1,7-heptanediol. "Pimelic acid" and "pimelate," "7-hydroxyheptanoic acid" and "7-hydroxyheptanoate," and "7-aminoheptanoic acid" 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 inter alia pH.
[0007] 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.
[0008] For compounds containing amine groups such as but not limited to organic amines, aminoacids and diamine, 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.
[0009] 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.
[0010] In the face of the optimality principle, it surprisingly has been discovered that appropriate non-natural metabolic pathways, feedstocks, cells (hosts or microorganisms), attenuation(s) to a cell's biochemical network, and/or cultivation strategies may be combined to efficiently produce 7-hydroxyheptanoate as a C7 (7-carbon) building block, or convert 7-hydroxyheptanoate to other C7 building blocks such as pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine, or 1,7-heptanediol.
[0011] In some embodiments, a terminal carboxyl group can be enzymatically formed using a thioesterase, an aldehyde dehydrogenase, a 6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, or a monooxgenase (e.g., in combination with an oxidoreductase and ferredoxin). See FIG. 1 and FIG. 2.
[0012] In some embodiments, a terminal amine group can be enzymatically formed using a .omega.-transaminase or a deacylase. See FIG. 4. The .omega.-transaminase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs.: 7-12.
[0013] In some embodiments, a terminal hydroxyl group can be enzymatically formed using an alcohol dehydrogenase. See FIG. 1 and FIG. 5.
[0014] In one aspect, this document features a method of producing 3-oxo-7-hydroxyheptanoyl-CoA. The method includes enzymatically converting 5-hydroxypentanoyl-CoA to 3-oxo-7-hydroxyheptanoyl-CoA using a .beta.-ketothiolase classified under EC. 2.3.1.- (e.g., EC 2.3.1.16 or EC 2.3.1.174). The .beta.-ketothiolase can have at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:13.
[0015] In one aspect, this document features a method of producing 3-oxo-7-hydroxyheptanoyl-CoA. The method includes enzymatically converting 5-hydroxypentanoyl-CoA to 3-oxo-7-hydroxyheptanoyl-CoA using a thiolase. The thiolase can have at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 15-36.
[0016] In one aspect, this document features a method of producing 5-hydroxypentanoyl-CoA. The method includes enzymatically converting 5-hydroxypentanoate to 5-hydroxypentanoyl-CoA using a 5-hydroxyvalerate CoA transferase. The 5-hydroxyvalerate CoA transferase can have at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 37.
[0017] In one aspect, this document features a method of producing 3-oxo-7-hydroxyheptanoyl-CoA from 5-hydroxypentanoate in a two-step enzymatic reaction catalyzed by a 5-hydroxyvalerate CoA transferase and a thiolase. The 5-hydroxyvalerate CoA transferase can have at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 37. The thiolase can have at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 15-36.
[0018] In one aspect, this document features a method for enzymatically converting 3-oxo-7-hydroxyheptanoyl-CoA to 7-hydroxyheptanoate using a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoA reductase, an enoyl-CoA hydratase, a trans-2-enoyl-CoA reductase, and a thioesterase or a CoA transferase. The 3-hydroxyacyl-CoA dehydrogenase or 3-oxoacyl-CoA reductase can be classified under EC 1.1.1.35, EC 1.1.1.36, EC 1.1.1.100, or EC 1.1.1.157. The enoyl-CoA hydratase can be classified under EC 4.2.1.17 or EC 4.2.1.119. The trans-2-enoyl-CoA reductase can be classified under EC 1.3.1.38, EC 1.3.1.44, or EC 1.3.1.8.
[0019] In one aspect, this document features a method for biosynthesizing 7-hydroxyheptanoate. The method includes enzymatically synthesizing 3-oxo-7-hydroxyheptanoyl-CoA from 5-hydroxypentanoyl-CoA using a .beta.-ketothiolase classified under EC. 2.3.1.- (e.g., EC 2.3.1.16 or EC 2.3.1.174) and enzymatically converting 3-oxo-7-hydroxyheptanoyl-CoA to 7-hydroxyheptanoate. The .beta.-ketothiolase can have at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:13. 3-oxo-7-hydroxyheptanoyl-CoA can be converted to 3-hydroxy-7-hydroxyheptanoyl-CoA using a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoA reductase, 3-hydroxy-7-hydroxyheptanoyl-CoA can be converted to 2,3-dehydro-7-hydroxyheptanoyl-CoA using an enoyl-CoA hydratase, 2,3-dehydro-7-hydroxyheptanoyl-CoA can be converted to 7-hydroxyheptanoyl-CoA using a trans-2-enoyl-CoA reductase, and 7-hydroxyheptanoyl-CoA can be converted to 7-hydroxyheptanoate using a thioesterase or a CoA transferase.
[0020] Any of the methods further can include enzymatically converting 7-hydroxyheptanoate to pimelic acid, 7-aminoheptanoate, heptamethylenediamine, or 1,7-heptanediol in one or more steps.
[0021] For example, 7-hydroxyheptanoate can be enzymatically converted to pimelic acid using one or more of a monooxygenase, an alcohol dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 5-hydroxyvalerate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 5-oxovalerate dehydrogenase, or an aldehyde dehydrogenase.
[0022] For example, 7-hydroxyheptanoate can be converted to 7-aminoheptanoate using one or more of an alcohol dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase, and a .omega.-transaminase. The .omega.-transaminase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs.: 7-12.
[0023] For example, 7-hydroxyheptanoate can be converted to heptamethylenediamine using one or more of a carboxylate reductase, a .omega.-transaminase, an alcohol dehydrogenase, an N-acetyltransferase, and an acetylputrescine deacylase. The co-transaminase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs.: 7-12.
[0024] For example, 7-hydroxyheptanoate can be converted to 1,7-heptanediol using a carboxylate reductase and an alcohol dehydrogenase. The carboxylate reductase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs.: 2-6.
[0025] In any of the methods, 5-hydroxypentanoyl-CoA can be enzymatically produced from 2-oxoadipate. For example, 5-hydroxypentanoyl-CoA can be enzymatically produced from 2-oxoadipate using one or more of a .alpha.-aminotransaminase; a 2-oxoadipate decarboxylase; a branch chain decarboxylase; a glutamate decarboxylase; a .omega.-transaminase; a CoA transferase, a CoA ligase, and an alcohol dehydrogenase.
[0026] In any of the methods, 5-hydroxypentanoyl-CoA can be enzymatically produced from malonyl-CoA. For example, 5-hydroxypentanoyl-CoA can be enzymatically produced from malonyl-CoA using one or more of a malonyl-CoA reductase; a 3-hydroxypropionate dehydrogenase; a 3-hydroxypropionyl-CoA synthase; a CoA-transferase; a .beta.-ketothiolase; a 3-hydroxyacyl-CoA dehydrogenase; a 3-oxoacyl-CoA reductase, an enoyl-CoA hydratase, and a trans-2-enoyl-CoA reductase.
[0027] In any of the methods described herein, pimelic acid can be produced by forming the second terminal functional group in pimelate semialdehyde (also known as 7-oxoheptanoate) using (i) an aldehyde dehydrogenase classified under EC 1.2.1.3, (ii) a 7-oxohexanoate dehydrogenase classified under EC 1.2.1.63 such as that encoded by ChnE or a 7-oxoheptanoate dehydrogenase classified under EC 1.2.1.- (e.g., the gene product of ThnG), or iii) a monooxgenase in the cytochrome P450 family.
[0028] In any of the methods described herein, 7-aminoheptanoic acid can be produced by forming the second terminal functional group in pimelate semialdehyde using a co-transaminase classified 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.
[0029] In any of the methods described herein, heptamethylenediamine can be produced by forming a second terminal functional group in (i) 7-aminoheptanal using a co-transaminase classified 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, or in (ii) N7-acetyl-1,7-diaminoheptane using a deacylase classified, for example, under EC 3.5.1.17.
[0030] In any of the methods described herein, 1,7-heptanediol can be produced by forming the second terminal functional group in 7-hydroxyheptanal using an alcohol dehydrogenase classified under EC 1.1.1.- (e.g., EC 1.1.1.1, 1.1.1.2, 1.1.1.21, or 1.1.1.184) such as that encoded by YMR318C, YqhD, or CAA81612.1.
[0031] In some embodiments, the biological feedstock can be 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.
[0032] In some embodiments, the non-biological feedstock can be or can derive from natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, benzoate, non-volatile residue (NVR) or a caustic wash waste stream from cycloheptane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.
[0033] In some embodiments, the host microorganism's tolerance to high concentrations of one or more C7 building blocks is improved through continuous cultivation in a selective environment.
[0034] In some embodiments, the host microorganism's biochemical network is attenuated or augmented to (1) ensure the intracellular availability of acetyl-CoA and 5-hydroxypentanoyl-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, central precursors leading to and including C7 building blocks, and/or (4) ensure efficient efflux from the cell.
[0035] In some embodiments, a cultivation strategy is used to achieve anaerobic, micro-aerobic, or aerobic cultivation conditions.
[0036] In some embodiments, the cultivation strategy includes limiting nutrients, such as limiting nitrogen, phosphate or oxygen.
[0037] In some embodiments, one or more C7 building blocks are produced by a single type of microorganism, e.g., a recombinant host containing one or more exogenous nucleic acids, using, for example, a fermentation strategy.
[0038] In some embodiments, one or more C7 building blocks are produced by a single type of microorganism having one or more exogenous nucleic acids which encode a polypeptide having a .beta.-ketothiolase activity, (ii) a thioesterase activity or a CoA transferase activity, and one or more of (iii) a 3-hydroxyacyl-CoA dehydrogenase activity or a 3-oxoacyl-CoA reductase activity, (iv) an enoyl-CoA hydratase activity, and (v) a trans-2-enoyl-CoA reductase activity, using, for example, a fermentation strategy.
[0039] In another aspect, this document features a recombinant host that includes at least one exogenous nucleic acid encoding (i) a .beta.-ketothiolase, (ii) a thioesterase or a CoA transferase, and one or more of (iii) a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoA reductase, (iv) an enoyl-CoA hydratase, and (v) a trans-2-enoyl-CoA reductase, the host producing 7-hydroxyheptanoate.
[0040] A host producing 7-hydroxyheptanoate further can include one or more of the following exogenous enzymes: a monooxygenase, an alcohol dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 5-hydroxyvalerate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 5-oxovalerate dehydrogenase, or an aldehyde dehydrogenase, the host further producing pimelic acid.
[0041] A host producing 7-hydroxyheptanoate further can include one or more of the following exogenous enzymes: a monooxygenase, a transaminase, a 6-hydroxyhexanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, and an alcohol dehydrogenase, the host further producing 7-aminoheptanoate.
[0042] A host producing 7-hydroxyheptanoate further can include one or more of the following exogenous enzymes: a carboxylate reductase, a .omega.-transaminase, a deacylase, a N-acetyl transferase, or an alcohol dehydrogenase, said host further producing heptamethylenediamine.
[0043] A host producing 7-hydroxyheptanoate further can include an exogenous carboxylate reductase and an exogenous alcohol dehydrogenase, the host further producing 1,7-heptanediol.
[0044] Any of the recombinant hosts described herein further can include one or more of the following exogenous enzymes: an alpha-aminotransaminase; a 2-oxoadipate decarboxylase; a branch-chain decarboxylase; a glutamate decarboxylase; a .omega.-transaminase; a CoA-ligase; a CoA-transferase; and an alcohol dehydrogenase.
[0045] Any of the recombinant hosts described herein further can include one or more of the following exogenous enzymes: a malonyl-CoA reductase; a 3-hydroxypropionate dehydrogenase; a 3-hydroxypropionyl-CoA synthase; a CoA-transferase; a .beta.-ketothiolase; a 3-hydroxyacyl-CoA dehydrogenase; a 3-oxoacyl-CoA reductase; an enoyl-CoA hydratase; and a trans-2-enoyl-CoA reductase.
[0046] Any of the recombinant hosts can be a prokaryote such as a prokaryote from a genus selected from the group consisting of Escherichia; Clostridia; Corynebacteria; Cupriavidus; Pseudomonas; Delftia; Bacilluss; Lactobacillus; Lactococcus; and Rhodococcus. For example, the prokaryote can be selected from the group consisting of 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. Such prokaryotes also can be sources of genes for constructing recombinant host cells described herein that are capable of producing C7 building blocks.
[0047] Any of the recombinant hosts can be a eukaryote such as a eukaryote from a genus selected from the group consisting of Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula, and Kluyveromyces. For example, the eukaryote can be selected from the group consisting of Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans, and Kluyveromyces lactis. Such eukaryotes also can be sources of genes for constructing recombinant host cells described herein that are capable of producing C7 building blocks.
[0048] Any of the recombinant hosts described herein further can include attenuation of one or more of the following enzymes: a polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, a phosphotransacetylase forming acetate, an acetate kinase, a lactate dehydrogenase, a menaquinol-fumarate oxidoreductase, an alcohol dehydrogenase forming ethanol, a triose phosphate isomerase, a pyruvate decarboxylase, a glucose-6-phosphate isomerase, NADH-consuming transhydrogenase, an NADH-specific glutamate dehydrogenase, a NADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoA dehydrogenase, an acyl-CoA dehydrogenase accepting C7 building blocks and central precursors as substrates, a butyryl-CoA dehydrogenase, or an adipyl-CoA synthetase.
[0049] Any of the recombinant hosts described herein further can overexpress one or more genes encoding: an acetyl-CoA synthetase; a 6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase; a glucose dehydrogenase; a fructose 1,6 diphosphatase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase; a formate dehydrogenase; a L-glutamine synthetase; a diamine transporter; a dicarboxylate transporter; and/or a multidrug transporter.
[0050] This document also features a biobased polymer comprising the bioderived pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine, or 1,7-heptanediol.
[0051] This document also features a biobased resin comprising the bioderived pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine, or 1,7-heptanediol, as well as a molded product obtained by molding a biobased resin.
[0052] In another aspect, this document features a process for producing a biobased polymer that includes chemically reacting the bioderived pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine, or 1,7-heptanediol, with itself or another compound in a polymer producing reaction.
[0053] In another aspect, this document features a process for producing a biobased resin that includes chemically reacting the bioderived pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine, or 1,7-heptanediol, with itself or another compound in a resin producing reaction.
[0054] Also, described herein is a biochemical network comprising a polypeptide having .beta.-ketothiolase activity, wherein the polypeptide having .beta.-ketothiolase activity enzymatically converts 5-hydroxypentanoyl-CoA to 3-oxo-7-hydroxyheptanoyl-CoA.
[0055] The biochemical network can further include a polypeptide having 3-hydroxyacyl-CoA dehydrogenase or a polypeptide having 3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoA hydratase activity, a polypeptide having thioesterase activity, a polypeptide having CoA transferase, and a polypeptide having trans-2-enoyl-CoA reductase activity. In one aspect, the biochemical network is a non-naturally occurring biochemical network comprising at least one substrate of FIG. 1, at least one exogenous nucleic acid encoding a polypeptide having the activity of at least one enzyme of FIG. 1 and at least one product of FIG. 1. In another aspect of the invention, the biochemical network is a non-naturally occurring biochemical network comprising a 3-hydroxypropionyl-CoA, an exogenous nucleic acid encoding a polypeptide having the activity of a .beta.-ketothiolase classified under EC. 2.3.1 and a 3-hydroxypropionyl-CoA.
[0056] In one aspect, this document features a method for producing a bioderived five and seven carbon compounds. The method for producing a bioderived five and seven carbon compounds can include culturing or growing a recombinant host as described herein under conditions and for a sufficient period of time to produce the bioderived five and seven carbon compounds, wherein, optionally, the bioderived carbon compound is pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine, or 1,7-heptanediol.
[0057] In one aspect, this document features composition comprising a bioderived five and seven carbon compounds as described herein and a compound other than the bioderived five and seven carbon compound, wherein the bioderived carbon compound is pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine, and 1,7-heptanediol. For example, the bioderived four carbon compound is a cellular portion of a host cell or an organism.
[0058] In one aspect, this document features nucleic acid constructs or expression vectors comprising a polynucleotide encoding a polypeptide having .beta.-ketothiolase 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 .beta.-ketothiolase activity is selected from the group consisting of: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NOs: 1 or 13; a polynucleotide encoding a polypeptide having .omega.-transaminase 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 .omega.-transaminase activity is selected from the group consisting of: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NOs: 7-12. Further, this document features compositions comprising the nucleic acid construct or expression vector as described above.
[0059] 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.
[0060] 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 application, including the written description and drawings and the claims. The word "comprising" in the claims may be replaced by "consisting essentially of" or with "consisting of," according to standard practice in patent law.
DESCRIPTION OF DRAWINGS
[0061] FIG. 1 is a schematic of exemplary biochemical pathways leading to 7-hydroxyheptanoate using 2-oxo-adipate and malonyl-CoA as central metabolites.
[0062] FIG. 2 is a schematic of exemplary biochemical pathways leading to pimelic acid using 7-hydroxyheptanoate as a central precursor.
[0063] FIG. 3 is a schematic of an exemplary biochemical pathway leading to 7-aminoheptanoate using 7-hydroxyheptanoate as a central precursor.
[0064] FIG. 4 is a schematic of exemplary biochemical pathways leading to heptamethylenediamine using 7-aminoheptanoate, 7-hydroxyheptanoate, pimelate semialdehyde, or 1,7-heptanediol as a central precursor.
[0065] FIG. 5 is a schematic of an exemplary biochemical pathway leading to 1,7-heptanediol using 7-hydroxyheptanoate as a central precursor.
[0066] FIG. 6 contains the amino acid sequences of a Cupriavidus necator .beta.-ketothiolase (see GenBank Accession No. AAC38322.1, SEQ ID NO: 1), a Mycobacterium marinum carboxylate reductase (see Genbank Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatis carboxylate reductase (see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), a Segniliparus rugosus carboxylate reductase (see Genbank Accession No. EFV11917.1, SEQ ID NO: 4), a Mycobacterium massiliense carboxylate reductase (see Genbank Accession No. EIV11143.1, SEQ ID NO: 5), a Segniliparus rotundus carboxylate reductase (see Genbank Accession No. ADG98140.1, SEQ ID NO: 6), a Chromobacterium violaceum .omega.-transaminase (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 7), a Pseudomonas aeruginosa .omega.-transaminase (see Genbank Accession No. AAG08191.1, SEQ ID NO: 8), a Pseudomonas syringae .omega.-transaminase (see Genbank Accession No. AAY39893.1, SEQ ID NO: 9), a Rhodobacter sphaeroides .omega.-transaminase (see Genbank Accession No. ABA81135.1, SEQ ID NO: 10), an Escherichia coli .omega.-transaminase (see Genbank Accession No. AAA57874.1, SEQ ID NO: 11), a Vibrio fluvialis .omega.-transaminase (See Genbank Accession No. AEA39183.1, SEQ ID NO: 12), an Escherichia coli .beta.-ketothiolase (see GenBank Accession No. AAC74479.1, SEQ ID NO: 13), a Mycobacterium smegmatis carboxylate reductase (see Genbank Accession No. ABK75684.1, SEQ ID NO: 14), a Pseudomonas putida thiolase (see Genbank Accession No. AAN70209.2, SEQ ID NO: 15), a Sphingomonas wittichii thiolase (see Genbank Accession No. ABQ69245.1, SEQ ID NO: 16), a Pseudomonas reinekei thiolase (see Genbank Accession No. ACZ63623.1, SEQ ID NO: 17), a Pseudomonas putida thiolase (see Genbank Accession No. AAA85138.1, SEQ ID NO: 18), a Burkholderia xenovorans thiolase (see Genbank Accession No. ABE28745.1, SEQ ID NO: 19), a Burkholderia xenovorans thiolase (see Genbank Accession No. ABE33819.1, SEQ ID NO: 20), a Rhodococcus jostii thiolase (see Genbank Accession No. ABG94668.1, SEQ ID NO: 21), a Bdellovibrio bacteriovorus thiolase (see Genbank Accession No. CAE79693.1, SEQ ID NO: 22), a Cronobacter turicensis thiolase (see Genbank Accession No. CBA32535.1, SEQ ID NO: 23), an Arthrobacter sp. thiolase (see Genbank Accession No. ABK03524.1, SEQ ID NO: 24), a Caulobacter segnis thiolase (see Genbank Accession No. ADG08907.1, SEQ ID NO: 25), a Dinoroseobacter shibae thiolase (see Genbank Accession No. ABV92581.1, SEQ ID NO: 26), a Burkholderia xenovorans thiolase (see Genbank Accession No. ABE36495.1, SEQ ID NO: 27), a Geobacillus kaustophilus thiolase (see Genbank Accession No. BAD75605.1, SEQ ID NO: 28), a Beijerinckia indica thiolase (see Genbank Accession No. ACB95386.1, SEQ ID NO: 29), a Citrobacter freundii thiolase (see Genbank Accession No. EKS55037.1, SEQ ID NO: 30), a Cupriavidus necator thiolase (see Genbank Accession No. AEI75849.1, SEQ ID NO: 31), a Gordonia bronchialis thiolase (see Genbank Accession No. ACY20886.1, SEQ ID NO: 32), a Burkholderia sp. thiolase (see Genbank Accession No. ADG18081.1, SEQ ID NO: 33), a Glutamicibacter arilaitensis thiolase (see Genbank Accession No. CBT74677.1, SEQ ID NO: 34), an Escherichia coli thiolase (see Genbank Accession No. AAC74479.1, SEQ ID NO: 35), a Cupriavidus necator thiolase (see Genbank Accession No. AAC38322.1, SEQ ID NO: 36), and a Clostridium viride 5-hydroxyvalerate CoA transferase (see NCBI Reference Sequence: NZ_KK211198.1, SEQ ID NO: 37).
[0067] FIG. 7 is a bar graph summarizing the change in absorbance at 340 nm after 20 minutes, which is a measure of the consumption of NADPH and activity of six carboxylate reductase preparations in enzyme only controls (no substrate).
[0068] FIG. 8 is a bar graph of the change in absorbance at 340 nm after 20 minutes, which is a measure of the consumption of NADPH and the activity of two carboxylate reductase preparations for converting pimelate to pimelate semialdehyde relative to the empty vector control.
[0069] FIG. 9 is a bar graph of the change in absorbance at 340 nm after 20 minutes, which is a measure of the consumption of NADPH and the activity of six carboxylate reductase preparations for converting 7-hydroxyheptanoate to 7-hydroxyheptanal relative to the empty vector control.
[0070] FIG. 10 is a bar graph of the change in absorbance at 340 nm after 20 minutes, which is a measure of the consumption of NADPH and the activity of three carboxylate reductase preparations for converting N7-acetyl-7-aminoheptanoate to N7-acetyl-7-aminoheptanal relative to the empty vector control.
[0071] FIG. 11 is a bar graph of the change in absorbance at 340 nm after 20 minutes, which is a measure of the consumption of NADPH and activity of a carboxylate reductase preparation for converting pimelate semialdehyde to heptanedial relative to the empty vector control.
[0072] FIG. 12 is a bar graph summarizing the percent conversion of pyruvate to L-alanine (mol/mol) as a measure of the .omega.-transaminase activity of the enzyme only controls (no substrate).
[0073] FIG. 13 is a bar graph of the percent conversion after 4 hours of pyruvate to L-alanine (mol/mol) as a measure of the .omega.-transaminase activity of four .omega.-transaminase preparations for converting 7-aminoheptanoate to pimelate semialdehyde relative to the empty vector control.
[0074] FIG. 14 is a bar graph of the percent conversion after 4 hours of L-alanine to pyruvate (mol/mol) as a measure of the .omega.-transaminase activity of three .omega.-transaminase preparations for converting pimelate semialdehyde to 7-aminoheptanoate relative to the empty vector control.
[0075] FIG. 15 is a bar graph of the percent conversion after 4 hours of pyruvate to L-alanine (mol/mol) as a measure of the .omega.-transaminase activity of six .omega.-transaminase preparations for converting heptamethylenediamine to 7-aminoheptanal relative to the empty vector control.
[0076] FIG. 16 is a bar graph of the percent conversion after 4 hours of pyruvate to L-alanine (mol/mol) as a measure of the .omega.-transaminase activity of six .omega.-transaminase preparations for converting N7-acetyl-1,7-diaminoheptane to N7-acetyl-7-aminoheptanal relative to the empty vector control.
[0077] FIG. 17 is a bar graph of the percent conversion after 4 hours of pyruvate to L-alanine (mol/mol) as a measure of the .omega.-transaminase activity of three .omega.-transaminase preparations for converting 7-aminoheptanol to 7-oxoheptanol relative to the empty vector control.
[0078] FIG. 18 is a bar graph of the production of 3-oxo-7-hydroxyheptanoyl-CoA after 3 hours and 9 hours in a two-step enzymatic reaction catalyzed by a 5-hydroxyvalerate CoA transferase and an enzyme from the thiolase family, relative to the empty vector control.
[0079] FIG. 19 contains a nucleic acid sequence (SEQ ID NO: 38) encoding a Pseudomonas putida thiolase, a nucleic acid sequence (SEQ ID NO: 39) encoding a Sphingomonas wittichii thiolase, a nucleic acid sequence (SEQ ID NO: 40) encoding a Pseudomonas reinekei thiolase, a nucleic acid sequence (SEQ ID NO: 41) encoding a Pseudomonas putida thiolase, a nucleic acid sequence (SEQ ID NO: 42) encoding a Burkholderia xenovorans thiolase, a nucleic acid sequence (SEQ ID NO: 43) encoding a Burkholderia xenovorans thiolase, a nucleic acid sequence (SEQ ID NO: 44) encoding a Rhodococcus jostii thiolase, a nucleic acid sequence (SEQ ID NO: 45) encoding a Bdellovibrio bacteriovorus thiolase, a nucleic acid sequence (SEQ ID NO: 46) encoding a Cronobacter turicensis thiolase, a nucleic acid sequence (SEQ ID NO: 47) encoding an Arthrobacter sp. thiolase, a nucleic acid sequence (SEQ ID NO: 48) encoding a Caulobacter segnis thiolase, a nucleic acid sequence (SEQ ID NO: 49) encoding a Dinoroseobacter shibae thiolase, a nucleic acid sequence (SEQ ID NO: 50) encoding a Burkholderia xenovorans thiolase, a nucleic acid sequence (SEQ ID NO: 51) encoding a Geobacillus kaustophilus thiolase, a nucleic acid sequence (SEQ ID NO: 52) encoding a Beijerinckia indica thiolase, a nucleic acid sequence (SEQ ID NO: 53) encoding a Citrobacter freundii thiolase, a nucleic acid sequence (SEQ ID NO: 54) encoding a Cupriavidus necator thiolase, a nucleic acid sequence (SEQ ID NO: 55) encoding a Gordonia bronchialis thiolase, a nucleic acid sequence (SEQ ID NO: 56) encoding a Burkholderia sp. thiolase, a nucleic acid sequence (SEQ ID NO: 57) encoding a Glutamicibacter arilaitensis thiolase, a nucleic acid sequence (SEQ ID NO: 58) encoding an Escherichia coli thiolase, a nucleic acid sequence (SEQ ID NO: 59) encoding a Cupriavidus necator thiolase, and a nucleic acid sequence (SEQ ID NO: 60) encoding a Clostridium viride 5-hydroxyvalerate CoA transferase.
DETAILED DESCRIPTION
[0080] In general, this document provides enzymes, non-natural pathways, cultivation strategies, feedstocks, host microorganisms and attenuations to the host's biochemical network, for producing 7-hydroxyheptanoate or one or more of pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine, or 1,7-heptanediol, all of which are referred to as C7 building blocks herein. As used herein, the term "central precursor" is used to denote any metabolite in any metabolic pathway shown herein leading to the synthesis of a C7 building block. The term "central metabolite" is used herein to denote a metabolite that is produced in all microorganisms to support growth.
[0081] Host microorganisms described herein can include endogenous pathways that can be manipulated such that 7-hydroxyheptanoate or one or more other C7 building blocks can be produced. In an endogenous pathway, the host microorganism naturally expresses all of the enzymes catalyzing the reactions within the pathway. In one aspect of the invention, 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.
[0082] 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 a 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. 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. 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.
[0083] 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.
[0084] 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 .beta.-ketothiolase: a 3-hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl-CoA reductase, an enoyl-CoA hydratase, a trans-2-enoyl-CoA reductase, a thioesterase, a CoA transferase, an aldehyde dehydrogenase, a monooxygenase, an alcohol dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a co transaminase, a 6-hydroxyhexanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a carboxylate reductase, a deacylase, an N-acetyl transferase, a .omega.-transaminase, or an amidohydrolase. In recombinant hosts expressing a carboxylate reductase, a phosphopantetheinyl transferase also can be expressed as it enhances activity of the carboxylate reductase. In recombinant hosts expressing a monooxygenase, an electron transfer chain protein such as an oxidoreductase or ferredoxin polypeptide also can be expressed.
[0085] For example, a recombinant host can include an exogenous .beta.-ketothiolase and produce 3-oxo-7-hydroxyheptanoyl-CoA, which can be converted to 7-hydroxyheptanoate.
[0086] For example, a recombinant host can include an exogenous enzyme from the thiolase family and produce 3-oxo-7-hydroxyheptanoyl-CoA, which can be converted to 7-hydroxyheptanoate.
[0087] For example, a recombinant host can include an exogenous .beta.-ketothiolase and an exogenous thioesterase or CoA-transferase, and one or more of the following exogenous enzymes: 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoA reductase, an enoyl-CoA hydratase, and a trans-2-enoyl-CoA reductase, and produce 7-hydroxyheptanoate. For example, a recombinant host can include an exogenous .beta.-ketothiolase, an exogenous thioesterase or CoA-transferase, an enoyl-CoA hydratase, an exogenous trans-2-enoyl-CoA reductase, and an exogenous 3-hydroxyacyl-CoA dehydrogenase or an exogenous 3-oxoacyl-CoA reductase, and produce 7-hydroxyheptanoate.
[0088] For example, a recombinant host producing 7-hydroxyheptanoate can include one or more of the following exogenous enzymes: a monooxygenase, an alcohol dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 5-oxovalerate dehydrogenase, a 6-oxohexanoate dehydrogenase, or an aldehyde dehydrogenase, and further produce pimelic acid. For example, a recombinant host producing 7-hydroxyheptanoate can include an exogenous monooxygenase and produce pimelic acid. For example, a recombinant host producing 7-hydroxyheptanoate can include an exogenous 6-hydroxyhexanoate dehydrogenase and an aldehyde dehydrogenase and produce pimelic acid. For example, a recombinant host producing 7-hydroxyheptanoate can include an exogenous alcohol dehydrogenase and one of the following exogenous enzymes: a 5-oxovalerate dehydrogenase, a 6-oxohexanoate dehydrogenase, or a 7-oxoheptanoate dehydrogenase, and produce pimelic acid.
[0089] For example, a recombinant host producing 7-hydroxyheptanoate can include one or more of the following exogenous enzymes: an alcohol dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, or a transaminase, and further produce 7-aminoheptanoate. For example, a recombinant host producing 7-hydroxyheptanoate can include an exogenous alcohol dehydrogenase and an exogenous transaminase and produce 7-aminoheptanoate. For example, a recombinant host producing 7-hydroxyheptanoate can include an exogenous 6-hydroxyhexanoate dehydrogenase and an exogenous transaminase and produce 7-aminoheptanoate.
[0090] For example, a recombinant host producing 7-hydroxyheptanoate can include one or more of the following exogenous enzymes: a carboxylate reductase, a .omega.-transaminase, a deacylase, an N-acetyl transferase, or an alcohol dehydrogenase, and produce heptamethylenediamine. For example, a recombinant host producing 7-hydroxyheptanoate can include an exogenous carboxylate reductase, an exogenous alcohol dehydrogenase, and one or more exogenous transaminases (e.g., one transaminase or two different transaminases), and produce heptamethylenediamine. For example, a recombinant host producing 7-hydroxyheptanoate can include an exogenous carboxylate reductase and one or more exogenous transaminases (e.g., one transaminase or two different transaminases) and produce heptamethylenediamine. For example, a recombinant host producing 7-hydroxyheptanoate can include an exogenous alcohol dehydrogenase, an exogenous carboxylate reductase, and one or more exogenous transaminases (e.g., one transaminase, or two or three different transaminases) and produce heptamethylenediamine. For example, a recombinant host producing 7-hydroxyheptanoate can include an exogenous alcohol dehydrogenase, an exogenous N-acetyl transferase, a carboxylate reductase, a deacylase, and one or more exogenous transaminases (e.g., one transaminase or two different transaminases) and produce heptamethylenediamine.
[0091] For example, a recombinant host producing 7-hydroxyheptanoate can include one or more of the following exogenous enzymes: a carboxylate reductase and an exogenous alcohol dehydrogenase, and further produce 1,7-heptanediol.
[0092] In any of the recombinant hosts, the recombinant host also can include one or more (e.g., one, two, three, or four) of the following exogenous enzymes used to convert 2-oxoadipate- to 5-hydroxypentanoyl-CoA: an alpha-aminotransaminase; a 2-oxoadipate decarboxylase; a branch-chain decarboxylase; a glutamate decarboxylase; a CoA-ligase; a CoA-transferase; a .omega.-transaminase; and an alcohol dehydrogenase. For example, a recombinant host can include an exogenous alpha-aminotransaminase; a glutamate decarboxylase; a CoA-ligase or a CoA-transferase; a .omega.-transaminase; and an alcohol dehydrogenase. For example, a recombinant host can include an exogenous 2-oxoadipate decarboxylase or a branch-chain decarboxylase; a CoA-ligase; a CoA-transferase; and an alcohol dehydrogenase.
[0093] In any of the recombinant hosts, the recombinant host also can include one or more (e.g., one, two, three, or four) of the following exogenous enzymes used to convert manonyl-CoA to 5-hydroxypentanoyl-CoA: a malonyl-CoA reductase; a 3-hydroxypropionate dehydrogenase; a 3-hydroxypropionyl-CoA synthase; a CoA-transferase; a .beta.-ketothiolase; a 3-hydroxyacyl-CoA dehydrogenase; a 3-oxoacyl-CoA reductase; an enoyl-CoA hydratase; and a trans-2-enoyl-CoA reductase. For example, a recombinant host can include an exogenous malonyl-CoA reductase; a 3-hydroxypropionate dehydrogenase; a 3-hydroxypropionyl-CoA synthase; a CoA-transferase; a .beta.-ketothiolase; a 3-hydroxyacyl-CoA dehydrogenase; a 3-oxoacyl-CoA reductase; an enoyl-CoA hydratase; and a trans-2-enoyl-CoA reductase.
[0094] Within an engineered pathway, the enzymes can be from a single source, i.e., from one species or genera, or can be from multiple sources, i.e., different species or genera. 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.
[0095] Any of the enzymes described herein that can be used for production of one or more C7 building blocks can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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) or on the basis of the immature enzyme (e.g., with any signal sequence included). It also will be appreciated that the initial methionine residue may or may not be present on any of the enzyme sequences described herein.
[0096] For example, a .beta.-ketothiolase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Cupriavidus necator (see GenBank Accession No. AAC38322.1, SEQ ID NO: 1) or an Escherichia coli (see GenBank Accession No. AAC74479.1, SEQ ID NO: 13) .beta.-ketothiolase. See FIG. 6.
[0097] For example, a carboxylate reductase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Mycobacterium marinum (see Genbank Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatis (see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), a Segnihparus rugosus (see Genbank Accession No. EFV11917.1, SEQ ID NO: 4), a Mycobacterium massiliense (see Genbank Accession No. EIV11143.1, SEQ ID NO: 5), or a Segnihparus rotundus (see Genbank Accession No. ADG98140.1, SEQ ID NO: 6) carboxylate reductase. See, FIG. 6.
[0098] For example, a .omega.-transaminase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Chromobacterium violaceum (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 7), a Pseudomonas aeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO: 8), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ ID NO: 9), a Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1, SEQ ID NO: 10), an Escherichia coli (see Genbank Accession No. AAA57874.1, SEQ ID NO: 11), or a Vibrio fluvialis (see Genbank Accession No. AEA39183.1, SEQ ID NO: 12) .omega.-transaminase. Some of these .omega.-transaminases are diamine .omega.-transaminases. See, FIG. 6.
[0099] For example, a thiolase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Pseudomonas putida thiolase (see Genbank Accession No. AAN70209.2, SEQ ID NO: 15), a Sphingomonas wittichii thiolase (see Genbank Accession No. ABQ69245.1, SEQ ID NO: 16), a Pseudomonas reinekei thiolase (see Genbank Accession No. ACZ63623.1, SEQ ID NO: 17), a Pseudomonas putida thiolase (see Genbank Accession No. AAA85138.1, SEQ ID NO: 18), a Burkholderia xenovorans thiolase (see Genbank Accession No. ABE28745.1, SEQ ID NO: 19), a Burkholderia xenovorans thiolase (see Genbank Accession No. ABE33819.1, SEQ ID NO: 20), a Rhodococcus jostii thiolase (see Genbank Accession No. ABG94668.1, SEQ ID NO: 21), a Bdellovibrio bacteriovorus thiolase (see Genbank Accession No. CAE79693.1, SEQ ID NO: 22), a Cronobacter turicensis thiolase (see Genbank Accession No. CBA32535.1, SEQ ID NO: 23), an Arthrobacter sp. thiolase (see Genbank Accession No. ABK03524.1, SEQ ID NO: 24), a Caulobacter segnis thiolase (see Genbank Accession No. ADG08907.1, SEQ ID NO: 25), a Dinoroseobacter shibae thiolase (see Genbank Accession No. ABV92581.1, SEQ ID NO: 26), a Burkholderia xenovorans thiolase (see Genbank Accession No. ABE36495.1, SEQ ID NO: 27), a Geobacillus kaustophilus thiolase (see Genbank Accession No. BAD75605.1, SEQ ID NO: 28), a Beijerinckia indica thiolase (see Genbank Accession No. ACB95386.1, SEQ ID NO: 29), a Citrobacter freundii thiolase (see Genbank Accession No. EKS55037.1, SEQ ID NO: 30), a Cupriavidus necator thiolase (see Genbank Accession No. AEI75849.1, SEQ ID NO: 31), a Gordonia bronchialis thiolase (see Genbank Accession No. ACY20886.1, SEQ ID NO: 32), a Burkholderia sp. thiolase (see Genbank Accession No. ADG18081.1, SEQ ID NO: 33), a Glutamicibacter arilaitensis thiolase (see Genbank Accession No. CBT74677.1, SEQ ID NO: 34), an Escherichia coli thiolase (see Genbank Accession No. AAC74479.1, SEQ ID NO: 35), or a Cupriavidus necator thiolase (see Genbank Accession No. AAC38322.1, SEQ ID NO: 36). See FIG. 6.
[0100] For example, a 5-hydroxyvalerate CoA transferase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Clostridium viride 5-hydroxyvalerate CoA transferase (see NCBI Reference Sequence: NZ_KK211198.1, SEQ ID NO: 37). See, FIG. 6.
[0101] The percent identity (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 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 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.
[0102] 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 (homology) 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 (homology) 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.
[0103] 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., bacteria or fungus) is obtained, using appropriate codon bias tables for that species.
[0104] Functional fragments of any of the enzymes described herein can also be used in the methods of the document. 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.
[0105] This document also provides (i) functional variants of the enzymes used in the methods of the document 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. In one aspect of the invention, 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.
[0106] 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., heptahistidine), 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 longer sequences than the full-length target proteins to which the heterologous sequences are attached.
[0107] 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 .beta.-ketothiolase, a dehydrogenase, a synthase, a decarboxylase, a reductase, a hydratase, a thioesterase, a monooxygenase, a thioesterase, amidohydrolase, and transaminase as described herein.
[0108] In addition, the production of 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 sources of the enzymes.
[0109] The reactions of the pathways described herein can be performed in one or more host 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 isolated from of the above types of host cells and used in a purified or semi-purified form. Moreover, such isolates or extracts include lysates (e.g. cell lysates) that can be used as sources of relevant enzymes. In the methods provided by the document, all the steps can be performed in host cells, all the steps can be performed using isolated or extracted enzymes, or some of the steps can be performed in cells and others can be performed using isolated or extracted enzymes.
Enzymes Generating 7-hydroxyheptanoate
[0110] As depicted in FIG. 1, 7-hydroxyheptanaote can be biosynthesized from 2-oxoadipate or malonyl-CoA through the intermediate 3-oxo-7-hydroxyheptanoyl-CoA, which can be produced from 5-hydroxypentanoyl-CoA using a .beta.-ketothiolase. 3-oxo-7-hydroxyheptanoyl-CoA can be converted to 7-hydroxyheptanoate using a 3-hydroxyacyl-CoA dehydrogenase or 3-oxoacyl-CoA dehydrogenase, an enoyl-CoA hydratase, a trans-2-enoyl-CoA reductase, and a thioesterase or a CoA transferase.
[0111] In some embodiments, a .beta.-ketothiolase may be classified under EC 2.3.1.16, such as the gene product of bktB, or may be classified under EC 2.3.1.174 such as the gene product of paaJ. The .beta.-ketothiolase encoded by bktB from Cupriavidus necator accepts acetyl-CoA and butanoyl-CoA as substrates, forming a 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). The .beta.-ketothiolase encoded by paaJ from Escherichia coli accepts succinyl-CoA and acetyl-CoA as substrates, forming a CoA-activated backbone (Nogales et al., Microbiology, 2007, 153, 357-365). See, for example, SEQ ID NO:1 and SEQ ID NO:13 in FIG. 6.
[0112] In some embodiments, a 3-hydroxyacyl-CoA dehydrogenase or 3-oxoacyl-CoA dehydrogenase can be classified under EC 1.1.1.-. For example, the 3-hydroxyacyl-CoA dehydrogenase can be classified under EC 1.1.1.35, such as the gene product of fadB; classified under EC 1.1.1.157, such as the gene product of hbd (also referred to as a 3-hydroxybutyryl-CoA dehydrogenase); or classified under EC 1.1.1.36, such as the acetoacetyl-CoA reductase gene product of phaB (Liu & Chen, Appl. Microbiol. Biotechnol., 2007, 76(5):1153-1159; Shen et al., Appl. Environ. Microbiol., 2011, 77(9):2905-2915; Budde et al., J. Bacteriol., 2010, 192(20):5319-5328).
[0113] In some embodiments, a 3-oxoacyl-CoA reductase can be classified under EC 1.1.1.100, such as the gene product of fabG (Budde et al., J. Bacteriol., 2010, 192(20):5319-5328; Nomura et al., Appl. Environ. Microbiol., 2005, 71(8):4297-4306).
[0114] In some embodiments, an enoyl-CoA hydratase can be classified under EC 4.2.1.17, such as the gene product of crt, or classified under EC 4.2.1.119, such as the gene product of phaJ (Shen et al., 2011, supra; Fukui et al., J. Bacteriol., 1998, 180(3):667-673).
[0115] In some embodiments, a trans-2-enoyl-CoA reductase can be classified under EC 1.3.1.38 or EC 1.3.1.44, such as 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), or EC 1.3.1.8 (Inui et al., Eur. J. Biochem., 1984, 142, 121-126).
[0116] In some embodiments, the terminal carboxyl group leading to the synthesis of 7-hydroxyheptanoate is enzymatically formed in 7-hydroxyheptanoyl-CoA by a thioesterase classified under EC 3.1.2.-, resulting in the production of 7-hydroxyheptanoate. The thioesterase can be the gene product of YciA or Acot13 (Cantu et al., Protein Science, 2010, 19, 1281-1295; Zhuang et al., Biochemistry, 2008, 47(9):2789-2796; Naggert et al., J. Biol. Chem., 1991, 266(17):11044-11050).
[0117] In some embodiments, the terminal carboxyl group leading to the synthesis of 7-hydroxyheptanoate is enzymatically formed in 7-hydroxyheptanoyl-CoA by a CoA-transferase classified under, for example, EC 2.8.3- such as the gene product of cat2 from Clostridium kluyveri, abfT from Clostridium aminobutyricum, or the 4-hydroxybutyrate CoA-transferase from Clostridium viride.
Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis of Pimelic Acid
[0118] As depicted in FIG. 2, the terminal carboxyl group leading to the production of pimelic acid can be enzymatically formed using an aldehyde dehydrogenase, a 5-oxovalerate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, or a monooxygenase.
[0119] In some embodiments, the second terminal carboxyl group leading to the synthesis of pimelic acid can be enzymatically formed in pimelate semialdehyde by an aldehyde dehydrogenase classified under EC 1.2.1.3 (Guerrillot & Vandecasteele, Eur. J. Biochem., 1977, 81, 185-192). See, FIG. 2.
[0120] In some embodiments, the second terminal carboxyl group leading to the synthesis of pimelic acid is enzymatically formed in pimelate semialdehyde by EC 1.2.1.- such as a 5-oxovalerate dehydrogenase classified, for example, under EC 1.2.1.20, such as the gene product of CpnE; a 6-oxohexanoate dehydrogenase classified, for example, EC 1.2.1.63 such as the gene product of ChnE from Acinetobacter sp.; or a 7-oxoheptanoate dehydrogenase such as the gene product of ThnG from Sphingomonas macrogolitabida (Iwaki et al., Appl. Environ. Microbiol., 1999, 65(11), 5158-5162; Lopez-Sanchez et al., Appl. Environ. Microbiol., 2010, 76(1), 110-118)). See, FIG. 2.
[0121] In some embodiments, the second terminal carboxyl group leading to the synthesis of pimelic acid is enzymatically formed in pimelate semialdehyde by a monooxygenase in the cytochrome P450 family such as CYP4F3B (see, e.g., Sanders et al., J. Lipid Research, 2005, 46(5):1001-1008; Sanders et al., The FASEB Journal, 2008, 22(6):2064-2071). See, FIG. 2.
Enzymes Generating the Terminal Amine Groups in the Biosynthesis of Heptamethylenediamine or 7-aminoheptanoate
[0122] As depicted in FIG. 3 and FIG. 4, terminal amine groups can be enzymatically formed using a .omega.-transaminase or a deacylase.
[0123] In some embodiments, a terminal amine group leading to the synthesis of 7-aminoheptanoic acid is enzymatically formed in pimelate semialdehyde by a co-transaminase classified, for example, under EC 2.6.1.- (e.g., 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 that obtained from Chromobacterium violaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO: 7), Pseudomonas aeruginosa (Genbank Accession No. AAG08191.1, SEQ ID NO: 8), Pseudomonas syringae (Genbank Accession No. AAY39893.1, SEQ ID NO: 9), Rhodobacter sphaeroides (Genbank Accession No. ABA81135.1, SEQ ID NO: 10), Vibrio fluvialis (Genbank Accession No. AEA39183.1, SEQ ID NO: 12), Streptomyces griseus, or Clostridium viride. See, FIG. 3.
[0124] An additional .omega.-transaminase that can be used in the methods and hosts described herein is from Escherichia coli (Genbank Accession No. AAA57874.1, SEQ ID NO: 11). Some of the .omega.-transaminases classified, for example, under EC 2.6.1.29 or EC 2.6.1.82 are diamine .omega.-transaminases (e.g., SEQ ID NO: 11).
[0125] The reversible .omega.-transaminase from Chromobacterium violaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO: 7) has demonstrated analogous activity accepting 7-aminoheptanoic acid as an amino donor, thus forming the first terminal amine group in pimelate semialdehyde (Kaulmann et al., Enzyme and Microbial Technology, 2007, 41, 628-637).
[0126] The reversible 4-aminobubyrate:2-oxoadipate transaminase from Streptomyces griseus has demonstrated activity for the conversion of 7-aminoheptanoate to pimelate semialdehyde (Yonaha et al., Eur. J. Biochem., 1985, 146, 101-106).
[0127] The reversible 5-aminovalerate transaminase from Clostridium viride has demonstrated activity for the conversion of 7-aminoheptanoate to pimelate semialdehyde (Barker et al., J. Biol. Chem., 1987, 262(19), 8994-9003).
[0128] In some embodiments, the second terminal amine group leading to the synthesis of heptamethylenediamine is enzymatically formed in 7-aminoheptanal by a diamine transaminase classified, for example, under EC 2.6.1.29; or classified, for example, under EC 2.6.1.82, such as the gene product of YgjG from E. coli (Genbank Accession No. AAA57874.1, SEQ ID NO: 12). The transaminases set forth in SEQ ID NOs:7-10 and 11 also can be used to produce heptamethylenediamine. See, FIG. 4.
[0129] The gene product of ygjG accepts a broad range of diamine carbon chain length substrates, such as putrescine, cadaverine, and spermidine (Samsonova et al., BMC Microbiology, 2003, 3:2).
[0130] The diamine transaminase from E. coli strain B has demonstrated activity for 1,7 diaminoheptane (Kim, The Journal of Chemistry, 1964, 239(3), 783-786).
[0131] In some embodiments, the second terminal amine group leading to the synthesis of heptamethylenediamine is enzymatically formed in N7-acetyl-1,7-diaminoheptane by a deacylase classified, for example, under EC 3.5.1.17 such as an acyl lysine deacylase.
Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis of 1,7 Heptanediol
[0132] As depicted in FIG. 5, the terminal hydroxyl group can be enzymatically formed using an alcohol dehydrogenase. For example, the second terminal hydroxyl group leading to the synthesis of 1,7 heptanediol can be enzymatically formed in 7-hydroxyheptanal by an alcohol dehydrogenase classified under EC 1.1.1.- (e.g., EC 1.1.1.1, 1.1.1.2, 1.1.1.21, or 1.1.1.184) such as the gene product of YMR318C or YqhD (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.
Biochemical Pathways
[0133] Pathways to 7-hydroxyheptanoate
[0134] In some embodiments, 5-hydroxypentanoyl-CoA is synthesized from the central metabolite, 2-oxoadipate, by conversion of 2-oxoadipate to 2-aminoadipate by a .alpha.-aminotransaminase classified, for example, under EC 2.6.1.39; followed by conversion of 2-aminoadipate to 5-aminopentanoate by a glutamate decarboxylase classified, for example, under EC 4.1.1.15; followed by conversion of 5-aminopentanoate to 5-oxopentanoate--by a .omega.-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, or EC 2.6.1.96 such as the gene product of gabT from Escherichia coli (Bartsch et al., J. Bacteriol., 1990, 172(12), 7035); followed by conversion of 5-oxopentanoate to 5-hydroxypentanoate by an alcohol dehydrogenase classified, for example, under EC 1.1.1.61 such as the gene product of gbd (e.g., from Sorangium cellulosum), gabD (Bartsch et al., J. Bacteriol., 1990, 172(12), 7035), or YihU (Saito et al., J. Biol. Chem., 2009, 284(24), 16442-16452), or a 5-hydroxyvalerate dehydrogenase such as the gene product of cpnD (see, for example, Iwaki et al., 2002, Appl. Environ. Microbiol., 68(11):5671-5684); followed by conversion of 5-hydroxypentanoate to 5-hydroxypentanoyl-CoA using a CoA-ligase classified under, for example, EC 6.2.1- (e.g., EC 6.2.1.40), or a CoA-transferase classified under, for example, EC 2.8.3.- such as the gene product of cat2 from Clostridium kluyveri, abfT from Clostridium aminobutyricum, or the 4-hydroxybutyrate CoA-transferase from Clostridium viride. See FIG. 1.
[0135] In some embodiments, 2-oxoadipate is converted to 5-oxopentanoate --using a 2-oxoadipate decarboxylase classified, for example, under EC 4.1.1.71 or a branch-chain decarboxylase classified, for example, under EC 4.1.1.72 such as the gene product of kdcA or kivD. 5-oxopentanoate--produced in this fashion can be converted to 5-hydroxypentanoyl-CoA as described above. See, FIG. 1.
[0136] In some embodiments, 5-hydroxypentanoyl-CoA is synthesized from the central metabolite, malonyl-CoA, by conversion of malonyl-CoA to malonate semialdehyde by a malonyl-CoA reductase classified, for example, under EC 1.2.1.75; followed by conversion of malonate semialdehyde to 3-hydroxypropanoate by a 3-hydroxypropionate dehydrogenase classified, for example, under EC 1.1.1.59; followed by conversion of 3-hydroxypropanoate to 3-hydroxypropanoyl-CoA by a 3-hydroxypropionyl-CoA synthase classified, for example, under EC 6.2.1.36, or a CoA-transferase classified, for example, under EC 2.8.3.1; followed by conversion of 3-hydropropanoyl-CoA to 3-oxo-5-hydroxypentanoyl-CoA using a .beta.-ketothiolase classified, for example, under EC 2.3.1.16 or EC 2.3.1.174 such as the gene product of bktB or paaJ (e.g., SEQ ID NO: 1 or 13); followed by conversion to 3-hydroxy-5-hydroxypentanoyl-CoA using a 3-hydroxyacyl-CoA dehydrogenase classified, for example, under EC 1.1.1.- such as EC 1.1.1.35 (e.g., the gene product of fadB), EC 1.1.1.36 (e.g., the gene product of phaB), or EC 1.1.1.157 (e.g., the gene product of hbd), or a 3-oxoacyl-CoA reductase classified, for example, under EC 1.1.1.100, such as the gene product of fabG; followed by conversion of 3-hydroxy-5-hydroxypentanoyl-CoA to 2,3-dehydro-5-hydroxypentanoyl-CoA using an enoyl-CoA hydratase 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 the gene product of phal; followed by conversion of 2,3-dehydro-5-hydroxypentanoyl-CoA to 5-hydroxypentanoyl-CoA by a trans-2-enoyl-CoA reductase classified, for example, under EC 1.3.1.38, EC 1.3.1.44, or EC 1.3.1.8 such as the gene product of ter or tdter. See FIG. 1.
[0137] In some embodiments, 7-hydroxyheptanoate is synthesized from the central precursor, 5-hydroxypentanoyl-CoA, by conversion of 5-hydroxypentanoyl-CoA to 3-oxo-7-hydroxyheptanoyl-CoA using .beta.-ketothiolase classified, for example, under EC 2.3.1.16 or EC 2.3.1.174 such as the gene product of bktB or paaJ (e.g., SEQ ID NO: 1 or 13); followed by conversion to 3-hydroxy-7-hydroxyheptanoyl-CoA using a 3-hydroxyacyl-CoA dehydrogenase classified, for example, under EC 1.1.1.- such as EC 1.1.1.35 (e.g., the gene product of fadB), EC 1.1.1.36 (e.g., the gene product of phaB), or EC 1.1.1.157 (e.g., the gene product of hbd), or a 3-oxoacyl-CoA reductase classified, for example, under EC 1.1.1.100 such as the gene product of fabG; followed by conversion of 3-hydroxy-7-hydroxyheptanoyl-CoA to 2,3-dehydro-7-hydroxyheptanoyl-CoA using an enoyl-CoA hydratase 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 the gene product of phal; followed by conversion of 2,3-dehydro-7-hydroxyheptanoyl-CoA to 7-hydroxyheptanoyl-CoA by a trans-2-enoyl-CoA reductase classified, for example, under EC 1.3.1.38, EC 1.3.1.44, or EC 1.3.1.8 such as the gene product of ter or tdter; followed by conversion of 7-hydroxyheptanoyl-CoA to 7-hydroxyheptanoate by a thioesterase classified, for example, under EC 3.1.2.- such as the gene product of YciA or Acot13, or a CoA-transferase classified, for example, under EC 2.8.3.-. See FIG. 1.
Pathways Using 7-hydroxyheptanoate as Central Precursor to Pimelic Acid
[0138] In some embodiments, pimelic acid is synthesized from 7-hydroxyheptanoate, by conversion of 7-hydroxyheptanoate to pimelate semialdehyde by an alcohol dehydrogenase classified under EC 1.1.1.- such as 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), cpnD (Iwaki et al., 2002, Appl. Environ. Microbiol., 68(11):5671-5684), or gabD (Lutke-Eversloh & Steinbuchel, 1999, FEMS Microbiology Letters, 181(1):63-71), or a 6-hydroxyheptanoate dehydrogenase classified, for example, under EC 1.1.1.258 such as the gene product of ChnD (Iwaki et al., Appl. Environ. Microbiol., 1999, 65(11):5158-5162); followed by conversion of pimelate semialdehyde to pimelic acid by a dehydrogenase classified, for example, under EC 1.2.1.- such as a 7-oxoheptanoate dehydrogenase (e.g., the gene product of ThnG), a 7-oxoheptanoate dehydrogenase (e.g., the gene product of ChnE), a glutarate semialdehyde dehydrogenase classified, for example, under EC 1.2.1.20, a 5-oxovalerate dehydrogenase such as the gene product of CpnE, or an aldehyde dehydrogenase classified under EC 1.2.1.3. See FIG. 2. The alcohol dehydrogenase encoded by YMR318C has broad substrate specificity, including the oxidation of C7 alcohols.
[0139] In some embodiments, pimelic acid is synthesized from the central precursor, 7-hydroxyheptanoate, by conversion of 7-hydroxyheptanoate to pimelate semialdehyde by a cytochrome P450 (Sanders et al., J. Lipid Research, 2005, 46(5), 1001-1008; Sanders et al., The FASEB Journal, 2008, 22(6), 2064-2071); followed by conversion of pimelate semialdehyde to pimelic acid by a monooxygenase in the cytochrome P450 family such as CYP4F3B. See FIG. 2.
Pathway Using 7-hydroxyheptanoate as Central Precursor to 7-aminoheptanoate-
[0140] In some embodiments, 7-aminoheptanoate is synthesized from the central precursor, 7-hydroxyheptanoate, by conversion of 7-hydroxyheptanoate to pimelate semialdehyde by an alcohol dehydrogenase classified, for example, under EC 1.1.1.2 such as the gene product of YMR318C, a 6-hydroxyhexanoate dehydrogenase classified, for example, under EC 1.1.1.258, a 5-hydroxypentanoate dehydrogenase classified, for example, under EC 1.1.1.- such as the gene product of cpnD, or a 4-hydroxybutyrate dehydrogenase classified, for example, under EC 1.1.1.- such as the gene product of gabD; followed by conversion of pimelate semialdehyde to 7-aminoheptanoate by a co-transaminase (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 one of SEQ ID NOs:7-10 or 12, see above). See FIG. 3.
Pathway using 7-aminoheptanoate, 7-hydroxyheptanoate, Pimelate Semialdehyde, or 1,7-heptanediol as a Central Precursor to Heptamethylenediamine
[0141] 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 the gene product of car 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 products of GriC and GriD from Streptomyces griseus (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion of 7-aminoheptanal to heptamethylenediamine by a .omega.-transaminase such as a .omega.-transaminase in EC 2.6.1.-, (e.g., EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, EC 2.6.1.82 such as SEQ ID NOs:7-12). 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 massiliense (Genbank Accession No. EIV11143.1, SEQ ID NO: 5), or Segniliparus rotundus (Genbank Accession No. ADG98140.1, SEQ ID NO: 6). See FIG. 4.
[0142] The carboxylate reductase encoded by the gene product of car and 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).
[0143] In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-hydroxyheptanoate (which can be produced as described in FIG. 1), by conversion of 7-hydroxyheptanoate to 7-hydroxyheptanal by a carboxylate reductase classified, for example, under EC 1.2.99.6 such as the gene product of car (see above) 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 & GriD (Suzuki et al., 2007, supra); followed by conversion of 7-hydroxyheptanal 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 SEQ ID NOs:7-12, 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 the gene product of YMR318C or YqhD (Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002, Biochem 361(Pt 1), 163-172; Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the protein having GenBank Accession No. CAA81612.1; 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 SEQ ID NOs:7-12, see above. See FIG. 4.
[0144] In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoate to N7-acetyl-7-aminoheptanoate by an 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 the gene product of car (see above, e.g., SEQ ID NO: 4, 5, or 6) 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 & 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, or EC 2.6.1.82 such as SEQ ID NOs:7-12, see above; followed by conversion to heptamethylenediamine by an acyl lysine deacylase classified, for example, under EC 3.5.1.17. See, FIG. 4.
[0145] 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 the gene product of car (see above, e.g., SEQ ID NO:6) 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 & GriD; followed by conversion to 7-aminoheptanal 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; 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 SEQ ID NOs:7-12. See FIG. 4.
[0146] In some embodiments, heptamethylenediamine is synthesized from 1,7-heptanediol by conversion of 1,7-heptanedion to 7-hydroxyheptanal using 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 the gene product of YMR318C or YqhD or the protein having GenBank Accession No. CAA81612.1; followed by conversion 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 SEQ ID NOs:7-12, 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 the gene product of YMR318C or YqhD or the protein having GenBank Accession No. CAA81612.1, 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 SEQ ID NOs:7-12. See FIG. 4.
Pathways Using 7-hydroxyheptanoate as Central Precursor to 1,7-heptanediol
[0147] 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 the gene product of car (see above, e.g., SEQ ID NO: 2, 3, 4, 5, or 6) 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 products of GriC and GriD from Streptomyces griseus (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion of 7-hydroxyheptanal to 1,7 heptanediol by an alcohol dehydrogenase (classified, for example, under EC 1.1.1.- such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the gene product of YMR318C or YqhD (from E. coli, GenBank Accession No. AAA69178.1) (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. 5.
Cultivation Strategy
[0148] In some embodiments, one or more C7 building blocks are biosynthesized in a recombinant host using anaerobic, aerobic, or micro-aerobic cultivation conditions. In some embodiments, the cultivation strategy entails nutrient limitation such as nitrogen, phosphate, or oxygen limitation.
[0149] 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.
[0150] 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.
[0151] In some embodiments, the biological feedstock can be 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.
[0152] 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 hpolytica (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).
[0153] 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, 2011, supra; Martin and Prather, J. Biotechnol., 2009, 139:61-67).
[0154] 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).
[0155] 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).
[0156] 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 microorganisms such as Escherichia coli, Corynebacterium glutamicum, Lactobacillus delbrueckii, and Lactococcus lactis (see, e.g., Hermann et al, J. Biotechnol., 2003, 104:155-172; Wee et al., Food Technol. Biotechnol., 2006, 44(2):163-172; Ohashi et al., J. Bioscience and Bioengineering, 1999, 87(5):647-654).
[0157] 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).
[0158] In some embodiments, the non-biological feedstock can be or can derive from natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, benzoate, non-volatile residue (NVR) or a caustic wash waste stream from cycloheptane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.
[0159] The efficient catabolism of methanol has been demonstrated for the methylotrophic yeast Pichia pastoris.
[0160] The efficient catabolism of ethanol has been demonstrated for Clostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008, 105(6) 2128-2133).
[0161] 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).
[0162] The efficient catabolism of syngas has been demonstrated for numerous microorganisms, such as Clostridium ljungdahlii and Clostridium autoethanogenum (Kopke et al., Applied and Environmental Microbiology, 2011, 77(15):5467-5475).
[0163] The efficient catabolism of the non-volatile residue waste stream from cycloheptane 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).
[0164] 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.
[0165] 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
[0166] The present document 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, 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.
[0167] 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, ten, or more of such steps, can be performed within a recombinant host. This document 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, 12 or more) recombinant forms of any of the enzymes recited in the document. 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.
[0168] In addition, this document 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.
[0169] Also, this document 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.
[0170] This document also recognizes that where an enzyme is shown to accept a particular co-factor, such as NADPH, or 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 document 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.
[0171] 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.
[0172] In some embodiments, the enzymes in the pathways outlined here can be gene dosed, i.e., overexpressed, into the resulting genetically modified organism via episomal or chromosomal integration approaches.
[0173] 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.
[0174] Attenuation strategies include, but are not limited to the use of transposons, homologous recombination (double cross-over approach), mutagenesis, enzyme inhibitors, and RNAi interference.
[0175] In some embodiments, fluxomic, metabolomic, 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.
[0176] 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.
[0177] 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 5-hydroxypentanoyl-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/central precursors leading to and including one or more C7 building blocks, and/or (4) ensure efficient efflux from the cell.
[0178] In some embodiments requiring intracellular availability of acetyl-CoA for C7 building block synthesis, endogenous enzymes catalyzing the hydrolysis of acetyl-CoA such as short-chain length thioesterases can be attenuated in the host organism.
[0179] 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).
[0180] 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.
[0181] 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 lactate dehydrogenase encoded by ldhA can be attenuated (Shen et al., 2011, supra).
[0182] In some embodiments, enzymes that catalyze anapleurotic reactions such as PEP carboxylase and/or pyruvate carboxylase can be overexpressed in the host organism.
[0183] 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 phophoenolpyruvate to succinate such as frdBC can be attenuated (see, e.g., Shen et al., 2011, supra).
[0184] 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).
[0185] 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).
[0186] In some embodiments, where pathways require excess NADH co-factor for C7 building block synthesis, a recombinant NADH-consuming transhydrogenase can be attenuated.
[0187] In some embodiments, an endogenous gene encoding an enzyme that catalyzes the degradation of pyruvate to ethanol such as pyruvate decarboxylase can be attenuated.
[0188] 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).
[0189] 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).
[0190] In some embodiments, carbon flux can be redirected into the pentose phosphate cycle to increase the supply of NADPH by overexpression of a 6-phosphogluconate dehydrogenase and/or a transketolase (Lee et al., 2003, Biotechnology Progress, 19(5), 1444-1449).
[0191] 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).
[0192] 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).
[0193] 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).
[0194] 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 zwf can be overexpressed in the host organisms (Lim et al., J. Bioscience and Bioengineering, 2002, 93(6), 543-549).
[0195] 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).
[0196] 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.
[0197] 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).
[0198] 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).
[0199] In some embodiments, an endogenous glutamate dehydrogenase (EC 1.4.1.3) that utilizes both NADH and NADPH as co-factors can be attenuated.
[0200] In some embodiments, a membrane-bound cytochrome P450 such as CYP4F3B can be solubilized by only expressing the cytosolic domain and not the N-terminal region that anchors the P450 to the endoplasmic reticulum (Scheller et al., J. Biol. Chem., 1994, 269(17): 12779-12783).
[0201] In some embodiments, an enoyl-CoA reductase can be solubilized via expression as a fusion protein with a small soluble protein, for example, the maltose binding protein (Gloerich et al., FEBS Letters, 2006, 580, 2092-2096).
[0202] In some embodiments using hosts that naturally accumulate polyhydroxyalkanoates, the endogenous polymer synthase enzymes can be attenuated in the host strain.
[0203] 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.
[0204] In some embodiments, a L-glutamate dehydrogenase, a L-glutamine synthetase, or a alpha-aminotransaminase can be overexpressed in the host to regenerate L-glutamate from 2-oxoadipate as an amino donor for .omega.-transaminase reactions.
[0205] In some embodiments, enzymes such as a pimeloyl-CoA dehydrogenase classified under, EC 1.3.1.62; an acyl-CoA dehydrogenase classified, for example, under EC 1.3.8.7, EC 1.3.8.1, or EC 1.3.99.-; and/or a butyryl-CoA dehydrogenase classified, for example, under EC 1.3.8.6 that degrade central metabolites and central precursors leading to and including C7 building blocks can be attenuated.
[0206] In some embodiments, endogenous enzymes activating C7 building blocks via Coenzyme A esterification such as CoA-ligases (e.g., an adipyl-CoA synthetase) classified under, for example, EC 6.2.1.- can be attenuated.
[0207] 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.
[0208] 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).
[0209] 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).
[0210] 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
[0211] 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, 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.
[0212] 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.
[0213] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1
[0214] Enzyme Activity of .omega.-transaminase Using Pimelate Semialdehyde as Substrate and Forming 7-aminoheptanoate
[0215] A nucleotide sequence encoding an N-terminal His-tag was added to the nucleic acid sequences from Chromobacterium violaceum, Pseudomonas syringae, Rhodobacter sphaeroides, and Vibrio fluvialis encoding the .omega.-transaminases of SEQ ID NOs: 7, 9, 10, and 12, respectively (see FIG. 6) such that N-terminal HIS tagged .omega.-transaminases could be produced. Each of the resulting modified genes was cloned into a pET21a expression vector under control of the T7 promoter and each expression vector was transformed into a BL21[DE3] E. coli host. The resulting recombinant E. coli strains were cultivated at 37.degree. C. in a 250 mL shake flask culture containing 50 mL LB media and antibiotic selection pressure, with shaking at 230 rpm. Each culture was induced overnight at 16.degree. C. using 1 mM IPTG.
[0216] The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and lysed via sonication. The cell debris was separated from the supernatant via centrifugation and the cell free extract was used immediately in enzyme activity assays.
[0217] Enzyme activity assays in the reverse direction (i.e., 7-aminoheptanoate to pimelate semialdehyde) were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM 7-aminoheptanoate, 10 mM pyruvate, and 100 .mu.M pyridoxyl 5' phosphate. Each enzyme activity assay reaction was initiated by adding cell free extract of the .omega.-transaminase gene product or the empty vector control to the assay buffer containing the 7-aminoheptanoate and incubated at 25.degree. C. for 4 h, with shaking at 250 rpm. The formation of L-alanine from pyruvate was quantified via RP-HPLC.
[0218] Each enzyme only control without 7-aminoheptanoate demonstrated low base line conversion of pyruvate to L-alanine. See FIG. 12. The gene products of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 12 accepted 7-aminoheptanote as substrate as confirmed against the empty vector control. See FIG. 13.
[0219] Enzyme activity in the forward direction (i.e., pimelate semialdehyde to 7-aminoheptanoate) was confirmed for the transaminases of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 12. Enzyme activity assays were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM pimelate semialdehyde, 10 mM L-alanine, and 100 .mu.M pyridoxyl 5' phosphate. Each enzyme activity assay reaction was initiated by adding a cell free extract of the .omega.-transaminase gene product or the empty vector control to the assay buffer containing the pimelate semialdehyde and incubated at 25.degree. C. for 4 h, with shaking at 250 rpm. The formation of pyruvate was quantified via RP-HPLC.
[0220] The gene products of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 12 accepted pimelate semialdehyde as substrate as confirmed against the empty vector control. See FIG. 14. The reversibility of the .omega.-transaminase activity was confirmed, demonstrating that the .omega.-transaminases of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 12 accepted pimelate semialdehyde as substrate and synthesized 7-aminoheptanoate as a reaction product.
Example 2
Enzyme Activity of Carboxylate Reductase Using Pimelate as Substrate and Forming Pimelate Semialdehyde
[0221] A nucleotide sequence encoding a HIS-tag was added to the nucleic acid sequences from Segniliparus rugosus and Segniliparus rotundus that encode the carboxylate reductases of SEQ ID NOs: 4 (EFV11917.1) and 6 (ADG98140.1), respectively (see FIG. 6), such that N-terminal HIS tagged carboxylate reductases could be produced. Each of the modified genes was cloned into a pET Duet expression vector along with a sfp gene encoding a HIS-tagged phosphopantetheine transferase from Bacillus subtilis, both under the T7 promoter. Each expression vector was transformed into a BL21[DE3] E. coli host and the resulting recombinant E. coli strains were cultivated at 37.degree. C. in a 250 mL shake flask culture containing 50 mL LB media and antibiotic selection pressure, with shaking at 230 rpm. Each culture was induced overnight at 37.degree. C. using an auto-induction media.
[0222] The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and lysed via sonication, and the cell debris was separated from the supernatant via centrifugation. The carboxylate reductases and phosphopantetheine transferases were purified from the supernatant using Ni-affinity chromatography, diluted 10-fold into 50 mM HEPES buffer (pH=7.5), and concentrated via ultrafiltration.
[0223] Enzyme activity assays (i.e., from pimelate to pimelate semialdehyde) were performed in triplicate in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 2 mM pimelate, 10 mM MgCl.sub.2, 1 mM ATP, and 1 mM NADPH. Each enzyme activity assay reaction was initiated by adding purified carboxylate reductase and phosphopantetheine transferase gene products or the empty vector control to the assay buffer containing the pimelate and then incubated at room temperature for 20 min. The consumption of NADPH was monitored by absorbance at 340 nm. Each enzyme only control without pimelate demonstrated low base line consumption of NADPH. See bars for EFV11917.1 and ADG98140.1 in FIG. 7.
[0224] The gene products of SEQ ID NO: 4 (EFV11917.1) and SEQ ID NO: 6 (ADG98140.1), enhanced by the gene product of sfp, accepted pimelate as substrate, as confirmed against the empty vector control (see FIG. 8), and synthesized pimelate semialdehyde.
Example 3
[0225] Enzyme Activity of Carboxylate Reductase Using 7-hydroxyheptanoate as Substrate and Forming 7-hydroxyheptanal
[0226] A nucleotide sequence encoding a His-tag was added to the nucleic acids from Mycobacterium marinum, Mycobacterium smegmatis, Segniliparus rugosus, Mycobacterium abscessus subsp. bolletii, Segnihparus rotundus, and Mycobacterium smegmatis that encode the carboxylate reductases of SEQ ID NOs: 2-6 and 14, respectively (GenBank Accession Nos. ACC40567.1, ABK71854.1, EFV11917.1, EIV11143.1, ADG98140.1, and ABK75684.1, respectively) (see FIG. 6) such that N-terminal HIS tagged carboxylate reductases could be produced. Each of the modified genes was cloned into a pET Duet expression vector alongside a sfp gene encoding a His-tagged phosphopantetheine transferase from Bacillus subtilis, both under control of the T7 promoter. Each expression vector was transformed into a BL21[DE3] E. coli host along with the expression vectors from Example 3. Each resulting recombinant E. coli strain was cultivated at 37.degree. C. in a 250 mL shake flask culture containing 50 mL LB media and antibiotic selection pressure, with shaking at 230 rpm. Each culture was induced overnight at 37.degree. C. using an auto-induction media.
[0227] The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and lysed via sonication. The cell debris was separated from the supernatant via centrifugation. The carboxylate reductases and phosphopantetheine transferase were purified from the supernatant using Ni-affinity chromatography, diluted 10-fold into 50 mM HEPES buffer (pH=7.5), and concentrated via ultrafiltration.
[0228] Enzyme activity (i.e., 7-hydroxyheptanoate to 7-hydroxyheptanal) assays were performed in triplicate in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 2 mM 7-hydroxyheptanal, 10 mM MgCl.sub.2, 1 mM ATP, and 1 mM NADPH. Each enzyme activity assay reaction was initiated by adding purified carboxylate reductase and phosphopantetheine transferase or the empty vector control to the assay buffer containing the 7-hydroxyheptanoate and then incubated at room temperature for 20 min. The consumption of NADPH was monitored by absorbance at 340 nm. Each enzyme only control without 7-hydroxyheptanoate demonstrated low base line consumption of NADPH. See FIG. 7.
[0229] The gene products of SEQ ID NOs: 2-6 and 14, enhanced by the gene product of sfp, accepted 7-hydroxyheptanoate as substrate as confirmed against the empty vector control (see FIG. 9), and synthesized 7-hydroxyheptanal.
Example 4
[0230] Enzyme Activity of .omega.-transaminase for 7-aminoheptanol, Forming 7-oxoheptanol
[0231] A nucleotide sequence encoding an N-terminal His-tag was added to the Chromobacterium violaceum, Pseudomonas syringae, and Rhodobacter sphaeroides nucleic acids encoding the .omega.-transaminases of SEQ ID NOs: 7, 9 and 10, respectively (see FIG. 6), such that N-terminal HIS tagged .omega.-transaminases could be produced. The modified genes were cloned into a pET21a expression vector under the T7 promoter. Each expression vector was transformed into a BL21[DE3] E. coli host. Each resulting recombinant E. coli strain was cultivated at 37.degree. C. in a 250 mL shake flask culture containing 50 mL LB media and antibiotic selection pressure, with shaking at 230 rpm. Each culture was induced overnight at 16.degree. C. using 1 mM IPTG.
[0232] The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and lysed via sonication. The cell debris was separated from the supernatant via centrifugation and the cell free extract was used immediately in enzyme activity assays.
[0233] Enzyme activity assays in the reverse direction (i.e., 7-aminoheptanol to 7-oxoheptanol) were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM 7-aminoheptanol, 10 mM pyruvate, and 100 .mu.M pyridoxyl 5' phosphate. Each enzyme activity assay reaction was initiated by adding cell free extract of the .omega.-transaminase gene product or the empty vector control to the assay buffer containing the 7-aminoheptanol and then incubated at 25.degree. C. for 4 h, with shaking at 250 rpm. The formation of L-alanine was quantified via RP-HPLC.
[0234] Each enzyme only control without 7-aminoheptanol had low base line conversion of pyruvate to L-alanine. See FIG. 12.
[0235] The gene products of SEQ ID NOs: 7, 9 & 10 accepted 7-aminoheptanol as substrate as confirmed against the empty vector control (see FIG. 17) and synthesized 7-oxoheptanol as reaction product. Given the reversibility of the .omega.-transaminase activity (see Example 1), it can be concluded that the gene products of SEQ ID NOs: 7, 9 & 10 accept 7-oxoheptanol as substrate and form 7-aminoheptanol.
Example 5
[0236] Enzyme Activity of .omega.-transaminase Using Heptamethylenediamine as Substrate and Forming 7-aminoheptanal
[0237] A nucleotide sequence encoding an N-terminal His-tag was added to the Chromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae, Rhodobacter sphaeroides, Escherichia coli, and Vibrio fluvialis nucleic acids encoding the .omega.-transaminases of SEQ ID NOs: 7-12, respectively (see FIG. 6) such that N-terminal HIS tagged .omega.-transaminases could be produced. The modified genes were cloned into a pET21a expression vector under the T7 promoter. Each expression vector was transformed into a BL21[DE3] E. coli host. Each resulting recombinant E. coli strain was cultivated at 37.degree. C. in a 250 mL shake flask culture containing 50 mL LB media and antibiotic selection pressure, with shaking at 230 rpm. Each culture was induced overnight at 16.degree. C. using 1 mM IPTG.
[0238] The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and lysed via sonication. The cell debris was separated from the supernatant via centrifugation and the cell free extract was used immediately in enzyme activity assays.
[0239] Enzyme activity assays in the reverse direction (i.e., heptamethylenediamine to 7-aminoheptanal) were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM heptamethylenediamine, 10 mM pyruvate, and 100 .mu.M pyridoxyl 5' phosphate. Each enzyme activity assay reaction was initiated by adding cell free extract of the .omega.-transaminase gene product or the empty vector control to the assay buffer containing the heptamethylenediamine and then incubated at 25.degree. C. for 4 h, with shaking at 250 rpm. The formation of L-alanine was quantified via RP-HPLC.
[0240] Each enzyme only control without heptamethylenediamine had low base line conversion of pyruvate to L-alanine. See FIG. 12.
[0241] The gene products of SEQ ID NOs: 7-12 accepted heptamethylenediamine as substrate as confirmed against the empty vector control (see FIG. 15) and synthesized 7-aminoheptanal as reaction product. Given the reversibility of the .omega.-transaminase activity (see Example 1), it can be concluded that the gene products of SEQ ID NOs: 7-12 accept 7-aminoheptanal as substrate and form heptamethylenediamine.
Example 6
[0242] Enzyme Activity of Carboxylate Reductase for N7-acetyl-7-aminoheptanoate, Forming N7-acetyl-7-aminoheptanal
[0243] The activity of each of the N-terminal His-tagged carboxylate reductases of SEQ ID NOs: 3, 5, and 6 (see Examples 2 and 3, and FIG. 6) for converting N7-acetyl-7-aminoheptanoate to N7-acetyl-7-aminoheptanal was assayed in triplicate in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 2 mM N7-acetyl-7-aminoheptanoate, 10 mM MgCl.sub.2, 1 mM ATP, and 1 mM NADPH. The assays were initiated by adding purified carboxylate reductase and phosphopantetheine transferase or the empty vector control to the assay buffer containing the N7-acetyl-7-aminoheptanoate then incubated at room temperature for 20 min. The consumption of NADPH was monitored by absorbance at 340 nm. Each enzyme only control without N7-acetyl-7-aminoheptanoate demonstrated low base line consumption of NADPH. See FIG. 7.
[0244] The gene products of SEQ ID NOs: 3, 5, and 6, enhanced by the gene product of sfp, accepted N7-acetyl-7-aminoheptanoate as substrate as confirmed against the empty vector control (see FIG. 10), and synthesized N7-acetyl-7-aminoheptanal.
Example 7
[0245] Enzyme Activity of .omega.-transaminase Using N7-acetyl-1,7-diaminoheptane, and Forming N7-acetyl-7-aminoheptanal
[0246] The activity of the N-terminal His-tagged .omega.-transaminases of SEQ ID NOs: 7-12 (see Example 5, and FIG. 6) for converting N7-acetyl-1,7-diaminoheptane to N7-acetyl-7-aminoheptanal was assayed using a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM N7-acetyl-1,7-diaminoheptane, 10 mM pyruvate, and 100 .mu.M pyridoxyl 5' phosphate. Each enzyme activity assay reaction was initiated by adding a cell free extract of the .omega.-transaminase or the empty vector control to the assay buffer containing the N7-acetyl-1,7-diaminoheptane then incubated at 25.degree. C. for 4 h, with shaking at 250 rpm. The formation of L-alanine was quantified via RP-HPLC.
[0247] Each enzyme only control without N7-acetyl-1,7-diaminoheptane demonstrated low base line conversion of pyruvate to L-alanine. See FIG. 12.
[0248] The gene products of SEQ ID NOs: 7-12 accepted N7-acetyl-1,7-diaminoheptane as substrate as confirmed against the empty vector control (see FIG. 16) and synthesized N7-acetyl-7-aminoheptanal as reaction product.
[0249] Given the reversibility of the .omega.-transaminase activity (see Example 1), the gene products of SEQ ID NOs: 7-12 accept N7-acetyl-7-aminoheptanal as substrate forming N7-acetyl-1,7-diaminoheptane.
Example 8
Enzyme Activity of Carboxylate Reductase Using Pimelate Semialdehyde as Substrate and Forming Heptanedial
[0250] The N-terminal His-tagged carboxylate reductase of SEQ ID NO: 6 (see Example 3 and FIG. 6) was assayed using pimelate semialdehyde as substrate. The enzyme activity assay was performed in triplicate in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 2 mM pimelate semialdehyde, 10 mM MgCl.sub.2, 1 mM ATP, and 1 mM NADPH. The enzyme activity assay reaction was initiated by adding purified carboxylate reductase and phosphopantetheine transferase or the empty vector control to the assay buffer containing the pimelate semialdehyde and then incubated at room temperature for 20 min. The consumption of NADPH was monitored by absorbance at 340 nm. The enzyme only control without pimelate semialdehyde demonstrated low base line consumption of NADPH. See FIG. 7.
[0251] The gene product of SEQ ID NO: 6, enhanced by the gene product of sfp, accepted pimelate semialdehyde as substrate as confirmed against the empty vector control (see FIG. 11) and synthesized heptanedial.
Example 9
[0252] Biotransformation of 5-hydroxypentanoate to 3-oxo-7-hydroxyheptanoyl-CoA in a Two-Step Enzymatic Reaction Catalyzed by a 5-hydroxyvalerate CoA Transferase and an Enzyme from the Thiolase Family
[0253] An enzyme with a 5-hydroxyvalerate CoA transferase activity was identified following the sequencing of Clostridium viride genome and subsequent analysis for putative CoA transferases. Five enzyme sequences were identified and two of these transferases acted upon 5-hydroxyvalerate. The CoA transferase selected for the reaction described in this Example is further called BDIGENE #246. The sequence is currently available publicly as NCBI Reference Sequence WP_027096059.1 (SEQ ID NO: 37). The gene (SEQ ID NO: 60) encoding the 5-hydroxyvalerate CoA transferase was codon-optimized for expression in E. coli.
[0254] Selected genes (SEQ ID NOs: 38-57) encoding various enzymes from thiolase family (see FIG. 18 for the list of the thiolases tested) were codon-optimized for expression in E. coli, synthesized and cloned into pET15b vector with the NdeI and BamHI restriction sites. Gene sequences were checked to ensure they did not contain the recognition sequences of these two restriction enzymes prior to cloning. The selection was supplemented by paaJ from E. coli which was cloned out of the respective genomic DNA. For P0C7L2 and Q0KBP1, the genes (SEQ ID NO: 58, and SEQ ID NO: 59, respectively) were cloned from naturally occurring sources. Specifically, the gene encoding P0C7L2 was from Escherichia coli, and the gene encoding Q0KBP1 was from Cupriavidus necator. The following chart shows the list of the genes expressed in this Example, as well as their corresponding thiolase designations as shown FIG. 18. See also FIG. 19.
TABLE-US-00001 SEQ ID NO Designation in FIG. 18 SEQ ID NO: 38 Q88E32 from Pseudomonas putida SEQ ID NO: 39 A5VAC9 from Sphingomonas wittichii SEQ ID NO: 40 D2I940 from Pseudomonas reinekei SEQ ID NO: 41 Q51956 from Pseudomonas putida SEQ ID NO: 42 Q146J4 from Burkholderia xenovorans SEQ ID NO: 43 Q13Q20 from Burkholderia xenovorans SEQ ID NO: 44 Q0SCR8 from Rhodococcus jostii SEQ ID NO: 45 Q6MM13 from Bdellovibrio bacteriovorus SEQ ID NO: 46 C9XWW6 from Cronobacter turicensis SEQ ID NO: 47 A0JWV4 from Arthrobacter sp. SEQ ID NO: 48 D5VE84 from Caulobacter segnis SEQ ID NO: 49 A8LRD1 from Dinoroseobacter shibae SEQ ID NO: 50 Q13HE4 from Burkholderia xenovorans SEQ ID NO: 51 GK1320 from Geobacillus kaustophilus SEQ ID NO: 52 B2ICW2 from Beijerinckia indica SEQ ID NO: 53 K8QJZ5 from Citrobacter freundii SEQ ID NO: 54 G0EVE5 from Cupriavidus necator SEQ ID NO: 55 D0L7M3 from Gordonia bronchialis SEQ ID NO: 56 D5WHY6 from Burkholderia sp. SEQ ID NO: 57 E1VSS4 from Arthrobacter arilaitensis SEQ ID NO: 58 P0C7L2 from Escherichia coli SEQ ID NO: 59 Q0KBP1 from Cupriavidus necator
[0255] For each culture, expression plasmids were freshly transformed into BL21(DE3) (Agilent). Colonies from fresh agar plates were used to inoculate 20 ml LB in 250 ml flask overnight pre-culture. For all constructs except 237, ampicillin was used, for 237 kanamycin was required. After incubation overnight at 37.degree. C. and 200 rpm, pre-cultures were used to inoculate (1:100) larger cultures, 350 ml LB, with respective antibiotic in 11 flasks. After 2.5 h of shaking with 200 rpm at 37.degree. C., cultures reached OD600 in the range 0.5-0.7 at which point they were induced with 1 mM IPTG and the temperature of the culture was changed to 25.degree. C. Incubation continued overnight. Cells were harvested the next day and stored at -20.degree. C. until protein purification was performed.
[0256] To purify an overexpressed protein, a bacterial pellet from a 350 ml culture was resuspended in up to total 20 ml of Binding Buffer (20 mM Sodium phosphate pH 7.4, 500 mM Sodium chloride, 20 mM imidazole, 5% glycerol) and sonicated for 2 min with a microtip (amplitude 50%, is pulse ON, 2 s OFF). Cell suspension was centrifuged with 20,000 G for 30 min at 4.degree. C. As all the tested proteins were His-tagged, the purification with Immobilized Metal Affinity Chromatography was sufficient to ensure both necessary purity and amount for the enzymatic assays. HisTrap (GE Healthcare) purification has been performed exactly according to manufacturer's protocol. Proteins were eluted with 2.times.60 .mu.l of Elution Buffer (20 mM Sodium phosphate pH 7.4, 500 mM Sodium chloride, 500 mM imidazole, 5% glycerol) and stored frozen at -20.degree. C.
[0257] Prior to tests with non-native substrates, enzymes were assayed for their native biological activity. Many of the selected enzymes are putative proteins, whose existence was inferred from homology to known thiolases and thus had not yet been biochemically investigated. In most cases there was no information available on what could be a possible native substrate. Therefore to assess their activity acetoacetyl-CoA was successfully used for all of them as a test substrate. The method from Slater (Slater et al., J. Bacteriol., 1998, 180(8):1979-1987) was used with minor modifications for assay of native enzyme activity. Assays were performed in disposable Corning UV-transparent 96-well plates without cover. Typically 200 .mu.l reaction mix contained 150 mM Tris-HCl pH 8.0, 50 mM MgCl.sub.2, 100 .mu.M CoA, with or without 40 .mu.M acetoacetyl-CoA (Sigma). Reaction was started by the addition of 10 .mu.l enzyme prepared as described above. Absorbance at 304 nm, corresponding to the disappearing acetoacetyl-CoA, was followed every minute for 2 hours. Negative controls with/without main substrate or with/without enzyme, as well as with empty vector were always included. The method showed that there was a good correlation between the solubility of the enzyme as judged from the SDS-PAGE gels and the apparent thiolase activity tested with this assay.
[0258] The set of thiolases was tested to investigate whether they would be able to accept 5-hydroxyvaleryl-CoA as a substrate to produce 3-oxo-7-hydroxyheptanoyl-CoA. Selected thiolases were tested in a coupled assay with BDIGENE #246 CoA transferase using 5-hydroxyvalerate and acetyl-CoA as substrates.
[0259] LC-MS based assay performed in 96-well format with 300 .mu.l total reaction volume in each well. Reaction mix contained 50 mM potassium phosphate pH 6.8, 1 mM acetyl-CoA (Applichem) and 1 mM of 5-hydroxyvalerate. Control samples without the substrate and/or without enzyme were always included. Samples with the empty vector control were included in each run as a reference. The reaction was initiated by the addition of 10 .mu.l of enzyme aliquote to 300 .mu.l reaction mix. The plate was covered with adhesive tape to minimize the evaporation and incubated at 30.degree. C. with gentle shaking (600 rpm in the Eppendorf thermomixer) for 3 hours. After the incubation samples were transferred with multichannel pipette to the 96-format filter (AcroPrep Advanced Filter Plate 0.2 .mu.m Supor) and filtered by centrifugation at 1500 G for 4 min to a clean 96-well plate before the LC-MS analysis. There were no analytical standards available for the reaction products hence the analysis relied exclusively on the presence of ions with expected molecular weight.
[0260] Analysis of acyl-CoAs was performed using an Agilent Technologies 1290 Series Infinity HPLC system coupled to an Agilent 6530 Series Q-ToF mass spectrometer equipped with an ESI interface operating in positive polarity with centroid data storage. QToF parameters were as follows: source temperature 350.degree. C., drying gas flow rate 13 l/min, nebulizer pressure 60 psig, sheath gas temperature 400.degree. C., sheath gas flow rate 12 l/min, VCap 3500 V, nozzle voltage 1000 V, fragmentor voltage 100 V, skimmer voltage 60 V, Octopole 1 RF voltage 750 V; scan range 50-1100 m/z, scan rate 2 spectra/sec; reference masses of 121.0509 m/z and 922.0098 m/z were infused directly into the source of the MS to ensure accurate mass correction of the instrument. Mobile phase A was 10 mM ammonium acetate and mobile phase B was acetonitrile.
[0261] For acyl-CoA screening a C18 column measuring 2.1 mm.times.50 mm with a 1.8 .mu.m particle size, 95 .ANG. pore size and 0.5 .mu.m frit was used (Agilent part no: 959757-902). Autosampler was kept at 4.degree. C. and 5 .mu.l of analyte was injected from each sample. Between injections the needle was washed with mobile phase in the flush port for 3 s to decrease the possibility of carryover contamination. Acyl-CoAs were eluted according to varying the percentage of mobile phase B as follows: 0-1 min, 3% B; 4-5.2 min, 100% B; 5.5-6.8 min, 3% B. Total run time was 6.8 minutes per sample. The intermediate product of the coupled reactions, 5-hydroxyvaleryl-CoA, was detected in all samples containing both substrates and BDIGENE #246. The final product, 3-oxo-7-hydroxyheptanoyl-CoA, was detected in several samples as illustrated in FIG. 18.
OTHER EMBODIMENTS
[0262] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Sequence CWU
1
1
611394PRTCupriavidus necator 1Met Thr Arg Glu Val Val Val Val Ser Gly Val
Arg Thr Ala Ile Gly 1 5 10
15 Thr Phe Gly Gly Ser Leu Lys Asp Val Ala Pro Ala Glu Leu Gly Ala
20 25 30 Leu Val
Val Arg Glu Ala Leu Ala Arg Ala Gln Val Ser Gly Asp Asp 35
40 45 Val Gly His Val Val Phe Gly
Asn Val Ile Gln Thr Glu Pro Arg Asp 50 55
60 Met Tyr Leu Gly Arg Val Ala Ala Val Asn Gly Gly
Val Thr Ile Asn 65 70 75
80 Ala Pro Ala Leu Thr Val Asn Arg Leu Cys Gly Ser Gly Leu Gln Ala
85 90 95 Ile Val Ser
Ala Ala Gln Thr Ile Leu Leu Gly Asp Thr Asp Val Ala 100
105 110 Ile Gly Gly Gly Ala Glu Ser
Met Ser Arg Ala Pro Tyr Leu Ala Pro 115 120
125 Ala Ala Arg Trp Gly Ala Arg Met Gly Asp Ala
Gly Leu Val Asp Met 130 135 140
Met Leu Gly Ala Leu His Asp Pro Phe His Arg Ile His Met Gly Val
145 150 155 160 Thr Ala
Glu Asn Val Ala Lys Glu Tyr Asp Ile Ser Arg Ala Gln Gln
165 170 175 Asp Glu Ala Ala Leu Glu
Ser His Arg Arg Ala Ser Ala Ala Ile Lys 180
185 190 Ala Gly Tyr Phe Lys Asp Gln Ile Val Pro
Val Val Ser Lys Gly Arg 195 200
205 Lys Gly Asp Val Thr Phe Asp Thr Asp Glu His Val Arg His
Asp Ala 210 215 220
Thr Ile Asp Asp Met Thr Lys Leu Arg Pro Val Phe Val Lys Glu Asn 225
230 235 240 Gly Thr Val Thr Ala
Gly Asn Ala Ser Gly Leu Asn Asp Ala Ala Ala 245
250 255 Ala Val Val Met Met Glu Arg Ala Glu Ala
Glu Arg Arg Gly Leu Lys 260 265
270 Pro Leu Ala Arg Leu Val Ser Tyr Gly His Ala Gly Val Asp
Pro Lys 275 280 285
Ala Met Gly Ile Gly Pro Val Pro Ala Thr Lys Ile Ala Leu Glu Arg 290
295 300 Ala Gly Leu Gln Val
Ser Asp Leu Asp Val Ile Glu Ala Asn Glu Ala 305 310
315 320 Phe Ala Ala Gln Ala Cys Ala Val Thr Lys
Ala Leu Gly Leu Asp Pro 325 330
335 Ala Lys Val Asn Pro Asn Gly Ser Gly Ile Ser Leu Gly His Pro
Ile 340 345 350 Gly
Ala Thr Gly Ala Leu Ile Thr Val Lys Ala Leu His Glu Leu Asn 355
360 365 Arg Val Gln Gly Arg
Tyr Ala Leu Val Thr Met Cys Ile Gly Gly Gly 370 375
380 Gln Gly Ile Ala Ala Ile Phe Glu Arg Ile
385 390 21174PRTMycobacterium marinum
2Met 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 31173PRTMycobacterium smegmatis 3Met
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 4 1148PRTSegniliparus rugosus 4Met 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 51185PRTMycobacterium
abscessusMycobacterium abscessus subsp. bolletii 5Met 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 6 1186PRTSegniliparus rotundus
6Met 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 7
459PRTChromobacterium violaceum 7Met 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 8468PRTPseudomonas
aeruginosa 8Met 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 9454PRTPseudomonas syringae
9Met 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
10467PRTRhodobacter sphaeroides 10Met 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 11459PRTEscherichia coli
11Met 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 12453PRTVibrio fluvialis 12Met 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 13401PRTEscherichia coli 13Met Arg Glu Ala Phe Ile Cys
Asp Gly Ile Arg Thr Pro Ile Gly Arg 1 5
10 15 Tyr Gly Gly Ala Leu Ser Ser Val Arg Ala Asp
Asp Leu Ala Ala Ile 20 25
30 Pro Leu Arg Glu Leu Leu Val Arg Asn Pro Arg Leu Asp Ala Glu
Cys 35 40 45 Ile
Asp Asp Val Ile Leu Gly Cys Ala Asn Gln Ala Gly Glu Asp Asn 50
55 60 Arg Asn Val Ala Arg Met
Ala Thr Leu Leu Ala Gly Leu Pro Gln Ser 65 70
75 80 Val Ser Gly Thr Thr Ile Asn Arg Leu Cys Gly
Ser Gly Leu Asp Ala 85 90
95 Leu Gly Phe Ala Ala Arg Ala Ile Lys Ala Gly Asp Gly Asp Leu Leu
100 105 110 Ile Ala
Gly Gly Val Glu Ser Met Ser Arg Ala Pro Phe Val Met Gly 115
120 125 Lys Ala Ala Ser Ala Phe
Ser Arg Gln Ala Glu Met Phe Asp Thr Thr 130 135
140 Ile Gly Trp Arg Phe Val Asn Pro Leu Met Ala
Gln Gln Phe Gly Thr 145 150 155
160 Asp Ser Met Pro Glu Thr Ala Glu Asn Val Ala Glu Leu Leu Lys Ile
165 170 175 Ser Arg
Glu Asp Gln Asp Ser Phe Ala Leu Arg Ser Gln Gln Arg Thr 180
185 190 Ala Lys Ala Gln Ser Ser
Gly Ile Leu Ala Glu Glu Ile Val Pro Val 195 200
205 Val Leu Lys Asn Lys Lys Gly Val Val Thr
Glu Ile Gln His Asp Glu 210 215 220
His Leu Arg Pro Glu Thr Thr Leu Glu Gln Leu Arg Gly Leu Lys
Ala 225 230 235 240 Pro
Phe Arg Ala Asn Gly Val Ile Thr Ala Gly Asn Ala Ser Gly Val
245 250 255 Asn Asp Gly Ala Ala Ala
Leu Ile Ile Ala Ser Glu Gln Met Ala Ala 260
265 270 Ala Gln Gly Leu Thr Pro Arg Ala Arg Ile
Val Ala Met Ala Thr Ala 275 280
285 Gly Val Glu Pro Arg Leu Met Gly Leu Gly Pro Val Pro Ala
Thr Arg 290 295 300
Arg Val Leu Glu Arg Ala Gly Leu Ser Ile His Asp Met Asp Val Ile 305
310 315 320 Glu Leu Asn Glu Ala
Phe Ala Ala Gln Ala Leu Gly Val Leu Arg Glu 325
330 335 Leu Gly Leu Pro Asp Asp Ala Pro His Val
Asn Pro Asn Gly Gly Ala 340 345
350 Ile Ala Leu Gly His Pro Leu Gly Met Ser Gly Ala Arg Leu
Ala Leu 355 360 365
Ala Ala Ser His Glu Leu His Arg Arg Asn Gly Arg Tyr Ala Leu Cys 370
375 380 Thr Met Cys Ile Gly
Val Gly Gln Gly Ile Ala Met Ile Leu Glu Arg 385 390
395 400 Val 141168PRTMycobacterium smegmatis
14Met 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
15392PRTPseudomonas putida 15Met Asn Asp Val Val Ile Val Ala Ala Thr Arg
Thr Ala Ile Gly Ser 1 5 10
15 Phe Gln Gly Ala Leu Ala Thr Val Pro Ala Val Asp Leu Gly Ala Ala
20 25 30 Val Ile
Lys Gln Leu Leu Lys Gln Thr Gly Leu Asp Pro Ala Gln Val 35
40 45 Asp Glu Val Ile Leu Gly Gln
Val Leu Thr Ala Gly Ala Gly Gln Asn 50 55
60 Pro Ala Arg Gln Ala Ala Ile Lys Ala Gly Leu Pro
Phe Ser Val Pro 65 70 75
80 Ala Leu Thr Leu Asn Lys Val Cys Gly Ser Gly Leu Lys Ala Leu His
85 90 95 Leu Ala Ala
Gln Ala Ile Arg Cys Gly Asp Ala Glu Val Val Ile Ala 100
105 110 Gly Gly Gln Glu Asn Met Ser
Leu Ala Pro Tyr Val Met Pro Ser Ala 115 120
125 Arg Thr Gly Gln Arg Met Gly His Gly Gln Leu
Ile Asp Ser Met Ile 130 135 140
Thr Asp Gly Leu Trp Asp Ala Phe Asn Asp Tyr His Met Gly Ile Thr
145 150 155 160 Ala Glu
Asn Leu Val Asp Lys Tyr Gly Leu Ser Arg Glu Gln Gln Asp
165 170 175 Ala Phe Ala Ala Glu Ser
Gln Arg Lys Ala Val Ala Ala Ile Glu Ala 180
185 190 Gly Arg Phe Asp Ala Glu Ile Thr Pro Ile
Val Leu Pro Gln Lys Lys 195 200
205 Gly Glu Pro Lys Val Phe Ala Arg Asp Glu Gln Pro Arg Pro
Asp Thr 210 215 220
Thr Ala Glu Ser Leu Ala Lys Leu Arg Pro Ala Phe Lys Lys Asp Gly 225
230 235 240 Ser Val Thr Ala Gly
Asn Ala Ser Ser Leu Asn Asp Gly Ala Ala Ala 245
250 255 Val Leu Leu Met Ser Ala Ala Lys Ala Glu
Ala Leu Gly Leu Pro Val 260 265
270 Leu Ala Lys Ile Ala Ala Tyr Ala Ser Ala Gly Val Asp Pro
Ala Ile 275 280 285
Met Gly Ile Gly Pro Val Ser Ala Thr Gln Arg Cys Leu Asp Lys Ala 290
295 300 Gly Trp Gln Leu Ala
Glu Leu Asp Leu Ile Glu Ala Asn Glu Ala Phe 305 310
315 320 Ala Ala Gln Ala Leu Ala Val Gly Asn Ala
Leu Ala Trp Asp Ala Ala 325 330
335 Arg Val Asn Val Asn Gly Gly Ala Ile Ala Leu Gly His Pro Ile
Gly 340 345 350 Ala
Ser Gly Cys Arg Val Leu Val Thr Leu Leu His Glu Met Ile Lys 355
360 365 Arg Asp Val Lys Lys
Gly Leu Ala Thr Leu Cys Ile Gly Gly Gly Gln 370 375
380 Gly Val Ala Leu Ala Ile Glu Arg 385
390 16393PRTSphingomonas wittichii 16Met Glu Asp Ile
Tyr Ile Val Gly Ala Ala Arg Thr Ala Ile Ala Asp 1 5
10 15 Phe Gly Gly Ala Leu Lys Asp Val Pro
Pro Ala Asp Leu Gly Val Ile 20 25
30 Val Ala Arg Ala Ala Leu Glu Arg Ala Gly Leu Glu Pro Gly
Asp Val 35 40 45
Gln Asn Val Val Met Gly Gln Val Met Pro Thr Glu Pro Arg Asp Ala 50
55 60 Tyr Leu Ala Arg Met
Val Gly Val Thr Ala Gly Val Pro Ile Glu Thr 65 70
75 80 Pro Ala Leu Thr Leu Asn Arg Leu Cys Gly
Ser Gly Val Glu Ala Ile 85 90
95 Val Thr Gly Ala Lys Ala Met Val Leu Gly Glu Ser Asp Ile Val
Leu 100 105 110 Ala
Gly Gly Ala Glu Val Met Ser Arg Val Pro His Val Val Lys Gly 115
120 125 Ala Arg Trp Gly Thr
Lys Met Gly Asn Val Glu Met Thr Asp Gly Leu 130 135
140 Ile Glu Ala Leu Ser Asp Pro Phe Asp Lys
Val His Met Gly Ile Thr 145 150 155
160 Ala Glu Asn Val Ala Glu Arg Tyr Gln Ile Thr Arg Glu Ala Gln
Asp 165 170 175 Ala
Leu Ala Leu Gln Gly His Gln Arg Ala Ala Arg Ala Ile Ala Glu
180 185 190 Gly Arg Phe Lys Ala
Gln Ile Val Pro Val Glu Val Lys Thr Arg Lys 195
200 205 Gly Val Val Ala Phe Asp Thr Asp Glu
His Val Arg Gly Asp Val Ser 210 215
220 Ala Glu Glu Leu Ala Lys Leu Arg Pro Val Phe Lys Lys
Asp Gly Thr 225 230 235
240 Val Thr Ala Ala Asn Ala Ser Gly Ile Asn Asp Gly Ala Ala Met Val
245 250 255 Val Leu Ala Thr
Lys Lys Ala Val Asp Ala Lys Gly Leu Lys Pro Leu 260
265 270 Ala Arg Ile Leu Ser Trp Gly His
Ala Gly Val Glu Pro Leu Tyr Met 275 280
285 Gly Ile Gly Pro Val Lys Ala Val Pro Ile Ala Leu
Glu Arg Ala Gly 290 295 300
Leu Thr Leu Ala Asp Ile Asp Val Ile Glu Ala Asn Glu Ala Phe Ala 305
310 315 320 Ala Gln Ala
Cys Ala Val Ala Gln Glu Leu Gly Phe Asp Pro Asp Lys 325
330 335 Val Asn Pro Asn Gly Ser Gly Val
Ala Leu Gly His Pro Val Gly Ala 340 345
350 Thr Gly Ala Ile Leu Thr Val Lys Thr Val Tyr Glu
Leu Glu Arg Ile 355 360 365
Gly Gly Arg Tyr Gly Leu Ile Thr Met Cys Ile Gly Gly Gly Gln Gly
370 375 380 Ile Ala Met
Val Val Glu Arg Cys Ala 385 390
17398PRTPseudomonas reinekei 17Met Lys Asn Ala Leu Ile Val Ser Pro Leu
Arg Thr Pro Ile Gly Lys 1 5 10
15 Phe Gly Gly Ala Leu Ala Pro Leu Thr Ala Glu His Leu Ala Ser
Phe 20 25 30 Met
Ile Ser Gln Val Met Ala Arg Thr Gly Val Pro Gly His Ser Leu 35
40 45 Asp Glu Val Ile Val Ala
Gln Ser Tyr Ala Ser Ser Glu Ala Pro Cys 50 55
60 Ile Gly Arg Tyr Ala Ala Leu Ser Ala Gly Leu
Pro Val Glu Val Pro 65 70 75
80 Gly Tyr Thr Leu Asp Arg Arg Cys Gly Ser Gly Leu Gln Ala Val Ile
85 90 95 Asp Ala
Ser Met Met Val Lys Thr Gly Asn Ala Glu Ala Val Leu Val 100
105 110 Val Gly Val Glu Ser Met
Ser Asn Ile Glu Tyr Tyr Ser Thr Asp Met 115 120
125 Arg Trp Gly Ala Arg Ala Gly Ser Val Arg
Phe His Asp Arg Leu Glu 130 135 140
Arg Gly Arg Glu Arg Ser Gln Pro Ser Glu Arg Phe Gly His Ile
Ser 145 150 155 160 Gly
Met Pro Glu Thr Ala Asp Asn Leu Ala Leu Asp Tyr Gly Ile Ser
165 170 175 Arg Glu Glu Ala Asp Ser
Phe Ser Val Arg Ser His Gln Asn Ala Ala 180
185 190 Ala Ala Trp Arg Glu Gly Arg Phe Ala Asp
Glu Val Val Ala Val Asp 195 200
205 Val Pro Gly Lys Arg Gly Ala Val Thr Arg Val Thr Ile Asp
Glu Gly 210 215 220
Ile Arg Glu Asp Ala Ser Leu Glu Ser Met Lys Ala Leu Arg Leu Ile 225
230 235 240 Arg Pro Glu Gly Val
Cys Thr Ala Gly Asn Ser Ser Gln Gln Asn Asp 245
250 255 Ala Ala Ala Gly Cys Leu Val Val Ser Pro
Glu Tyr Ala Ala Arg His 260 265
270 Gly Leu Thr Pro Met Ala Arg Leu Val Asp Trp Ala Ala Ala
Gly Cys 275 280 285
Glu Pro Ser Arg Met Gly Ile Gly Pro Val Pro Ala Thr Gln Lys Leu 290
295 300 Leu Met Arg Thr Gly
Leu Ser Leu Ala Glu Leu Asp Leu Ile Glu Leu 305 310
315 320 Asn Glu Ala Phe Ala Ala Gln Ala Leu Ala
Val Leu Lys Thr Trp Gly 325 330
335 Leu Asp Asp Leu Ser Arg Val Asn Val Asn Gly Ser Gly Ile Ser
Leu 340 345 350 Gly
His Pro Ile Gly Ala Thr Gly Val Arg Ile Met Thr Thr Leu Leu 355
360 365 His Glu Met Arg Arg
Arg Glu Ala Arg Tyr Gly Leu Glu Thr Met Cys 370 375
380 Ile Gly Gly Gly Gln Gly Leu Ala Ala Leu
Phe Glu Arg Val 385 390 395
18400PRTPseudomonas putida 18Met Arg Asp Val Phe Ile Cys Asp Ala Ile Arg
Thr Pro Ile Gly Arg 1 5 10
15 Phe Gly Gly Ala Leu Ala Gly Val Arg Ala Asp Asp Leu Ala Ala Val
20 25 30 Pro Leu
Lys Ala Leu Ile Glu Pro Asn Pro Ala Val Gln Trp Asp Gln 35
40 45 Val Asp Glu Val Phe Phe Gly
Cys Ala Asn Gln Ala Gly Glu Asp Asn 50 55
60 Arg Asn Val Ala Arg Met Ala Leu Leu Leu Ala Gly
Leu Pro Glu Ser 65 70 75
80 Ile Pro Gly Val Thr Leu Asn Arg Leu Cys Ala Ser Gly Met Asp Ala
85 90 95 Ile Gly Thr
Ala Phe Arg Ala Ile Ala Ser Gly Glu Met Glu Leu Ala 100
105 110 Ile Ala Gly Gly Val Glu Ser
Met Ser Arg Ala Pro Phe Val Met Gly 115 120
125 Lys Ala Glu Ser Gly Tyr Ser Arg Asn Met Lys
Leu Glu Asp Thr Thr 130 135 140
Ile Gly Trp Arg Phe Ile Asn Pro Leu Met Lys Ser Gln Tyr Gly Val
145 150 155 160 Asp Ser
Met Pro Glu Thr Ala Asp Asn Val Ala Asp Asp Tyr Gln Val
165 170 175 Ser Arg Ala Asp Gln Asp
Ala Phe Ala Leu Arg Ser Gln Gln Lys Ala 180
185 190 Ala Ala Ala Gln Ala Ala Gly Phe Phe Ala
Glu Glu Ile Val Pro Val 195 200
205 Arg Ile Ala His Lys Lys Gly Glu Thr Ile Val Glu Arg Asp
Glu His 210 215 220
Leu Arg Pro Glu Thr Thr Leu Glu Ala Leu Thr Lys Leu Lys Pro Val 225
230 235 240 Asn Gly Pro Asp Lys
Thr Val Thr Ala Gly Asn Ala Ser Gly Val Asn 245
250 255 Asp Gly Ala Ala Ala Leu Ile Leu Ala Ser
Ala Glu Ala Val Lys Lys 260 265
270 His Gly Leu Thr Pro Arg Ala Arg Val Leu Gly Met Ala Ser
Gly Gly 275 280 285
Val Ala Pro Arg Val Met Gly Ile Gly Pro Val Pro Ala Val Arg Lys 290
295 300 Leu Thr Glu Arg Leu
Gly Val Ala Val Ser Asp Phe Asp Val Ile Glu 305 310
315 320 Leu Asn Glu Ala Phe Ala Ser Gln Gly Leu
Ala Val Leu Arg Glu Leu 325 330
335 Gly Val Ala Asp Asp Ala Pro Gln Val Asn Pro Asn Gly Gly Ala
Ile 340 345 350 Ala
Leu Gly His Pro Leu Gly Met Ser Gly Ala Arg Leu Val Leu Thr 355
360 365 Ala Leu His Gln Leu
Glu Lys Ser Gly Gly Arg Lys Gly Leu Ala Thr 370 375
380 Met Cys Val Gly Val Gly Gln Gly Leu Ala
Leu Ala Ile Glu Arg Val 385 390 395
400 19402PRTBurkholderia xenovorans 19Met Ser Glu Thr His Met
Ser Gly Thr Lys Ala Asp Pro Ile Val Ile 1 5
10 15 Val Gly Val Ala Arg Thr Pro Met Ala Ala Phe
Gln Gly Asp Phe Ala 20 25
30 Thr Leu Ser Ala Pro Gln Leu Gly Ser Val Ala Ile Gln Ala Ala
Val 35 40 45 Gln
Arg Ala Gly Leu Lys Pro Glu Gln Ile Asp Glu Val Val Met Gly 50
55 60 Cys Val Leu Pro Ala Gly
Leu Gly Gln Ala Pro Ala Arg Gln Ala Ala 65 70
75 80 Leu Gly Ala Gly Leu Pro Leu Ala Thr Gly Ser
Thr Thr Val Asn Lys 85 90
95 Met Cys Gly Ser Gly Met Arg Ala Ala Met Phe Ala His Asp Met Leu
100 105 110 Ala Ala
Gly Ser Val Asp Val Ile Val Ala Gly Gly Met Glu Ser Met 115
120 125 Thr Asn Ala Pro Tyr Leu
Leu Pro Lys Ala Arg Ala Gly Met Arg Met 130 135
140 Gly His Gly Gln Val Ile Asp His Met Phe Tyr
Asp Gly Leu Glu Asp 145 150 155
160 Ala Tyr Glu Lys Gly Arg Leu Met Gly Ser Phe Ala Glu Glu Cys Ala
165 170 175 Ala Ser
Phe Asp Phe Thr Arg Glu Ala Gln Asp Ala Phe Ala Val Glu 180
185 190 Ser Leu Ala Arg Ala Lys
Arg Ala Asn Glu Asp Gly Ser Phe Ala Trp 195 200
205 Glu Ile Ala Pro Val Lys Val Glu Ser Arg
Lys Gly Glu Val Thr Ile 210 215 220
Asp Arg Asp Glu Gln Pro Phe Lys Ala Asn Ile Glu Lys Ile Pro
Thr 225 230 235 240 Leu
Lys Pro Ala Phe Ser Lys Thr Gly Thr Val Thr Ala Ala Asn Ser
245 250 255 Ser Ser Ile Ser Asp Gly
Ala Ala Ala Leu Val Met Met Arg Glu Ser 260
265 270 Thr Ala Lys Arg Leu Gly Val Gln Pro Ile
Ala Arg Val Val Gly His 275 280
285 Ser Thr Leu Ala Gln Glu Pro Ala Lys Phe Thr Thr Ala Pro
Val Gly 290 295 300
Ala Ile Arg Lys Leu Phe Glu Lys Asn Gly Trp Arg Ala Asp Glu Val 305
310 315 320 Asp Leu Phe Glu Val
Asn Glu Ala Phe Ala Val Val Thr Met Ala Ala 325
330 335 Met Lys Glu His His Leu Pro His Glu Lys
Val Asn Val Asn Gly Gly 340 345
350 Ala Cys Ala Leu Gly His Pro Ile Gly Ala Ser Gly Ala Arg
Ile Leu 355 360 365
Val Thr Leu Ile Gly Ala Leu Lys Lys Arg Gly Gly Lys Arg Gly Val 370
375 380 Ala Thr Leu Cys Ile
Gly Gly Gly Glu Ala Thr Ala Met Gly Ile Glu 385 390
395 400 Leu Val 20400PRTBurkholderia xenovorans
20Met Thr Glu Ala Phe Leu Cys Asp Ala Ile Arg Thr Pro Ile Gly Arg 1
5 10 15 Tyr Ala Gly Ala
Leu Ser Ser Val Arg Ala Asp Asp Leu Gly Ala Val 20
25 30 Pro Leu Lys Ala Leu Met Glu Arg Asn
Lys Glu Val Asp Trp Asn Ala 35 40
45 Ile Asp Asp Val Ile Tyr Gly Cys Ala Asn Gln Ala Gly Glu
Asp Asn 50 55 60
Arg Asn Val Ala Arg Met Ser Leu Leu Leu Ala Gly Leu Pro Gln Gly 65
70 75 80 Val Pro Gly Thr Thr
Val Asn Arg Leu Cys Gly Ser Gly Met Asp Ala 85
90 95 Val Gly Ile Ala Ala Arg Ala Ile Lys Ser
Gly Glu Ala Ala Leu Met 100 105
110 Val Ala Gly Gly Val Glu Ser Met Ser Arg Ala Pro Phe Val
Thr Gly 115 120 125
Lys Ala Thr Ser Ala Phe Ser Arg Gln Ala Glu Ile Tyr Asp Thr Thr 130
135 140 Ile Gly Trp Arg Phe
Val Asn Pro Leu Met Lys Lys Leu Tyr Gly Val 145 150
155 160 Asp Ser Met Pro Glu Thr Gly Glu Asn Val
Ala Thr Asp Tyr Asn Ile 165 170
175 Ser Arg Ala Asp Gln Asp Ala Phe Ala Leu Arg Ser Gln Gln Lys
Ala 180 185 190 Ala
Arg Ala Gln Arg Asp Gly Thr Leu Ala Gln Glu Ile Val Gly Val 195
200 205 Thr Ile Ala Gln Lys
Lys Gly Asp Pro Val Thr Val Ser Gln Asp Glu 210 215
220 His Pro Arg Glu Thr Ser Leu Asp Ala Leu
Ala Lys Leu Lys Gly Val 225 230 235
240 Val Arg Pro Asp Gly Thr Val Thr Ala Gly Asn Ala Ser Gly Val
Asn 245 250 255 Asp
Gly Ala Ala Ala Leu Leu Leu Ala Asn Glu Glu Thr Ala Arg Arg
260 265 270 Phe Gly Leu Thr Pro
Arg Ala Arg Val Leu Gly Ile Ala Thr Ala Gly 275
280 285 Val Ala Pro Arg Val Met Gly Ile Gly
Pro Ala Pro Ala Thr Gln Lys 290 295
300 Leu Leu Ala Arg Leu Asn Met Ser Leu Asp Gln Phe Asp
Val Ile Glu 305 310 315
320 Leu Asn Glu Ala Phe Ala Ser Gln Gly Ile Ala Val Leu Arg Ala Leu
325 330 335 Gly Val Ala Asp
Asp Asp Thr Arg Val Asn Pro Asn Gly Gly Ala Ile 340
345 350 Ala Leu Gly His Pro Leu Gly Met
Ser Gly Ala Arg Leu Val Thr Thr 355 360
365 Ala Met Tyr Gln Leu His Arg Thr Gln Gly Arg Phe
Ala Leu Cys Thr 370 375 380
Met Cys Ile Gly Val Gly Gln Gly Ile Ala Ile Ala Ile Glu Arg Val 385
390 395 400
21406PRTRhodococcus jostii 21Met Ala Glu Val Phe Leu Val Asp Gly Ala Arg
Thr Pro Gln Gly Arg 1 5 10
15 Tyr Gly Gly Ala Leu Ala Gly Val Arg Pro Asp Asp Leu Ala Gly Leu
20 25 30 Val Val
Ala Glu Ala Ala Arg Arg Ala Gly Ile Pro Gly Asp Ala Val 35
40 45 Asp Glu Val Ile Leu Gly Ala
Ala Asn Gln Ala Gly Glu Asp Asn Arg 50 55
60 Asp Val Ala Arg Met Ala Val Leu Leu Ala Gly Leu
Pro Asp Ser Val 65 70 75
80 Pro Gly Tyr Thr Val Asn Arg Leu Cys Ala Ser Gly Leu Thr Ala Val
85 90 95 Ala Ser Ala
Ala His Thr Ile Arg Ser Gly Glu Ala Asp Ile Val Ile 100
105 110 Ala Gly Gly Val Glu Ser Met
Thr Arg Ala Pro Trp Val Met Ala Lys 115 120
125 Pro Gly Thr Pro Trp Ala Arg Pro Gly Glu Val
Ala Asp Thr Ser Leu 130 135 140
Gly Trp Arg Phe Thr Asn Pro Arg Phe Thr Ala Ala Asp Arg Asp Val
145 150 155 160 Pro Ala
Gly Ala Gly Pro Asp Val Arg Lys Val Thr Leu Ser Met Gly
165 170 175 Glu Thr Ala Glu Glu Val
Ala Ala Leu Glu Gly Val Thr Arg Ala Glu 180
185 190 Ser Asp Ala Phe Ala Leu Arg Ser Gln Glu
Arg Ala Ile Ala Ala Val 195 200
205 Asp Ala Gly Arg Phe Glu Arg Glu Ile Val Pro Val Pro Val
Arg Asp 210 215 220
Gly Glu Leu Ala Ala Asp Glu Thr Pro Arg Arg Gly Thr Thr Leu Glu 225
230 235 240 Lys Leu Gly Ser Leu
Lys Pro Val Phe Arg Thr Gly Gly Ile Val Thr 245
250 255 Ala Gly Ser Ser Ser Ser Leu Ser Asp Gly
Ala Ala Ala Leu Val Val 260 265
270 Ala Ser Glu Ala Ala Val Glu Lys Tyr Gly Leu Thr Val Arg
Gly Arg 275 280 285
Ile Val Thr Ser Ala Ser Ala Gly Ile Ala Pro Asn Val Met Gly Leu 290
295 300 Gly Pro Val Pro Ala
Thr Arg Lys Ala Leu Ala Arg Ala Asn Trp Ser 305 310
315 320 Ile Ser Asp Leu Gly Ala Ala Glu Leu Asn
Glu Ala Phe Ala Ala Gln 325 330
335 Ser Leu Gly Val Ile Arg Gln Leu Lys Leu Asp Glu Ser Ile Val
Asn 340 345 350 Ala
Asp Gly Gly Ala Ile Ala Leu Gly His Pro Leu Gly Cys Ser Gly 355
360 365 Ala Arg Ile Leu Leu
Thr Leu Leu Gly Arg Met Glu Arg Glu Gly Ala 370 375
380 Arg Arg Gly Leu Ala Thr Leu Cys Val Gly
Val Gly Gln Gly Val Ala 385 390 395
400 Met Leu Ile Glu Ala Pro 405
22447PRTBdellovibrio bacteriovorus 22Met Lys Ser Pro Arg Asp Val Val Leu
Val Glu Gly Val Arg Thr Pro 1 5 10
15 Phe Ala Lys Ala Gly Thr Lys Leu Lys Lys Val His Pro Ala
Glu Leu 20 25 30
Gly Lys Val Ala Leu Lys Gln Val Ile Ala Gln Thr Asn Leu Asp Val
35 40 45 Asn Leu Val Asp
Glu Val Ile Ile Gly Asn Thr Gly Asn Pro Pro Asp 50
55 60 Ser Val Asn Ile Ser Arg Val Val
Ala Leu Asn Ala Gly Ile Pro Leu 65 70
75 80 Lys Thr Ser Ala Tyr Thr Val His Arg Asn Cys Ala
Ser Ala Leu Glu 85 90
95 Ser Ile Ser Asn Gly Tyr Glu Lys Ile Lys Ser Gly Thr Met Asp Val
100 105 110 Ile Leu
Ala Gly Gly Thr Glu Asn Met Ser Gln Met Pro Thr Leu Pro 115
120 125 Pro Lys Lys Phe Gln Glu
Ile Tyr Glu Lys Leu Phe Ala Ala Lys Gly 130 135
140 Pro Lys Gln Ala Leu Pro Leu Leu Trp Ser Leu
Phe Lys Ala Asp Val 145 150 155
160 Lys Gln Ile Lys Ala Leu Leu Ser Gly Asn Met Arg Asp Glu Tyr Phe
165 170 175 Pro Val
Ile Ser Val Met Met Gly Leu Thr Asp Pro Phe Val Gly Ile 180
185 190 Asn Met Gly Gln Thr Ala
Glu Ile Leu Ala Lys Glu Trp Gly Leu Ser 195 200
205 Arg Glu Thr Gln Asp Lys Phe Ala Leu Arg
Ser His Gln Leu Ala Ser 210 215 220
Lys Ala Met Lys Glu Gly Arg Met Arg Glu Glu Ile Ala Pro Val
Tyr 225 230 235 240 Leu
Ala Pro Glu Tyr Lys Glu Val Ile Ser Glu Asp Ile Gly Pro Arg
245 250 255 Asp Thr Gln Thr Met Glu
Ala Leu Ala Lys Leu Lys Pro Phe Phe Asp 260
265 270 Lys Ala Thr Gly Ser Ile Thr Ala Gly Asn
Ser Cys Pro Ile Thr Asp 275 280
285 Gly Ala Ala Met Val Leu Met Met Ser Arg Glu Lys Ala Glu
Ala Leu 290 295 300
Gly Tyr Lys Pro Leu Ala Thr Ile Arg Ser Tyr Gly Phe Ala Gly Leu 305
310 315 320 Glu Pro Glu Arg Met
Gly Leu Gly Pro Val Tyr Ser Thr Pro Val Ala 325
330 335 Leu Lys Arg Ala Gly Leu Ser Met Lys Asp
Ile Gly Leu Val Glu Leu 340 345
350 Asn Glu Ala Phe Ala Ala Gln Val Leu Ser Cys Gln Lys Ala
Phe Asp 355 360 365
Ser Asp Lys Phe Gly Gln Glu Lys Leu Gly Leu Ser Ser Lys Ile Gly 370
375 380 Glu Ile Arg Asp Asp
Ile Leu Asn Val Asn Gly Gly Ala Ile Ala Leu 385 390
395 400 Gly His Pro Val Gly Ala Thr Gly Thr Arg
Ile Val Leu Thr Leu Ala 405 410
415 Lys Glu Met Lys Arg Arg Asn Thr Gln Phe Gly Leu Ala Thr Leu
Cys 420 425 430 Ile
Gly Gly Gly Gln Gly Gly Ser Met Ile Leu Glu Asn Glu Gly 435
440 445 23445PRTCronobacter turicensis
23Met Phe Ser Leu Leu Gln Gly Asn Val Met Ser Gln Ala Leu Pro Leu 1
5 10 15 Val Thr Arg Gln
Gly Asp Arg Ile Ala Ile Val Ser Gly Leu Arg Thr 20
25 30 Pro Phe Ala Arg Gln Ala Thr Ala Tyr
His Gly Val Pro Ala Val Asp 35 40
45 Leu Gly Lys Met Val Val Gly Glu Leu Leu Ala Arg Ser Glu
Ile Pro 50 55 60
Pro Asp Val Ile Glu Gln Leu Val Phe Gly Gln Val Val Gln Met Pro 65
70 75 80 Glu Ala Pro Asn Ile
Ala Arg Glu Ile Val Leu Gly Thr Gly Met Ser 85
90 95 Val His Thr Asp Ala Tyr Ser Val Ser Arg
Ala Cys Ala Thr Ser Phe 100 105
110 Gln Ala Val Ala Asn Val Ala Glu Ser Leu Met Ala Gly Thr
Ile Arg 115 120 125
Ala Gly Ile Ala Gly Gly Ala Asp Ser Ser Ser Val Leu Pro Ile Gly 130
135 140 Val Ser Lys Lys Leu
Ala Arg Thr Leu Val Asp Ala Asn Lys Ala Arg 145 150
155 160 Thr Ala Gly Gln Arg Leu Lys Leu Phe Ser
Arg Leu Arg Leu Arg Asp 165 170
175 Leu Leu Pro Val Pro Pro Ala Val Ala Glu Tyr Ser Thr Gly Leu
Arg 180 185 190 Met
Gly Asp Thr Ala Glu Gln Met Ala Lys Thr His Gly Ile Thr Arg 195
200 205 Glu Gln Gln Asp Ala
Leu Ala His Arg Ser His Gln Leu Ala Ala Gln 210 215
220 Ala Trp Ala Glu Gly Lys Leu Arg Glu Glu
Val Met Thr Ala Tyr Thr 225 230 235
240 Pro Pro Tyr Arg Glu Pro Leu Ser Glu Asp Asn Asn Ile Arg Lys
Asn 245 250 255 Ser
Ser Leu Ala Asp Tyr Thr Lys Leu Arg Pro Ala Phe Asp Arg Lys
260 265 270 His Gly Thr Val Thr
Ala Ala Asn Ser Thr Pro Leu Thr Asp Gly Ala 275
280 285 Ala Ala Val Ile Leu Met Thr Glu Ser
Arg Ala Arg Glu Leu Gly Leu 290 295
300 Thr Pro Leu Gly Tyr Leu Arg Ser Tyr Ala Phe Thr Ala
Val Asp Val 305 310 315
320 Trp Gln Asp Met Leu Leu Gly Pro Ala Trp Ser Thr Pro Leu Ala Leu
325 330 335 Glu Arg Ala Gly
Leu Thr Met Ala Asp Leu Thr Leu Ile Asp Met His 340
345 350 Glu Ala Phe Ala Ser Gln Thr Leu
Ala Asn Leu Lys Leu Leu Ala Ser 355 360
365 Asp Arg Phe Ala Arg Glu Val Leu Gly Arg Ser Gln
Ala Thr Gly Glu 370 375 380
Val Asp Glu Ser Lys Phe Asn Val Leu Gly Gly Ser Ile Ala Tyr Gly 385
390 395 400 His Pro Phe
Ala Ala Thr Gly Ala Arg Met Ile Thr Gln Thr Leu Asn 405
410 415 Glu Leu Arg Arg Arg Gly Gly Gly
Phe Gly Leu Val Thr Ala Cys Ala 420 425
430 Ala Gly Gly Leu Gly Ala Ala Met Val Leu Glu Ala
Glu 435 440 445
24456PRTArthrobacter sp. 24Met Ser Phe Asn Gly Gln Ser Ala Thr Gly Pro
Asp Glu Ser Ala Ala 1 5 10
15 Ala Pro Ala Ala Thr Pro Gly Ala Gly Leu Leu Arg Lys Ala Val Val
20 25 30 Val Gly
Gly Asn Arg Ile Pro Phe Ala Arg Thr Gly Gly Ala Tyr Thr 35
40 45 Lys Ser Ser Asn Gln Asp Met
Leu Thr Ala Ala Leu Asp Gly Leu Ile 50 55
60 Ala Arg Phe Gly Leu Ala Asp Glu Arg Ile Gly Glu
Val Ala Ala Gly 65 70 75
80 Ala Val Leu Lys His Ser Arg Asp Phe Asn Leu Thr Arg Glu Ala Val
85 90 95 Leu Gly Ser
Ala Leu Ser Ala Glu Thr Pro Ala Tyr Asp Leu Gln Gln 100
105 110 Ala Cys Ala Thr Gly Leu Glu
Thr Val Leu Gly Leu Ala Asn Lys Ile 115 120
125 Lys Leu Gly Gln Ile Asp Ser Ala Ile Ala Gly
Gly Val Asp Ser Ala 130 135 140
Ser Asp Ala Pro Ile Ala Val Ser Glu Gly Leu Arg Glu Val Leu Leu
145 150 155 160 Asp Leu
Asn Arg Ala Lys Thr Leu Pro Gln Arg Leu Lys Val Leu Gly
165 170 175 Arg Leu Arg Pro Lys Asp
Leu Ala Pro Asp Ala Pro Asn Thr Gly Glu 180
185 190 Pro Arg Thr Gly Leu Ser Met Gly Glu His
Gln Ala Leu Thr Thr Ala 195 200
205 Gln Trp Lys Ile Thr Arg Glu Ala Gln Asp Glu Leu Ala Tyr
Asn Ser 210 215 220
His Arg Asn Leu Ala Ala Ala Tyr Asp Ala Gly Phe Phe Asp Asp Leu 225
230 235 240 Leu Thr Pro Tyr Arg
Gly Leu Asn Arg Asp Ser Asn Leu Arg Ala Asp 245
250 255 Thr Thr Arg Glu Lys Leu Ser Thr Leu Lys
Pro Val Phe Gly Lys Asn 260 265
270 Leu Gly Ala Glu Ala Thr Met Thr Ala Gly Asn Ser Thr Pro
Leu Thr 275 280 285
Asp Gly Ala Ser Thr Val Leu Leu Ala Ser Glu Glu Trp Ala Asp Ala 290
295 300 His Glu Leu Pro Lys
Leu Ala Thr Val Val Asp Gly Glu Ala Ala Ala 305 310
315 320 Val Asp Phe Val His Gly Lys Asp Gly Leu
Leu Met Ala Pro Ala Phe 325 330
335 Ala Val Pro Arg Leu Leu Ala Arg Asn Gly Leu Thr Leu Asp Asp
Ile 340 345 350 Asp
Phe Phe Glu Ile His Glu Ala Phe Ala Gly Thr Val Leu Ser Thr 355
360 365 Leu Ala Ala Trp Glu
Asp Glu Glu Phe Gly Arg Thr Arg Leu Gly Leu 370 375
380 Asp Gly Pro Leu Gly Ser Ile Asp Arg Ala
Lys Leu Asn Val Asn Gly 385 390 395
400 Ser Ser Leu Ala Ala Gly His Pro Phe Ala Ala Thr Gly Gly Arg
Ile 405 410 415 Val
Ala Thr Leu Ala Lys Met Leu His Asp Lys Gly Gln Val Asp Gly
420 425 430 Arg Pro Ala Arg Gly
Leu Ile Ser Ile Cys Ala Ala Gly Gly Gln Gly 435
440 445 Val Val Ala Ile Leu Glu Ala Ser
450 455 25429PRTCaulobacter segnis 25Met Ala Thr Ala
Ser Ser Ser Ala Ala Ser Ser Ser Gly Val Trp Leu 1 5
10 15 Ala Ala Gly Val Arg Ser Pro Phe Ala
Lys Val Asp Gly Ala Leu Ala 20 25
30 Gly His Asp Ala Ile Gly Leu Ser Val Pro Val Val Lys Ala
Met Leu 35 40 45
Ala Arg Ala Lys Pro Asp Phe Ala Val Trp Gly Thr Val Ile Pro Asn 50
55 60 Leu Thr Trp Ser Asn
Leu Ala Arg Glu Val Leu Leu Asp Ala Gly Gly 65 70
75 80 Asp Pro Thr Ile Pro Ala Phe Ser Thr Val
Met Ala Cys Ser Thr Ser 85 90
95 Met Ile Gly Ala Ile Glu Ala Ala Gly Met Val Asp Gly Arg Gly
Arg 100 105 110 Asp
Leu Ala Leu Val Gly Gly Val Glu Ser Met Ser Arg Val Gln Leu 115
120 125 Gly Leu Ser Val Ala
Leu Ser Asp Trp Ile Arg Lys Phe Gln Asn Ala 130 135
140 Lys Thr Gly Gln Gln Arg Leu Ala Ala Leu
Gly Ala Leu Asn Leu Lys 145 150 155
160 Asp Val Arg Leu Phe Ile Pro Lys Val Val Asn Arg Val Thr Gly
Leu 165 170 175 Ser
Met Gly Glu His Thr Glu Ile Thr Ala Lys Glu Trp Asn Leu Ser
180 185 190 Arg Ala Asp Gln Asp
Ala Ile Ala Leu Ala Ser His Gln Gly Ala Val 195
200 205 Lys Gly Trp Glu Ser Gly Phe Phe Asp
Asp Leu Val Ile Pro Val Gly 210 215
220 Glu Val Lys Arg Asp Gly Ile Pro Arg Lys Asp Thr Ser
Leu Glu Lys 225 230 235
240 Leu Ala Lys Leu Gly Pro Ala Phe Asp Lys Thr Ser Gly Lys Gly Thr
245 250 255 Leu Thr Ala Gly
Asn Ser Ser Pro Leu Thr Asp Gly Ala Ala Ala Val 260
265 270 Trp Val Gly Ser Ala Ala Gly Met
Ala Arg Leu Pro Gly Glu Thr Pro 275 280
285 Lys Val Arg Leu Val Asp Tyr Glu Val Thr Ser Ile
Asp Leu Arg His 290 295 300
Glu Gly Leu Leu Met Ala Pro Ala Tyr Gly Val Pro Arg Met Leu Ala 305
310 315 320 Arg Asn Gly
Leu Thr Tyr Ala Asp Val Gly Leu Trp Glu Ile His Glu 325
330 335 Ala Phe Ala Ala Gln Val Leu Ser
His Ile Ala Ala Trp Glu Ser Ala 340 345
350 Lys Phe Leu Ser Glu Lys Ala Gly Val Thr Thr Pro
Met Gly Ala Phe 355 360 365
Pro Arg Glu Arg Met Asn Pro Asn Gly Gly Ser Leu Ala Leu Gly His
370 375 380 Pro Phe Gly
Ala Thr Gly Ala Arg Ile Ile Ser Gln Thr Val Lys Glu 385
390 395 400 Leu Ala Ala Arg Pro Lys Gly
Glu Arg Ala Ile Val Ser Ile Cys Ala 405
410 415 Asp Gly Gly Gln Gly Thr Met Met Leu Leu Glu
Ser Ala 420 425
26403PRTDinoroseobacter shibae 26Met Thr Glu Ala Tyr Ile Tyr Asp Ala Ile
Arg Ser Pro Arg Gly Lys 1 5 10
15 Gly Arg Lys Asp Gly Ser Leu His Glu Val Thr Ala Val Ser Leu
Ser 20 25 30 Ala
Gln Thr Leu Asn Ala Ile Lys Asp Arg Asn Gly Leu Thr Gly His 35
40 45 Ala Val Glu Asp Val Ile
Trp Gly Asn Val Thr Gln Val Met Glu Gln 50 55
60 Gly Gly Cys Leu Ala Arg Thr Ala Val Leu Ala
Ser Asp Leu Asp Glu 65 70 75
80 Ser Ile Pro Gly Leu Ala Ile Asn Arg Phe Cys Ala Ser Gly Leu Glu
85 90 95 Ala Val
Asn Leu Ala Ala Asn Gln Val Arg Gly Gly Gly Gly Gln Ala 100
105 110 Tyr Ile Ala Gly Gly Val
Glu Met Met Gly Arg Val Pro Met Gly Ser 115 120
125 Asp Gly Ala Ala Ile Ala Ala Asp Pro Ser
Val Ala Met Lys Thr Tyr 130 135 140
Phe Val Pro Gln Gly Ile Ser Ala Asp Ile Ile Ala Thr Glu Tyr
Gly 145 150 155 160 Ile
Ser Arg Asp Asp Ala Asp Ala Leu Ala Val Ala Ser Gln Arg Arg
165 170 175 Ala Lys Ala Ala Trp Asp
Glu Asn Arg Phe Asn Gly Ser Val Phe Thr 180
185 190 Val Arg Asp Gln Asn Gly Leu Asn Ile Leu
Asp His Asp Glu Tyr Met 195 200
205 Arg Pro Glu Thr Asp Met Gln Ser Leu Gly Ala Leu Lys Pro
Ala Phe 210 215 220
Lys Asp Met Gly Glu Gln Met Pro Gly Phe Asp Lys Ile Ala Leu Met 225
230 235 240 Lys Tyr Pro His Leu
Glu Lys Ile Glu His Ile His His Ala Gly Asn 245
250 255 Ser Ser Gly Ile Val Asp Gly Ser Ala Ala
Leu Leu Ile Gly Asn Lys 260 265
270 Ala Phe Gly Glu Ala His Gly Leu Lys Pro Arg Ala Val Ile
Lys Ala 275 280 285
Thr Ala Lys Ile Gly Thr Asp Pro Thr Ile Met Leu Thr Gly Pro Val 290
295 300 Pro Ala Thr Glu Lys
Ile Leu Ala Asp Ser Gly Met Ser Ile Ser Asp 305 310
315 320 Ile Asp Leu Phe Glu Val Asn Glu Ala Phe
Ser Ser Val Val Leu Arg 325 330
335 Phe Met Gln Ala Phe Asp Val Asp His Asp Lys Val Asn Val Asn
Gly 340 345 350 Gly
Ala Ile Ala Met Gly His Pro Leu Gly Ala Thr Gly Ala Met Ile 355
360 365 Leu Gly Thr Leu Leu
Asp Glu Leu Glu Arg Thr Gly Lys Gly Thr Gly 370 375
380 Leu Ala Thr Leu Cys Val Ala Ser Gly Met
Gly Ala Ala Thr Ile Ile 385 390 395
400 Glu Arg Val 27394PRTBurkholderia xenovorans 27Met Thr Arg
Asp Thr Arg Asp Val Val Ile Val Asp Ala Val Arg Thr 1 5
10 15 Pro Ile Gly Lys Phe Arg Gly Ala
Leu Ala Gly Val Arg Ala Asp His 20 25
30 Leu Gly Ala Leu Val Ile Asp Glu Leu Ile Arg Arg Ala
Gly Val Lys 35 40 45
Pro Gln Ala Val Asn Asp Val Val Phe Gly Cys Val Thr Gln Ile Gly 50
55 60 Glu Gln Ser Ala
Asn Ile Ala Arg Thr Ser Val Leu Gly Ala Gly Trp 65 70
75 80 Pro Glu Thr Ile Pro Gly Leu Thr Ile
Asp Arg Lys Cys Gly Ser Gly 85 90
95 Glu Glu Ala Val His Ile Ala Ala Gly Leu Ile Ala Phe Gly
Ala Ala 100 105 110
Asp Val Ile Val Ala Gly Gly Ala Glu Ser Met Ser Arg Val Pro Met
115 120 125 Gly Ser Asn Arg
Asp Leu His Gly Glu Ala Phe Gly Trp Met Ala Ser 130
135 140 Glu Arg Phe Glu Leu Thr Ser Gln
Gly Glu Ala Ala Glu Arg Leu Cys 145 150
155 160 Asp Cys Trp Ala Leu Thr Arg Ala Gln Leu Asp Ala
Tyr Ser Val Glu 165 170
175 Ser His Arg Arg Ala Ala Ala Ala Ala Ala Glu Gly Trp Phe Ala Arg
180 185 190 Glu Ile
Val Pro Val Pro Val Gly Gln Val Arg Glu Lys Ser Leu Glu 195
200 205 Gly Glu Ala Ala Leu Phe
Ala Ala Asp Glu Thr Ile Arg Pro Gly Thr 210 215
220 Asn Ala Asp Lys Leu Ala Thr Leu Lys Ser Ser
Phe Arg Ser Asp Gly 225 230 235
240 Arg Leu Thr Ala Gly Asn Ser Ser Gln Ile Ser Asp Gly Ala Ala Ala
245 250 255 Leu Leu
Leu Met Ser Ser Asp Lys Ala Arg Glu Leu Gly Val Lys Ala 260
265 270 Arg Ala Arg Val Arg Ala
Val Thr Thr Val Gly Ser Asp Pro Thr Leu 275 280
285 Met Leu Thr Gly Pro Ile Leu Ala Thr Cys
Gln Val Leu Glu Lys Ala 290 295 300
Gly Leu Gly Leu Ser Asp Ile Asp Leu Phe Glu Ile Asn Glu Ala
Phe 305 310 315 320 Ala
Pro Val Pro Leu Val Trp Met Lys Glu Phe Gly Val Pro His Ala
325 330 335 Lys Leu Asn Val Asn Gly
Gly Ala Ile Ala Leu Gly His Pro Leu Gly 340
345 350 Ala Ser Gly Ala Arg Ile Met Thr Ser Met
Leu His Glu Leu Glu Arg 355 360
365 Arg Gly Ala Arg Tyr Gly Leu Gln Ala Ile Cys Cys Ala Gly
Gly Met 370 375 380
Gly Thr Ala Thr Leu Ile Glu Arg Leu Asp 385 390
28382PRTGeobacillus kaustophilus 28Met Arg Glu Ala Val Ile Val Glu
Ala Val Arg Thr Pro Val Gly Lys 1 5 10
15 Arg Asn Gly Val Phe Arg Asp Val His Pro Val His Leu
Ala Ala Val 20 25 30
Val Leu Asp Glu Val Val Arg Arg Ala Gly Met Asp Lys Gly Ala Val
35 40 45 Glu Asp Ile Val
Met Gly Cys Val Thr Pro Val Ala Glu Gln Gly Tyr 50
55 60 Asn Ile Gly Arg Leu Ala Ala Leu
Glu Ala Gly Phe Pro Ile Glu Val 65 70
75 80 Pro Ala Val Gln Ile Asn Arg Met Cys Gly Ser Gly
Gln Gln Ala Ile 85 90
95 His Phe Ala Ala Gln Glu Ile Arg Ser Gly Asp Met Asp Val Thr Ile
100 105 110 Ala Ala
Gly Val Glu Ser Met Thr Lys Val Pro Ile Leu Ser Asp Gly 115
120 125 Asn Glu Arg Thr Ile Pro
Pro Ser Leu His Glu Lys Tyr Glu Phe Ile 130 135
140 His Gln Gly Val Ser Ala Glu Arg Ile Ala Lys
Lys Tyr Gly Leu Thr 145 150 155
160 Arg Glu Glu Leu Asp Ala Tyr Ala Tyr Glu Ser His Gln Arg Ala Leu
165 170 175 Ala Ala
Leu Arg Glu Gly Lys Phe Arg Ala Glu Ile Val Pro Val Lys 180
185 190 Gly Leu Asp Arg Asp Gly
Arg Glu Ile Leu Val Thr Asp Asp Glu Gly 195 200
205 Pro Arg Ala Asp Thr Ser Pro Glu Ala Leu
Ala Ala Leu Lys Pro Val 210 215 220
Phe Gln Glu Asp Gly Leu Ile Thr Ala Gly Asn Ala Ser Gln Met
Ser 225 230 235 240 Asp
Gly Ala Ala Ala Val Leu Leu Met Glu Arg Glu Ala Ala Arg Arg
245 250 255 Phe Gly Leu Lys Pro Lys
Ala Arg Ile Val Ala Gln Thr Val Val Gly 260
265 270 Ser Asp Pro Thr Tyr Met Leu Asp Gly Val
Ile Pro Ala Thr Arg Gln 275 280
285 Val Leu Lys Lys Ala Gly Leu Ser Ile Asp Asp Ile Asp Leu
Ile Glu 290 295 300
Ile Asn Glu Ala Phe Ala Pro Val Val Leu Ala Trp Gln Lys Glu Ile 305
310 315 320 Gly Ala Pro Leu Glu
Lys Val Asn Val Asn Gly Gly Ala Ile Ala Leu 325
330 335 Gly His Pro Leu Gly Ala Thr Gly Ala Lys
Leu Met Thr Ser Leu Val 340 345
350 His Glu Leu Glu Arg Arg Gly Gly Arg Tyr Gly Leu Leu Thr
Ile Cys 355 360 365
Ile Gly His Gly Met Ala Thr Ala Thr Ile Ile Glu Arg Glu 370
375 380 29378PRTBeijerinckia indica 29Met
Thr Lys Val Val Ile Ala Gly Tyr Ile Arg Ser Pro Phe Thr Leu 1
5 10 15 Ala Lys Lys Gly Glu Leu
Ala Thr Val Arg Pro Asp Asp Leu Ala Ala 20
25 30 Gln Val Val Lys Gly Leu Ile Lys Lys Thr
Gly Ile Pro Ala Glu Asp 35 40
45 Ile Glu Asp Leu Leu Leu Gly Cys Ala Phe Pro Glu Gly Glu
Gln Gly 50 55 60
Phe Asn Val Ala Arg Leu Val Ser Phe Leu Ala Gly Leu Pro Leu Ser 65
70 75 80 Val Gly Ala Ser Thr
Val Asn Arg Phe Cys Gly Ser Ser Met Thr Thr 85
90 95 Val His Met Ala Ala Gly Ala Ile Gln Met
Asn Ala Gly Asn Ala Phe 100 105
110 Ile Ala Ala Gly Val Glu Ser Met Ser Arg Val Pro Met Met
Gly Phe 115 120 125
Asn Pro Leu Pro Asn Pro Glu Leu Ala Ala Thr Met Pro Gly Ala Tyr 130
135 140 Met Gly Met Gly Asp
Thr Ala Glu Asn Val Ala Ala Lys Trp Thr Ile 145 150
155 160 Ser Arg Lys Glu Gln Glu Glu Phe Ala Leu
Arg Ser His Gln Arg Ala 165 170
175 Thr Ala Ala Gln Lys Glu Gly Arg Leu Thr Gly Glu Ile Ile Pro
Ile 180 185 190 Thr
Gly Arg Lys Gly Thr Ile Thr Thr Asp Gly Cys Ile Arg Pro Asp 195
200 205 Thr Thr Leu Glu Gly
Leu Ala Glu Leu Lys Pro Ala Phe Ser Ala Asn 210 215
220 Gly Val Val Thr Ala Gly Thr Ser Ser Pro
Leu Thr Asp Gly Ala Ala 225 230 235
240 Ala Val Leu Val Cys Ser Glu Asp Tyr Ala Lys His His His Leu
Asp 245 250 255 Val
Leu Ala Ser Val Lys Ala Ile Ala Val Ser Gly Cys Ser Pro Glu
260 265 270 Ile Met Gly Ile Gly
Pro Val Ala Ala Ser Arg Lys Ala Leu Ala Arg 275
280 285 Ala Gly Leu Glu Ala Gly Gln Ile Asp
Ile Val Glu Leu Asn Glu Ala 290 295
300 Phe Ala Ser Gln Ser Ile Ala Cys Met Arg Glu Leu Asn
Leu Ser Pro 305 310 315
320 Asp Arg Val Asn Ile Asp Gly Gly Ala Ile Ala Leu Gly His Pro Leu
325 330 335 Gly Ala Thr Gly
Ala Arg Ile Val Gly Lys Ala Ala Ser Leu Leu Lys 340
345 350 Arg Glu Lys Gly Lys Tyr Ala Leu
Ala Thr Gln Cys Ile Gly Gly Gly 355 360
365 Gln Gly Ile Ala Thr Val Leu Glu Ala Phe 370
375 30387PRTCitrobacter freundii 30Met Glu Gln
Val Val Ile Val Asp Ala Ile Arg Thr Pro Met Gly Arg 1 5
10 15 Ser Lys Gly Gly Ala Phe Arg Asn
Val Arg Ala Glu Asp Leu Ser Ala 20 25
30 His Leu Met Arg Ser Leu Leu Ala Arg Asn Pro Ala Leu
Asp Pro Thr 35 40 45
Ala Leu Asp Asp Ile Tyr Trp Gly Cys Val Gln Gln Thr Leu Glu Gln 50
55 60 Gly Phe Asn Ile
Ala Arg Asn Ala Ala Leu Leu Ala Glu Ile Pro His 65 70
75 80 Ser Val Pro Ala Val Thr Val Asn Arg
Leu Cys Gly Ser Ser Met Gln 85 90
95 Ala Leu His Asp Ala Ala Arg Met Ile Met Thr Gly Asp Ala
Gln Ala 100 105 110
Cys Leu Ile Gly Gly Val Glu His Met Gly His Val Pro Met Ser His
115 120 125 Gly Val Asp Phe
His Pro Gly Met Ser Arg Asn Val Ala Lys Ala Ala 130
135 140 Gly Met Met Gly Leu Thr Ala Glu
Met Leu Ser Arg Met His Gly Ile 145 150
155 160 Ser Arg Glu Met Gln Asp Ala Phe Ala Ala Arg Ser
His Ala Arg Ala 165 170
175 Trp Ala Ala Thr Gln Ser Gly Ala Phe Lys Asn Glu Ile Ile Pro Thr
180 185 190 Gly Gly
His Asp Ala Asp Gly Val Leu Lys Gln Phe Asn Tyr Asp Glu 195
200 205 Val Ile Arg Pro Glu Thr
Thr Val Glu Ala Leu Ser Thr Leu Arg Pro 210 215
220 Ala Phe Asp Pro Val Ser Gly Thr Val Thr Ala
Gly Thr Ser Ser Ala 225 230 235
240 Leu Ser Asp Gly Ala Ala Ala Met Leu Val Met Ser Glu Ser Arg Ala
245 250 255 Arg Glu
Leu Gly Leu Thr Pro Arg Ala Arg Ile Arg Ser Met Ala Val 260
265 270 Val Gly Cys Asp Pro Ser
Ile Met Gly Tyr Gly Pro Val Pro Ala Ser 275 280
285 Lys Leu Ala Leu Lys Lys Ala Gly Leu Ser
Thr Ser Asp Ile Gly Leu 290 295 300
Phe Glu Met Asn Glu Ala Phe Ala Ala Gln Ile Leu Pro Cys Ile
Lys 305 310 315 320 Asp
Leu Gly Leu Met Glu Gln Ile Asp Glu Lys Ile Asn Leu Asn Gly
325 330 335 Gly Ala Ile Ala Leu Gly
His Pro Leu Gly Cys Ser Gly Ala Arg Ile 340
345 350 Ser Thr Thr Leu Leu Asn Leu Met Glu Arg
Lys Asp Val Gln Phe Gly 355 360
365 Leu Ala Thr Met Cys Ile Gly Leu Gly Gln Gly Ile Ala Thr
Val Phe 370 375 380
Glu Arg Val 385 31398PRTCupriavidus necator 31Met Lys Gln Leu Gln
Asp Ala Tyr Ile Val Ala Ala Thr Arg Ser Pro 1 5
10 15 Ile Gly Lys Ala Pro Lys Gly Ala Phe Lys
Asn Thr Arg Pro Asp Asp 20 25
30 Leu Leu Ala Thr Ile Leu Lys Ala Ala Val Ala Gln Val Pro Asn
Leu 35 40 45 Asp
Pro Lys Leu Ile Glu Asp Ala Ile Val Gly Cys Ala Ile Pro Glu 50
55 60 Ala Gln Gln Gly Leu Asn
Val Ala Arg Ile Gly Ala Leu Leu Ser Gly 65 70
75 80 Leu Pro Asn Thr Val Gly Gly Ile Thr Val Asn
Arg Phe Cys Ala Ser 85 90
95 Gly Val Ser Ala Val Ala Met Ala Ala Asp Arg Ile Arg Val Gly Glu
100 105 110 Ser Asp
Val Met Ile Ala Ala Gly Val Glu Ser Met Ser Met Val Pro 115
120 125 Met Met Gly Asn Ser Pro
Ser Met Ser Pro Glu Ile Phe Thr Arg Asp 130 135
140 Glu Asn Val Gly Ile Ala Tyr Gly Met Gly Leu
Thr Ala Glu Lys Val 145 150 155
160 Ala Gln Gln Trp Gln Val Ser Arg Glu Asp Gln Asp Ala Phe Ser Leu
165 170 175 Ala Ser
His Gln Lys Ala Ile Ala Ala Gln Gln Ala Gly Glu Phe Lys 180
185 190 Asp Glu Ile Thr Pro Ile
Glu Ile Val Glu Arg Phe Pro Asp Leu Ala 195 200
205 Ser Gly Gln Val Asn Val Lys Thr Arg Thr
Ile Ser Leu Asp Glu Gly 210 215 220
Pro Arg Pro Glu Thr Ser Leu Glu Gly Leu Gly Lys Leu Arg Pro
Val 225 230 235 240 Phe
Ala Asn Lys Gly Ser Val Thr Ala Gly Asn Ser Ser Gln Thr Ser
245 250 255 Asp Gly Ala Gly Ala Leu
Ile Leu Val Ser Glu Lys Ile Leu Lys Gln 260
265 270 Phe Asn Leu Val Pro Leu Ala Arg Phe Val
Ser Phe Ala Val Arg Gly 275 280
285 Val Pro Pro Glu Ile Met Gly Ile Gly Pro Lys Glu Ala Ile
Pro Ala 290 295 300
Ala Leu Lys Ala Ala Gly Leu Thr Gln Asp Gln Leu Asp Trp Ile Glu 305
310 315 320 Leu Asn Glu Ala Phe
Ala Ala Gln Ser Leu Ala Val Met Arg Asp Leu 325
330 335 Gln Leu Asp Pro Ala Lys Val Asn Arg Met
Gly Gly Ala Ile Ala Leu 340 345
350 Gly His Pro Leu Gly Ala Thr Gly Ala Ile Arg Ser Ala Thr
Val Val 355 360 365
His Ala Leu Arg Arg His Asn Leu Lys Tyr Gly Met Val Thr Met Cys 370
375 380 Val Gly Thr Gly Met
Gly Ala Ala Gly Ile Phe Glu Arg Val 385 390
395 32415PRTGordonia bronchialis 32Met Ala Pro Cys Ser Val
Lys Ala Met Pro Glu Ala Val Ile Val Ala 1 5
10 15 His Ala Arg Ser Pro Ile Gly Arg Ala Gly Lys
Gly Ser Leu Lys Asp 20 25
30 Val Arg Pro Asp Glu Leu Ser Arg Gln Met Val Ala Ala Ala Leu
Ala 35 40 45 Lys
Val Pro Glu Leu Ala Pro Ser Asp Ile Glu Asp Ile His Trp Gly 50
55 60 Ile Gly Gln Pro Gly Gly
Gln Gly Gly Tyr Asn Ile Ala Arg Val Ile 65 70
75 80 Ala Val Glu Leu Gly Tyr Asp His Ile Pro Gly
Val Thr Val Asn Arg 85 90
95 Tyr Cys Ser Ser Ser Leu Gln Thr Thr Arg Met Ala Leu His Ala Ile
100 105 110 Lys Ala
Gly Glu Ala Asp Val Leu Ile Ser Gly Gly Val Glu Ser Val 115
120 125 Ser Ser Phe Gly Ile Ser
Gly Gly Ala Asp Gly Ala Pro Asp Ser Lys 130 135
140 Asn Pro Val Phe Asp Asp Ala Gln Ala Arg Thr
Ala Lys Ala Ala Glu 145 150 155
160 Gly Gly Ala Pro Ala Trp Thr Asp Pro Arg Glu Gln Gly Leu Ile Pro
165 170 175 Asp Val
Tyr Ile Ala Met Gly Gln Thr Ala Glu Asn Val Ala Ser Phe 180
185 190 Thr Gly Ile Ser Arg Glu
Asp Gln Asp Arg Trp Ser Val Leu Ser Gln 195 200
205 Asn Arg Ala Glu Glu Ala Ile Asn Ala Gly
Phe Phe Glu Arg Glu Ile 210 215 220
Asp Pro Val Thr Leu Pro Asp Gly Ser Thr Val Asn Thr Asp Asp
Gly 225 230 235 240 Pro
Arg Ala Gly Thr Thr Tyr Glu Lys Val Ser Gln Leu Lys Pro Val
245 250 255 Phe Arg Pro Asp Gly Thr
Val Thr Ala Gly Asn Ala Cys Pro Leu Asn 260
265 270 Asp Gly Ala Ala Ala Leu Val Ile Met Ser
Asp Ser Lys Ala Lys Gln 275 280
285 Leu Gly Leu Thr Pro Leu Ala Arg Val Val Ala Thr Ala Ala
Thr Gly 290 295 300
Leu Ser Pro Glu Ile Met Gly Leu Gly Pro Ile Glu Ala Ile Arg Lys 305
310 315 320 Val Leu Arg Ile Ser
Gly Met Ser Leu Ser Asp Ile Asp Leu Val Glu 325
330 335 Ile Asn Glu Ala Phe Ala Val Gln Val Leu
Gly Ser Ala Asn Glu Leu 340 345
350 Gly Ile Asp His Asp Lys Leu Asn Val Ser Gly Gly Ala Ile
Ala Leu 355 360 365
Gly His Pro Phe Gly Met Thr Gly Ala Arg Ile Thr Thr Thr Leu Leu 370
375 380 Asn Asn Leu Gln Thr
Arg Asp Lys Thr Phe Gly Ile Glu Ser Met Cys 385 390
395 400 Val Gly Gly Gly Gln Gly Met Ala Met Val
Leu Glu Arg Leu Ser 405 410
415 33393PRTBurkholderia sp. 33Met Arg Glu Ala Val Ile Val Ser Thr Ala
Arg Thr Pro Leu Thr Lys 1 5 10
15 Ala His Arg Gly Glu Phe Asn Ile Thr Pro Gly Pro Thr Leu Ala
Ser 20 25 30 Phe
Ala Val Arg Ala Ala Val Glu Arg Ser Gly Val Asp Pro Asp Ile 35
40 45 Ile Glu Asp Ala Ile Leu
Gly Cys Gly Tyr Pro Glu Gly Thr Thr Gly 50 55
60 Arg Asn Val Ala Arg Gln Ser Val Ile Arg Ala
Gly Leu Pro Leu Ser 65 70 75
80 Ile Ala Gly Thr Thr Val Asn Arg Phe Cys Ala Ser Gly Leu Gln Ala
85 90 95 Ile Ala
Met Ala Ala Gly Arg Ile Val Val Asp Gly Ala Pro Ala Met 100
105 110 Ile Ala Gly Gly Val Glu
Ser Ile Ser Asn Ile Gln Thr Arg Glu Asp 115 120
125 Gly Val Ser Gly Leu Asp Pro Trp Ile Val
Glu His Lys Pro Ser Leu 130 135 140
Tyr Thr Ala Met Ile Asp Thr Ala Asp Ile Val Ala Arg Arg Tyr
Gly 145 150 155 160 Ile
Ser Arg Glu Ala Gln Asp Gln Phe Ser Val Glu Ser Gln Arg Arg
165 170 175 Thr Ala Glu Ala Gln Gln
Ala Gly Arg Tyr Ala Asp Glu Ile Ile Pro 180
185 190 Val Thr Thr Thr Met Ala Ile Thr Asp Lys
Glu Thr Arg Ala Val Ser 195 200
205 Tyr Arg Glu Val Thr Val Ser Ala Asp Asn Cys Asn Arg Pro
Gly Thr 210 215 220
Thr Tyr Glu Ala Leu Ala Lys Leu Ala Pro Val Lys Gly Pro Asp Gln 225
230 235 240 Phe Ile Thr Ala Gly
Asn Ala Ser Gln Asn Ala Asp Gly Ala Ser Ala 245
250 255 Cys Val Leu Met Glu Ala Lys Ala Ala Glu
Arg Ala Asn Phe Ala Pro 260 265
270 Leu Gly Ala Phe Arg Gly Leu Ala Leu Ala Gly Cys Glu Pro
Asp Glu 275 280 285
Met Gly Ile Gly Pro Val Leu Ala Val Pro Lys Leu Leu Ala Arg His 290
295 300 Gly Leu Thr Val Asp
Asp Ile Gly Leu Trp Glu Leu Asn Glu Ala Phe 305 310
315 320 Ala Ser Gln Ala Val Tyr Cys Gln Lys Arg
Leu Glu Ile Pro Ser Glu 325 330
335 Arg Leu Asn Val Asn Gly Gly Ala Ile Ser Ile Gly His Pro Phe
Gly 340 345 350 Met
Thr Gly Ser Arg Leu Val Gly His Val Leu Ile Glu Gly Arg Arg 355
360 365 Arg Gly Val Lys Tyr
Ala Val Val Thr Met Cys Met Ala Gly Gly Met 370 375
380 Gly Ala Ala Gly Leu Phe Glu Ile Tyr 385
390 34403PRTGlutamicibacter arilaitensis
34Met Gln Gln Ala Tyr Leu Tyr Asp Ala Ile Arg Thr Pro Phe Gly Lys 1
5 10 15 Ile Gly Gly Ala
Leu Ser Ser His Arg Pro Asp Asp Leu Ala Ala His 20
25 30 Val Val Arg Glu Leu Val Ala Arg Ser
Pro Lys Leu Asp Val Ala Asp 35 40
45 Ile Asp Glu Ser Ile Phe Gly Asn Ala Asn Gly Ala Gly Glu
Glu Asn 50 55 60
Arg Asn Val Ala Arg Met Ala Thr Leu Leu Ala Gly Leu Pro Thr Ser 65
70 75 80 Leu Pro Gly Thr Thr
Met Asn Arg Leu Cys Gly Ser Ser Leu Asp Ala 85
90 95 Ser Ile Ala Ala Ser Arg Gln Ile Ala Thr
Gly Asp Ala Asp Leu Val 100 105
110 Leu Val Gly Gly Val Glu Ser Met Ser Arg Ala Pro Trp Val
Leu Pro 115 120 125
Lys Thr Glu Arg Pro Phe Pro Met Ser Asn Leu Glu Leu Ala Asn Thr 130
135 140 Thr Leu Gly Trp Arg
Leu Val Asn Pro Ala Met Pro Gly Glu Trp Thr 145 150
155 160 Val Ser Leu Gly Glu Ala Thr Glu Gln Leu
Arg Glu Lys His Gly Ile 165 170
175 Ser Arg Glu Asp Gln Asp Glu Phe Ser Ala Ala Ser His Gln Arg
Ala 180 185 190 Ala
Ala Ala Trp Gln Ala Gly Lys Tyr Asp Asn Leu Val Val Pro Val 195
200 205 Pro Pro Ala Asn Lys
Arg Gly Thr Glu Val Thr Arg Asp Glu Thr Ile 210 215
220 Arg Ala Asp Ser Thr Ala Gln Thr Leu Ser
Lys Leu Arg Thr Val Phe 225 230 235
240 Arg Thr Gly Glu Asn Ala Thr Val Thr Ala Gly Asn Ala Ser Pro
Met 245 250 255 Ser
Asp Gly Ala Ser Ala Ala Phe Ile Gly Ser Glu Arg Gly Gly Glu
260 265 270 Leu Leu Gly Ala Ala
Pro Ile Ala Arg Ile Ala Ser Asn Gly Ala Ala 275
280 285 Ala Leu Asp Pro Gln Phe Phe Gly Phe
Ala Pro Val Glu Ala Ala Asn 290 295
300 Lys Ala Leu Ala Lys Ala Gly Leu Lys Trp Ser Asp Ile
Ala Ala Val 305 310 315
320 Glu Leu Asn Glu Ala Phe Ala Ala Gln Ser Leu Ala Cys Ile Arg Ala
325 330 335 Trp Asp Ile Asp
Pro Ala Ile Val Asn Ala Trp Gly Gly Ala Ile Ser 340
345 350 Ile Gly His Pro Leu Gly Ala Ser
Gly Leu Arg Ile Leu Gly Thr Val 355 360
365 Ala Arg Arg Leu Ala Glu Ser Gly Glu Arg Tyr Gly
Leu Ala Ala Ile 370 375 380
Cys Ile Gly Val Gly Gln Gly Leu Ala Val Val Val Glu Asn Ile Asn 385
390 395 400 Ala Thr Lys
35401PRTEscherichia coli 35Met Arg Glu Ala Phe Ile Cys Asp Gly Ile Arg
Thr Pro Ile Gly Arg 1 5 10
15 Tyr Gly Gly Ala Leu Ser Ser Val Arg Ala Asp Asp Leu Ala Ala Ile
20 25 30 Pro Leu
Arg Glu Leu Leu Val Arg Asn Pro Arg Leu Asp Ala Glu Cys 35
40 45 Ile Asp Asp Val Ile Leu Gly
Cys Ala Asn Gln Ala Gly Glu Asp Asn 50 55
60 Arg Asn Val Ala Arg Met Ala Thr Leu Leu Ala Gly
Leu Pro Gln Ser 65 70 75
80 Val Ser Gly Thr Thr Ile Asn Arg Leu Cys Gly Ser Gly Leu Asp Ala
85 90 95 Leu Gly Phe
Ala Ala Arg Ala Ile Lys Ala Gly Asp Gly Asp Leu Leu 100
105 110 Ile Ala Gly Gly Val Glu Ser
Met Ser Arg Ala Pro Phe Val Met Gly 115 120
125 Lys Ala Ala Ser Ala Phe Ser Arg Gln Ala Glu
Met Phe Asp Thr Thr 130 135 140
Ile Gly Trp Arg Phe Val Asn Pro Leu Met Ala Gln Gln Phe Gly Thr
145 150 155 160 Asp Ser
Met Pro Glu Thr Ala Glu Asn Val Ala Glu Leu Leu Lys Ile
165 170 175 Ser Arg Glu Asp Gln Asp
Ser Phe Ala Leu Arg Ser Gln Gln Arg Thr 180
185 190 Ala Lys Ala Gln Ser Ser Gly Ile Leu Ala
Glu Glu Ile Val Pro Val 195 200
205 Val Leu Lys Asn Lys Lys Gly Val Val Thr Glu Ile Gln His
Asp Glu 210 215 220
His Leu Arg Pro Glu Thr Thr Leu Glu Gln Leu Arg Gly Leu Lys Ala 225
230 235 240 Pro Phe Arg Ala Asn
Gly Val Ile Thr Ala Gly Asn Ala Ser Gly Val 245
250 255 Asn Asp Gly Ala Ala Ala Leu Ile Ile Ala
Ser Glu Gln Met Ala Ala 260 265
270 Ala Gln Gly Leu Thr Pro Arg Ala Arg Ile Val Ala Met Ala
Thr Ala 275 280 285
Gly Val Glu Pro Arg Leu Met Gly Leu Gly Pro Val Pro Ala Thr Arg 290
295 300 Arg Val Leu Glu Arg
Ala Gly Leu Ser Ile His Asp Met Asp Val Ile 305 310
315 320 Glu Leu Asn Glu Ala Phe Ala Ala Gln Ala
Leu Gly Val Leu Arg Glu 325 330
335 Leu Gly Leu Pro Asp Asp Ala Pro His Val Asn Pro Asn Gly Gly
Ala 340 345 350 Ile
Ala Leu Gly His Pro Leu Gly Met Ser Gly Ala Arg Leu Ala Leu 355
360 365 Ala Ala Ser His Glu
Leu His Arg Arg Asn Gly Arg Tyr Ala Leu Cys 370 375
380 Thr Met Cys Ile Gly Val Gly Gln Gly Ile
Ala Met Ile Leu Glu Arg 385 390 395
400 Val 36394PRTCupriavidus necator 36Met Thr Arg Glu Val Val
Val Val Ser Gly Val Arg Thr Ala Ile Gly 1 5
10 15 Thr Phe Gly Gly Ser Leu Lys Asp Val Ala Pro
Ala Glu Leu Gly Ala 20 25
30 Leu Val Val Arg Glu Ala Leu Ala Arg Ala Gln Val Ser Gly Asp
Asp 35 40 45 Val
Gly His Val Val Phe Gly Asn Val Ile Gln Thr Glu Pro Arg Asp 50
55 60 Met Tyr Leu Gly Arg Val
Ala Ala Val Asn Gly Gly Val Thr Ile Asn 65 70
75 80 Ala Pro Ala Leu Thr Val Asn Arg Leu Cys Gly
Ser Gly Leu Gln Ala 85 90
95 Ile Val Ser Ala Ala Gln Thr Ile Leu Leu Gly Asp Thr Asp Val Ala
100 105 110 Ile Gly
Gly Gly Ala Glu Ser Met Ser Arg Ala Pro Tyr Leu Ala Pro 115
120 125 Ala Ala Arg Trp Gly Ala
Arg Met Gly Asp Ala Gly Leu Val Asp Met 130 135
140 Met Leu Gly Ala Leu His Asp Pro Phe His Arg
Ile His Met Gly Val 145 150 155
160 Thr Ala Glu Asn Val Ala Lys Glu Tyr Asp Ile Ser Arg Ala Gln Gln
165 170 175 Asp Glu
Ala Ala Leu Glu Ser His Arg Arg Ala Ser Ala Ala Ile Lys 180
185 190 Ala Gly Tyr Phe Lys Asp
Gln Ile Val Pro Val Val Ser Lys Gly Arg 195 200
205 Lys Gly Asp Val Thr Phe Asp Thr Asp Glu
His Val Arg His Asp Ala 210 215 220
Thr Ile Asp Asp Met Thr Lys Leu Arg Pro Val Phe Val Lys Glu
Asn 225 230 235 240 Gly
Thr Val Thr Ala Gly Asn Ala Ser Gly Leu Asn Asp Ala Ala Ala
245 250 255 Ala Val Val Met Met Glu
Arg Ala Glu Ala Glu Arg Arg Gly Leu Lys 260
265 270 Pro Leu Ala Arg Leu Val Ser Tyr Gly His
Ala Gly Val Asp Pro Lys 275 280
285 Ala Met Gly Ile Gly Pro Val Pro Ala Thr Lys Ile Ala Leu
Glu Arg 290 295 300
Ala Gly Leu Gln Val Ser Asp Leu Asp Val Ile Glu Ala Asn Glu Ala 305
310 315 320 Phe Ala Ala Gln Ala
Cys Ala Val Thr Lys Ala Leu Gly Leu Asp Pro 325
330 335 Ala Lys Val Asn Pro Asn Gly Ser Gly Ile
Ser Leu Gly His Pro Ile 340 345
350 Gly Ala Thr Gly Ala Leu Ile Thr Val Lys Ala Leu His Glu
Leu Asn 355 360 365
Arg Val Gln Gly Arg Tyr Ala Leu Val Thr Met Cys Ile Gly Gly Gly 370
375 380 Gln Gly Ile Ala Ala
Ile Phe Glu Arg Ile 385 390
37427PRTClostridium viride 37Met Ala Gln Phe Val Thr Ala Gln Glu Ala Val
Lys His Ile Pro Asn 1 5 10
15 Gly Ser Arg Val Val Leu Ala His Ser Thr Gly Glu Pro Arg Thr Leu
20 25 30 Val Lys
Ala Met Val Glu Asn Tyr Glu Gln Tyr Lys Asp Val Glu Val 35
40 45 Cys His Met Leu Gly Leu Gly
Pro Tyr Glu Tyr Thr Asn Pro Glu Met 50 55
60 Lys Gly His Leu Trp His Asn Ser Leu Phe Met Gly
Pro Gly Gly Arg 65 70 75
80 Lys Ala Phe Asn Glu Asn Arg Leu Asp Phe Thr Pro Gly Tyr Phe Cys
85 90 95 Asp Ser Ile
Lys Phe Phe Arg Glu Gly Tyr Leu Pro Val Asp Val Leu 100
105 110 Met Met Thr Val Ser Pro Pro
Asp Lys His Gly Tyr Val Thr Cys Gly 115 120
125 Ile Thr Cys Asp Phe Thr Met Pro Ala Phe Glu
Cys Ala Lys Met Val 130 135 140
Ile Val Gln Val Asn Lys Asn Met Pro Arg Thr Phe Gly Gln Thr Ala
145 150 155 160 Ile His
Leu Asp Asp Ile Asp Phe Ala Val Glu Ala Asp Asp Pro Leu
165 170 175 Tyr Gly Ser Val Pro Gly
Glu Leu Thr Asp Ile Asp Arg Lys Ile Gly 180
185 190 Glu His Cys Ala Ser Leu Ile Asn Asp Gly
Ala Cys Leu Gln Leu Gly 195 200
205 Ile Gly Gly Ile Pro Asn Ala Val Leu Thr Tyr Leu Thr Glu
Lys Asn 210 215 220
Asp Met Gly Ile His Ser Glu Met Leu Ser Asp Gly Ile Leu Gln Leu 225
230 235 240 Ile Lys Ala Gly Asn
Ile Asn Asn Ser Lys Lys Gln Ile His Val Gly 245
250 255 Lys Ser Ala Val Thr Phe Leu Asn Gly Ser
Gln Glu Leu Tyr Asp Tyr 260 265
270 Val Asp Asp Asn Pro Ser Val Glu Phe Tyr Pro Val Asp Tyr
Ile Asn 275 280 285
Asp Pro Tyr Val Ile Gly Lys Asn Asp Asn Met Val Ser Val Asn Ser 290
295 300 Ala Leu Ser Val Asp
Leu Met Gly Gln Ile Val Ala Asp Asn Leu Ser 305 310
315 320 Ala Thr Arg Gln Ile Ser Gly Ala Gly Gly
Phe Val Asp Phe Val Arg 325 330
335 Gly Ala Thr Ile Ser Lys Gly Gly Ile Ser Ile Val Ala Met Pro
Ser 340 345 350 Thr
Ala Ala Gly Gly Lys Ala Ser Arg Ile Glu Met Met Phe Asp Ala 355
360 365 Gly Arg Pro Ile Thr
Leu Thr Arg Phe Glu Ser Phe Tyr Val Val Thr 370 375
380 Glu Tyr Gly Ile Ala Lys Met Arg Gly Asn
Ser Leu Arg Thr Arg Ala 385 390 395
400 Arg Gln Leu Ile Glu Ile Ala His Pro Asp Phe Arg Asp Glu Met
Lys 405 410 415 Glu
Phe Tyr Glu Lys Arg Phe Gly Glu Lys Tyr 420
425 381179DNAPseudomonas putida 38atgaacgatg tggtcattgt
ggctgcaacg cgcactgcaa ttggcagctt tcagggagcg 60ttggctacgg tgccagccgt
agatctggga gctgccgtca tcaaacagct cctgaaacag 120accggcttag acccagcaca
ggtagatgag gtaatcctgg ggcaagtgct cactgctggt 180gccggccaga atccggcacg
ccaagcggca atcaaagctg gactgccgtt ttcggttccg 240gcattaaccc tgaacaaagt
gtgtggctct ggtctgaaag cactgcatct ggcagcacaa 300gcgattcgct gtggtgatgc
ggaggtagtt atcgcaggtg gccaggagaa catgtccttg 360gccccttatg tgatgcctag
cgcgcgtacc gggcaacgca tgggccatgg ccagctcatt 420gatagcatga ttaccgacgg
tttatgggat gcgttcaatg actaccacat gggcattacg 480gccgagaacc tggtggacaa
gtacggcctg tctcgcgaac agcaggatgc ttttgcggct 540gaatcgcagc gcaaagcggt
cgcggctatc gaagcaggcc ggtttgacgc ggagatcacg 600cccattgtgt tgccgcagaa
gaaaggggaa ccgaaagtgt tcgcacgtga tgaacaaccg 660cgtccggata ccacagccga
atcgcttgcc aaattacgtc ctgcgttcaa gaaagatggc 720agtgtaacag ccgggaacgc
gtcaagcctg aatgatggcg ctgccgccgt tctgctgatg 780agcgccgcta aagcggaagc
gctggggtta cccgttttgg cgaaaatcgc cgcttatgcg 840tcagccggtg tcgatccggc
gattatgggt atcggtccag tgtccgccac tcagcgttgc 900ttggacaaag ccggttggca
gctggcggaa cttgacctga ttgaagcgaa cgaagccttt 960gcggcgcaag cactggccgt
tggcaatgca cttgcgtggg atgcggcacg cgtgaacgtt 1020aatggcggtg cgattgccct
tggacatccg attggggcta gtggttgccg tgtcctggtt 1080accctcctgc acgaaatgat
caaacgggac gtcaagaaag gcctggcgac cctgtgcatt 1140ggtggtggtc aaggcgttgc
gctggccatt gaacgctaa 1179391182DNASphingomonas
wittichii 39atggaggaca tttacattgt tggcgctgcc cgtacggcaa ttgcggactt
tggaggcgcc 60ttaaaggacg ttccaccagc tgatcttggc gtcattgtgg cacgtgctgc
tctggaacgc 120gctgggctcg aaccgggtga tgttcagaac gtagtaatgg gccaggtgat
gcctaccgaa 180ccgcgtgatg cctacttagc tcgcatggtg ggtgtgactg ctggtgtccc
gatcgaaacc 240ccagccctca cactgaatcg cctgtgtgga agcggagttg aggcaatcgt
taccggcgca 300aaagccatgg ttctgggaga atcggatatt gtccttgcgg gtggcgcgga
agtcatgagc 360cgtgttcctc acgtggtaaa aggtgcgcgt tggggtacca aaatggggaa
tgtcgagatg 420accgatggtc tgatcgaggc gttgtccgat ccgttcgaca aagtgcacat
gggcattacc 480gcggaaaacg tcgccgaacg gtaccagatc actcgcgaag cacaggatgc
tcttgctctg 540cagggtcatc aacgtgcggc acgcgcgatc gccgagggtc gcttcaaagc
ccagattgtc 600cctgtggaag tgaaaacgcg caaaggcgtt gtggcgttcg ataccgacga
gcacgttcgc 660ggggatgtgt ctgcggaaga actggcgaaa ctgcgtccgg tctttaagaa
ggatggcacc 720gtaacagccg ccaatgcgtc aggcatcaac gatggcgcag caatggtggt
cttggcaacg 780aagaaagccg tcgacgcgaa agggttgaaa cccttagccc gcatcttgtc
gtggggtcat 840gcaggggtag aaccgctgta tatgggcatt ggccccgtaa aagctgttcc
gattgcgctg 900gaacgcgcag gcttaactct ggcggatatc gacgtgattg aggccaatga
agcctttgcc 960gcgcaagcat gcgcagttgc gcaggaactc gggtttgacc cggataaagt
gaacccgaac 1020ggcagtggcg ttgcgctggg ccatccggta ggtgcgacag gtgcgatcct
gaccgtgaaa 1080acggtgtatg agctggaacg gattggaggt cgctatggtc tgatcacgat
gtgcattggc 1140ggcggtcaag ggattgccat ggtggtggaa cgttgtgcgt aa
1182401197DNAPseudomonas reinekei 40atgaagaacg cgttgattgt
ctctcctctg cgtactccga tcgggaagtt tggcggtgca 60ctggctccgc ttaccgcgga
acatctcgcc agtttcatga tctcgcaagt gatggcgcgt 120accggcgtgc caggccattc
gctggatgag gtgattgtgg cccagtctta tgcgagctcc 180gaagccccgt gcattggtcg
ctatgcggcg ttgagtgccg gcttaccggt ggaagtaccc 240ggatataccc tggatcgccg
ctgtggttca gggctgcagg ctgtcattga tgccagcatg 300atggtcaaaa ccggtaatgc
cgaagccgtt ctggtggtag gggttgaaag catgtcgaat 360atcgagtact actcaaccga
tatgcgctgg ggtgctcgcg ccggtagtgt ccggtttcat 420gatcggttgg agcgtggtcg
cgagcgcagt caaccgagcg aacgctttgg ccacatttcg 480gggatgccag aaacggcgga
caatctggcc ctcgactatg gcatctcacg ggaagaggcc 540gatagcttca gcgttcgtag
ccaccagaat gcagctgcgg cgtggcgtga gggtcgtttt 600gcggatgaag tggtggcagt
ggacgtacct ggtaaacgtg gcgctgtgac acgcgtcacg 660attgatgagg gtattcgcga
agatgcctct ctggagtcca tgaaagcttt acgcttgatc 720cgtccggaag gcgtttgcac
tgcgggcaac agttcgcagc agaacgatgc ggcggcaggc 780tgtctggttg tatccccgga
atacgcagct cgccatggcc tgactccgat ggctcgtctg 840gtcgactggg cggcagcagg
ctgtgaacca tcccgcatgg ggattggccc cgttcctgcg 900acccagaaac tgctgatgcg
tacagggtta tctctggcag aactggacct catcgagctt 960aacgaagcgt ttgcagccca
agcgctcgcc gtactgaaaa cgtggggtct ggatgacctg 1020tcccgcgtta acgtcaacgg
atcaggcatt agcttaggcc atccgatcgg tgcaacgggt 1080gttcgcatca tgaccaccct
tctgcacgaa atgcgtcgtc gtgaagcccg ctatggcctg 1140gaaacgatgt gcattggagg
tggccaggga ttagcggcac ttttcgaacg cgtgtaa 1197411203DNAPseudomonas
putida 41atgcgcgatg tctttatctg cgacgccatc cgtacgccga ttggtcggtt
tggcggtgca 60ctggcgggtg ttcgcgcaga tgatctggcg gccgttccgt tgaaagccct
tatcgaaccg 120aatccagccg tacagtggga tcaggttgac gaggtgttct tcggttgtgc
gaaccaagcg 180ggagaggaca atcgcaacgt cgcgcgcatg gccctgctct tagctgggct
gccagagagc 240attccgggag tcacgctgaa tcgtctgtgc gctagtggca tggatgcgat
cggtactgcg 300tttcgtgcta ttgcctctgg cgaaatggaa ctggcgattg caggtggggt
cgaatcgatg 360tcgcgtgcac cctttgtgat gggcaaagcg gaatccggtt atagccgcaa
catgaagctt 420gaggatacca ctattggttg gcgcttcatc aacccgctga tgaaatccca
gtatggagta 480gactccatgc ccgaaactgc ggacaacgtt gccgatgact accaggttag
ccgtgcggat 540caagatgcgt tcgcactgcg ctcacaacag aaagccgcag ctgcacaagc
agcggggttc 600tttgccgaag aaatcgtgcc ggtccgcatt gctcacaaga aaggcgaaac
cattgtggaa 660cgcgacgaac atctgcggcc ggaaaccacg ttagaggcgt tgaccaagct
caaaccggtg 720aatggcccgg ataaaaccgt tacggctggg aatgcaagcg gcgtgaacga
tggtgctgca 780gccttgatcc tggcgtctgc tgaggccgtg aagaaacacg gtctgacacc
acgtgcgcgt 840gtacttggca tggccagtgg cggtgtggct cctcgcgtca tgggaattgg
cccggttcct 900gcggtgcgca aactgacaga acgcttaggc gtggcagtga gcgactttga
tgtgattgaa 960ctgaacgaag cgtttgcgag tcaggggttg gcagtactgc gcgaactggg
tgttgcggat 1020gatgccccac aagtcaaccc taatggcggt gcaattgccc tgggtcatcc
gctcggcatg 1080tcaggggccc gtctggtttt aaccgcgctg catcagctgg agaaatcggg
cggccgtaaa 1140ggcttagcca ccatgtgtgt gggcgtaggt cagggactcg cgcttgctat
cgaacgtgtc 1200taa
1203421209DNABurkholderia xenovorans 42atgagcgaaa cgcacatgag
cggcaccaaa gccgatccca tcgtgattgt cggagtggct 60cgcaccccta tggcagcctt
tcagggcgac tttgcgacct tgtcggcacc gcagttaggg 120tcagtggcca ttcaagccgc
tgttcagcgt gccggactga aaccggaaca gatcgatgaa 180gtcgtaatgg ggtgtgtact
gccagctggt ctgggtcagg ctcctgcacg gcaagctgcg 240cttggcgcgg gattaccgct
ggccactggc tcaacgacgg tcaacaaaat gtgcggctct 300ggcatgcgtg cagcgatgtt
tgcccacgac atgctcgctg ccggctcagt tgacgtaatt 360gtagcgggcg gtatggaatc
catgacaaat gcgccgtatc tgcttcccaa ggcgcgtgcg 420ggaatgcgca tggggcatgg
gcaggtgatt gaccacatgt tctatgatgg tctggaagat 480gcatacgaga aaggtcgcct
tatgggcagc tttgccgagg aatgtgcagc gagtttcgac 540ttcacccgtg aagcgcaaga
tgcttttgcg gtggaatcgc tggcccgtgc gaaacgtgca 600aatgaagatg gctcgtttgc
ctgggaaatt gccccagtta aagtcgaatc tcgcaaaggt 660gaagtgacga tcgatcggga
tgagcagccg tttaaagcga acatcgagaa aatcccgacc 720ttaaagccgg cgttcagcaa
aacggggact gttacggcgg ccaacagcag ttccatttcc 780gatggtgcag ctgcgctggt
tatgatgcgc gaaagcactg ccaaacgcct cggcgttcag 840ccgattgcgc gcgttgtcgg
ccattctacc ctggcacaag aaccggcgaa atttaccacc 900gccccagttg gtgctatccg
caagctgttc gagaagaatg gctggcgtgc ggatgaagtc 960gacctgttcg aagtgaacga
agcgtttgcg gtggtgacaa tggcggcaat gaaagaacac 1020catctgcctc atgagaaagt
gaacgtcaat ggtggtgcct gcgcattagg ccatccgatt 1080ggcgcaagtg gagcacgcat
cttggtaacc ttgattggtg ccctgaagaa acgcggtggt 1140aaacgtggtg tggctactct
gtgcattggc ggtggcgagg caacagcgat ggggatcgag 1200ctcgtgtaa
1209431203DNABurkholderia
xenovorans 43atgacggaag cgttcctgtg tgatgcgatc cgcacgccga ttggccggta
tgcgggcgcg 60cttagctccg tacgcgcaga tgatctgggt gcggtaccgt tgaaagccct
tatggagcgt 120aacaaagagg ttgactggaa tgccatcgac gacgttatct acgggtgcgc
aaaccaagct 180ggagaggata accgcaacgt tgcccggatg tcgctcctct tagctggcct
gccgcaaggt 240gttcctggaa cgacagtgaa tcgcttgtgt ggcagtggca tggacgcggt
tggcattgca 300gcacgtgcca tcaagtcagg ggaagcagct ctgatggttg caggcggggt
ggaaagcatg 360tcacgtgcgc cgtttgtgac gggaaaagcc accagcgcct tttcccggca
ggcagaaatc 420tacgatacca ccattggttg gcgctttgtc aacccgctga tgaagaaact
ctatggcgtc 480gatagcatgc cggaaaccgg tgagaatgtt gccaccgact acaacattag
ccgcgcagat 540caagacgctt tcgccttacg ctctcagcag aaagcggcac gtgcgcagcg
tgatggcact 600ctggcacagg aaatcgtggg tgttaccatt gcccagaaga aaggcgatcc
agtcaccgtg 660tcgcaggacg aacatccgcg cgaaaccagt cttgacgccc tggctaaact
gaaaggtgtg 720gtgcgtcccg atgggaccgt aactgctggc aacgctagtg gggtcaacga
tggggcagcg 780gcgttgctgc tggcgaatga ggaaacagcc cgtcgtttcg gtttgactcc
ccgtgcacgc 840gtgcttggta ttgccaccgc aggtgtagct cctcgcgtga tgggcattgg
accagcccct 900gcgacgcaga aactgttagc gcgtctgaat atgtccctgg atcagttcga
tgtcatcgaa 960ctgaacgaag cgtttgcgtc gcaaggcatt gcggtcctgc gcgcgttagg
agtcgctgat 1020gacgataccc gtgtgaatcc gaatggcggt gccattgccc tcggtcatcc
actgggtatg 1080tctggtgcgc gcctggtgac tacggcgatg tatcaactgc accgcacaca
aggccgcttt 1140gctctgtgca cgatgtgcat cggcgtgggt cagggcattg cgattgccat
tgaacgcgta 1200taa
1203441221DNARhodococcus jostii 44atggccgaag tgttcctcgt
ggatggtgcg cgtaccccac aaggtcgcta tggaggtgcc 60ctggcgggtg ttcgccctga
cgatctggca ggcttggtgg ttgcggaagc cgcgcgtcgc 120gcaggaattc cgggagatgc
ggtggacgaa gtgatccttg gggcagcgaa tcaagcaggc 180gaggataacc gcgatgtggc
tcgtatggct gtactcctgg cgggcctgcc cgatagcgtc 240ccaggctata ccgtcaatcg
cctgtgtgcg tcgggcctta ctgctgtggc gagtgccgcg 300cacaccattc gcagcggcga
agctgacatc gtcattgcgg gaggggtgga atcaatgacg 360cgtgctcctt gggtaatggc
caaaccgggt accccatggg cacgtccggg tgaagttgcg 420gacacgagct tgggctggcg
tttcacgaat ccccgcttta ccgccgctga tcgcgatgta 480ccggccggtg ctgggcctga
tgtccgcaaa gtaaccctgt cgatgggcga aactgcggag 540gaagttgccg ccctggaagg
cgtaacccgt gcggaatccg acgcatttgc tctccgttct 600caggaacgtg cgatcgcagc
ggttgatgcc ggtcggtttg aacgggagat tgttccggtg 660ccggttcgcg atggcgaact
ggcagcggac gagacaccgc gtcgtggtac gacactggag 720aaattaggga gtctgaaacc
cgtgtttcgt acgggcggta ttgtcactgc agggtccagc 780agttctctgt ctgatggcgc
ggcagcctta gttgtcgcca gcgaagcagc ggtggaaaag 840tacggcctga ccgtccgcgg
ccgcattgtc acgtcggcct cagcgggcat tgcaccgaac 900gtgatgggtc ttggtccggt
gccagcgact cgcaaagcgc tggcacgggc gaactggtcc 960atcagcgatc tgggtgctgc
ggagttgaac gaagcgttcg cagcacagag tctgggcgtt 1020atccgccagt taaagctgga
cgagtcgatc gtaaatgctg acggtggggc cattgctctc 1080ggacatccgt tgggctgctc
aggtgcccgc atcttactga ccctgttagg gcgcatggaa 1140cgcgaaggcg cccgtcgcgg
tcttgccaca ctgtgcgtgg gtgttggcca gggcgtggcc 1200atgctgattg aggccccgta a
1221451344DNABdellovibrio
bacteriovorus 45atgaagtcac cacgcgacgt tgtgctcgtc gaaggtgttc ggacaccgtt
cgcaaaagcg 60ggtaccaaac tgaaaaaagt ccacccagct gaactgggca aagtggcgct
taaacaggtc 120attgcgcaaa cgaacctgga tgtcaacctt gtggatgaag tgatcatcgg
gaatactgga 180aacccaccag attcggtgaa catctctcgt gttgttgccc tgaatgccgg
tattccgctc 240aaaacctcag cgtacacggt acaccgcaat tgcgcatctg cccttgaaag
cattagtaac 300ggctatgaga aaatcaaatc cggcacgatg gatgttattc tggcgggtgg
taccgagaat 360atgtcacaaa tgccgacttt accgccgaag aaattccagg aaatctatga
gaaactgttt 420gctgcaaaag gcccgaaaca agctctgccc ctgctgtgga gtctgttcaa
agcggatgtg 480aagcagatca aagccctgtt aagtgggaat atgcgtgacg agtacttccc
ggtgatttcg 540gtgatgatgg gcttaaccga tccctttgtg ggcattaaca tgggccagac
tgccgaaatc 600ttggcgaaag aatggggcct gtctcgcgaa acccaggata agttcgccct
gcgttcccat 660cagctggcga gcaaagccat gaaagagggt cgtatgcggg aagaaattgc
accggtgtat 720ctcgccccgg aatacaagga agttattagc gaggatatcg gtcctcgtga
tacccagacg 780atggaagcgc tggccaaact gaaaccgttc tttgacaaag cgacaggctc
tattaccgca 840ggcaacagct gtcccattac cgatggagcg gccatggttc tgatgatgtc
gcgcgagaaa 900gcagaagcgc tgggctataa gcctttggca accattcgct cctatgggtt
tgcgggcttg 960gaaccggaac gcatgggttt aggtcctgta tactccacac ctgtagctct
caaacgcgca 1020ggattaagca tgaaagacat tgggttggtc gaattgaatg aagcgtttgc
tgctcaagtc 1080ttatcgtgcc agaaagcgtt tgatagcgac aagtttggcc aggaaaagct
tggtctgagt 1140agcaaaatcg gcgaaatccg cgacgacatt ctgaacgtga atggcggtgc
tattgcactg 1200ggtcatccgg taggtgccac cggtacgcgc atcgttctga cgctggcgaa
agagatgaaa 1260cgtcgtaaca cccagtttgg gttggccact ctgtgtattg gcggaggcca
aggaggtagc 1320atgattctcg aaaatgaggg gtaa
1344461338DNACronobacter turicensis 46atgttttcgc ttctgcaggg
gaacgtcatg tctcaagctc tgccgcttgt cacgcgtcag 60ggcgatcgca ttgcgatcgt
ttccggcttg cgcaccccat ttgcccgcca agctaccgcg 120tatcacgggg ttcctgccgt
ggatctgggc aaaatggtag ttggggagct gctggcccgt 180tcggaaattc caccggatgt
cattgagcag ctggtattcg gtcaggtagt acagatgcca 240gaagcgccga acattgcacg
ggagattgtg ctgggtacgg gtatgtccgt tcacacagat 300gcgtatagcg tcagccgcgc
ctgtgcgact agcttccagg cagtggccaa tgtggcggaa 360agtctgatgg ctggcacgat
tcgtgcagga atcgcaggcg gagccgatag ctcaagtgtg 420ctgccgattg gtgtcagcaa
gaaactggct cgtaccctgg tggacgcgaa taaagcccgt 480acagccggcc agcgcctgaa
actgttctct cgcttacgcc tgcgtgatct gttgccggtt 540cctccggcgg ttgctgagta
ctcaacgggt ttacgcatgg gagatacggc cgaacagatg 600gcgaaaacgc atgggatcac
gcgtgaacag caagatgcac ttgcgcatcg ttctcaccaa 660ctcgcagccc aagcgtgggc
agagggcaaa ctccgcgaag aagttatgac cgcttacacc 720ccaccgtatc gtgaaccgct
gagtgaggac aataacatcc ggaagaattc gagcttggcg 780gactacacca aactgcgtcc
ggcatttgac cggaaacatg gcactgtgac agctgcaaac 840tccacccctc ttacggatgg
cgccgcggca gtgatcctga tgacagaatc acgcgcccgc 900gaactggggt tgaccccgtt
gggttatctg cgctcgtacg ctttcaccgc tgtcgatgtc 960tggcaggaca tgttgttagg
tcccgcctgg agtactcccc tcgcgctgga acgcgcgggt 1020ttaaccatgg ccgatctgac
gctgatcgac atgcatgaag cgtttgcctc tcagaccctt 1080gcgaacctca aactgctcgc
gtcagatcgc tttgcacgcg aagtgttagg tcgttcccaa 1140gctaccggcg aagtagacga
gagcaagttc aatgtgttgg gtggcagcat tgcgtatggc 1200catccgtttg cggcgactgg
tgcgcgcatg attacccaga ctctgaacga actgcgtcgt 1260cgcggtggag gctttggctt
agttaccgca tgcgcagcgg gcggtttagg ggcagccatg 1320gtgctggaag ccgaataa
1338471371DNAArthrobacter sp.
47atgtccttta acgggcaatc cgcaaccggt ccggatgaaa gtgcagcggc gcctgctgcc
60acacctggtg cgggtctgtt gcgcaaagcg gtagttgtgg gcggaaaccg catcccattt
120gcccgtacgg gtggtgctta tacgaagagc agcaatcagg acatgctgac agcggcgctg
180gacggcctga ttgctcgctt tggcctggcg gatgaacgca ttggggaagt ggcggctggt
240gcggttctga agcattcacg cgacttcaac ttaacgcggg aagccgtgtt aggcagtgcc
300ctctctgccg aaaccccggc atacgatctg cagcaggcat gtgcgacagg actggaaacg
360gtactcggtc tggccaacaa aatcaaatta ggccagattg attccgccat tgccggtgga
420gttgattctg cgtctgacgc cccgattgcg gttagcgagg gtctgcgcga agtcctgctg
480gacctgaatc gcgcgaaaac cctgccgcaa cgcctcaaag tgctggggcg cttacgtccc
540aaagatctgg ccccagatgc gccgaatacg ggagaacccc gtaccggact gtccatgggc
600gaacatcagg ccctgacaac tgcacagtgg aagattaccc gcgaagcaca ggacgagttg
660gcatacaaca gccatcgcaa cctcgcggct gcgtatgacg caggcttctt tgacgacctc
720ttgacgccgt atcgtggtct taaccgtgac tcgaacttac gcgcggatac cacgcgtgag
780aaattgtcaa cgctgaaacc ggtctttggc aagaatctgg gcgctgaagc gactatgacc
840gccggcaact cgaccccgct taccgatggc gccagtaccg tgcttctggc gagcgaggaa
900tgggctgatg cgcacgaatt accgaaactg gccactgtgg tggatggcga agcagcagcg
960gtcgattttg tccatggcaa agacggtctg ttaatggcgc cagcgttcgc ggtacctcgg
1020ctgttggcac gcaatggcct tactctcgac gatatcgatt tcttcgagat tcatgaggcc
1080tttgctggca ccgtgttgag tacccttgca gcttgggaag atgaagagtt cggtcgtacc
1140cgtctgggcc ttgatggtcc actggggtcg attgatcgtg caaaactgaa tgtgaatggg
1200tcgtcattgg cagctgggca cccgtttgcg gcgactggcg gccgtatcgt agccaccctg
1260gccaaaatgc tgcacgataa agggcaagtt gatggtcggc cggctcgcgg tctgatcagc
1320atctgcgcag ccggcggtca aggcgtcgtt gccattctgg aagctagcta a
1371481290DNACaulobacter segnis 48atggccacag cgagttcctc ggcggcatcc
tcttccggtg tctggttagc tgcgggtgtt 60cgcagcccat ttgccaaagt agatggtgcg
ttagccggtc atgacgccat tgggttatcg 120gtgcccgtgg tcaaagcgat gttagcacgt
gccaaaccgg actttgcggt ttggggtact 180gtgatcccga acctcacgtg gagtaacctg
gcccgtgagg tcctgttgga tgccggtggt 240gatccgacga ttccggcgtt ttccacggtt
atggcttgca gcaccagcat gattggcgcg 300attgaagcag ccggcatggt ggatggccgt
ggtcgcgatc tggcgctggt aggcggcgtg 360gaaagcatgt cacgtgtgca gttgggcctg
agtgttgcac tgagcgattg gattcgcaag 420ttccagaacg cgaaaaccgg ccaacagcgg
ctcgcggctc tgggagccct taacctgaaa 480gatgtacgct tattcatccc gaaggtcgtt
aaccgtgtta cgggactgtc aatgggagaa 540cataccgaga ttaccgcgaa agagtggaat
ttgtctcgtg cagaccaaga tgccattgct 600ctggcttcgc atcagggtgc cgtgaaaggc
tgggaaagcg gattctttga cgacctggtc 660attccagtgg gcgaagtgaa acgcgatgga
atcccgcgca aagacacatc tctggagaag 720ctcgcaaaac tgggcccagc ctttgacaaa
accagtggca aaggcaccct gaccgcgggg 780aattcctcac cccttaccga tggtgcggct
gcggtttggg taggttctgc agctggcatg 840gcgcgtttac cgggtgagac accgaaggtt
cgtctggtcg actacgaagt caccagcatc 900gatctgcgtc atgaaggcct gttgatggcg
ccggcttatg gggtaccgcg catgctcgcc 960cgcaatgggc tgacttatgc cgatgttggg
ctgtgggaaa tccacgaagc tttcgcagca 1020caggtgttga gccacattgc ggcatgggaa
tcggccaagt tcctgagcga gaaagccggc 1080gtgactacgc ctatgggtgc atttcctcgc
gaacgcatga acccgaatgg cggctctctg 1140gctcttggtc acccatttgg tgcaactggc
gcgcgcatca tttcgcagac cgtgaaagaa 1200ctggcagcgc gccctaaagg ggaacgggcg
atcgtgagta tctgtgcgga tggtgggcaa 1260ggcacgatga tgcttctgga atcagcctaa
1290491212DNADinoroseobacter shibae
49atgacggaag cctacatcta tgacgcgatt cgctcaccac gtggtaaagg gcgcaaagat
60ggctccctgc atgaagtcac tgcggtcagc ctgagtgccc aaaccctgaa tgcgattaaa
120gaccgcaatg gcctgaccgg gcatgcggtt gaagatgtga tttggggtaa cgtcacccag
180gtaatggaac agggtggatg tctggcacgt acggcggtac ttgcgtctga tctggatgag
240agcattcctg gcttagcgat caaccgcttt tgcgccagcg gtttagaagc cgtcaatttg
300gcggccaatc aagtgcgtgg aggcggtgga caggcataca ttgctggcgg cgttgagatg
360atgggtcgcg ttcccatggg tagcgatggc gccgcaatcg cagccgaccc gtctgtggcg
420atgaaaacct actttgtgcc gcaaggtatt tccgccgata tcatcgcaac ggaatatggc
480attagccgcg atgatgcgga tgccctggct gtagcgtcgc aacgtcgggc taaagctgcg
540tgggatgaga accgcttcaa tgggtccgtg tttaccgttc gcgatcagaa cggtctgaac
600attctggacc acgacgagta tatgcgtcct gaaactgaca tgcagagcct gggtgcactg
660aaaccggctt tcaaagatat gggtgaacag atgcccggtt tcgacaagat tgcgctcatg
720aagtatccgc atttagagaa aatcgaacac attcatcacg ccggtaactc gtcaggcatt
780gttgatggga gtgcagcgct ccttatcggc aataaagcct tcggcgaagc tcatggcctt
840aaaccacgtg cagtgatcaa agccacagcc aagatcggaa ccgatccgac gattatgctg
900actggcccgg ttccagcgac cgaaaagatt ttggcagatt ctggcatgag tatctcagac
960attgatctgt tcgaagtgaa cgaggccttt tcgtcggtcg tgttgcggtt tatgcaggcg
1020tttgatgtcg accacgacaa agtgaacgtg aatggcggtg cgattgcaat ggggcatccg
1080ttgggtgcaa caggcgcgat gattctgggc accctgctcg atgagctgga acgtacgggc
1140aaagggaccg ggttagctac actgtgcgtt gctagtggaa tgggcgcggc tacgatcatc
1200gaacgcgtat aa
1212501185DNABurkholderia xenovorans 50atgactcgcg atacgcgcga cgtggtgatc
gtagatgcgg ttcgcacacc aattggcaaa 60ttccgtggtg ccttggctgg tgtacgtgcc
gatcatctgg gtgcactcgt cattgacgaa 120ctgatccgcc gtgccggtgt caaaccgcag
gcggtgaacg atgtggtctt tgggtgcgtt 180acccagatcg gtgagcagtc agcgaacatt
gcccgcacct ctgtgttagg ggcgggatgg 240ccggaaacca tcccaggcct gaccattgac
cgcaagtgtg gtagtgggga agaagcagtg 300cacattgccg ccggcctgat cgcgtttggc
gcagcggatg tgatcgttgc cggtggcgct 360gaatctatgt cgcgtgtccc gatgggtagc
aatcgggatc tgcatggcga agcattcggg 420tggatggctt cagagcgctt cgagctgacc
tcgcaaggcg aggccgccga acgtctgtgc 480gattgttggg cgctgacacg tgcgcaactt
gacgcgtact cggtagagtc tcatcgccgc 540gctgcagcgg ctgcggctga aggctggttt
gcgcgcgaga ttgtgcccgt tccagttggt 600caggtgcgcg aaaagtccct cgaaggcgaa
gccgcgttat ttgccgctga tgaaacgatt 660cgccctggca ccaatgcgga caaactggcg
accttgaaat cctcgtttcg cagtgatggt 720cgcctgacag ccgggaatag ctcacagatt
agcgatggtg cggcagcact tctgctgatg 780agcagtgaca aagcacgtga actcggagtc
aaagctcgtg cccgggttcg tgcggttacc 840acggtgggta gtgatccgac cctcatgctt
acgggaccga ttcttgcgac ttgtcaggta 900ctggagaaag cgggcttagg tttgagcgat
atcgacctgt tcgagatcaa cgaagcgttt 960gcgcctgtac cgctggtctg gatgaaggaa
ttcggtgttc cccacgcaaa actgaacgtg 1020aatggcgggg ccattgcact gggccatccg
ttaggcgcat ccggcgcacg tatcatgacg 1080agcatgttgc acgaactgga acgccgggga
gcacgttatg gcctgcaagc catttgctgc 1140gctggaggta tgggcactgc cacgctgatt
gaacgcttag attaa 1185511149DNAGeobacillus kaustophilus
51atgcgtgaag ctgtgattgt agaagccgta cgcacacctg tcgggaaacg gaatggcgta
60ttccgcgatg tgcatcccgt acatctggca gcggtcgtgt tggatgaagt cgtccgtcgc
120gcagggatgg ataaaggtgc ggttgaggac atcgtgatgg ggtgtgtgac ccctgtggca
180gagcaaggtt acaacattgg ccgtttagca gctctggagg ccggcttccc gattgaggtg
240ccagcggttc agattaaccg catgtgcggc agtggacagc aggccatcca ttttgccgcc
300caagaaatcc gcagtggcga tatggatgtc acgattgcgg ccggtgtgga atcgatgacc
360aaagtcccca ttctcagcga tgggaatgag cgcaccattc cgccaagcct ccacgaaaag
420tacgagttca tccaccaagg tgtatctgcg gaacgcattg cgaagaaata cggactgaca
480cgcgaagagt tggacgcgta tgcgtatgaa agccaccagc gtgcccttgc tgcactgcgt
540gaaggcaaat ttcgggcgga aatcgttccg gtaaaaggcc ttgatcgtga tggccgcgaa
600atcctggtga ccgacgatga aggtccgcgt gctgatacta gcccggaagc cttagcagcg
660ctgaaaccgg tgtttcagga agatggcctg attaccgcgg gtaatgcttc ccagatgtct
720gatggcgcag ccgccgttct ccttatggaa cgtgaagcgg cacgtcgctt tggcctgaaa
780cctaaagctc gcattgtcgc gcaaaccgtt gtgggatcag acccgaccta tatgctggat
840ggtgttattc cggcaactcg tcaggtcctg aagaaggccg ggctgtcgat cgacgacatt
900gacctgatcg aaatcaacga ggcgtttgcg ccggttgttc tggcttggca gaaagaaatt
960ggcgccccgc tggagaaagt gaacgtgaat ggtggtgcga ttgccttagg ccatccactg
1020ggtgcaactg gcgcgaaact gatgacgtcc ttggttcacg agttagaacg gcgcggtgga
1080cgctatggcc tgttgacgat ctgcattggg catggtatgg cgacggctac gatcattgaa
1140cgcgaataa
1149521137DNABeijerinckia indica 52atgaccaaag tggtcattgc aggctacatt
cgctcaccgt ttaccctggc gaagaaaggg 60gaactggcca cggtgcgtcc tgacgatttg
gcagctcaag tcgtgaaagg cctgatcaag 120aaaaccggca ttccggccga ggacatcgaa
gatctgcttc tggggtgtgc gttcccagaa 180ggtgaacagg gcttcaacgt agctcgcctg
gtttcgtttc tggcgggtct gccgctgtcg 240gtgggcgcaa gcaccgtgaa tcgcttttgt
ggctcttcga tgactaccgt tcacatggca 300gcaggagcca ttcagatgaa cgcaggaaac
gcctttatcg cggctggtgt cgaaagcatg 360agccgcgtac cgatgatggg cttcaatcct
ctgccgaatc ccgaattggc cgcgacaatg 420cctggcgcct atatgggcat gggtgatacg
gctgagaatg tggctgccaa atggaccatt 480tcccggaaag aacaggaaga attcgccctg
cgtagccatc aacgtgcgac tgccgcgcag 540aaggaaggac gtttgaccgg tgaaattatc
ccgattaccg gccggaaagg gacgatcacg 600acggatggct gcattcgtcc cgataccaca
cttgagggtc tggcagaact caaaccggcg 660ttctcagcta atggagtcgt aaccgcgggt
acttcctctc cgctgacgga tggcgccgct 720gccgttctcg tgtgcagtga ggactatgcg
aaacatcacc acttagacgt tttagcgagc 780gtgaaagcta tcgcggtaag cggttgctct
ccggagatta tggggattgg tccagttgcg 840gcctcacgca aggccttagc gcgtgcaggc
ctggaagcgg gtcagatcga tattgtcgaa 900ttgaacgaag cttttgccag tcagtccatt
gcgtgtatgc gcgaactgaa cctgagtccg 960gaccgcgtta acatcgatgg tggcgcgatt
gcactgggcc atccactcgg ggcgacaggg 1020gcgcgcatcg tgggaaaagc agcatcgctg
ctcaaacgcg agaaaggcaa atacgcctta 1080gcgactcagt gcattggtgg tggccaaggt
atcgccaccg ttcttgaggc attttaa 1137531164DNACitrobacter freundii
53atggaacagg tcgtgatcgt cgatgccatt cgcacgccaa tgggccgttc aaaaggtgga
60gcctttcgca atgtgcgcgc cgaagatctg tctgcacatc tgatgcgctc tctgctggcc
120cgtaaccccg cattagatcc gacagcgctg gacgacatct attggggctg tgtgcagcag
180acactggaac aagggttcaa cattgcgcgt aatgctgctc tgctggcgga aatccctcac
240agtgtgccag cagtaacggt taatcgcttg tgtggctcca gcatgcaagc tctccatgat
300gctgcacgca tgatcatgac cggcgatgcg caagcgtgcc tcattggtgg tgtagaacac
360atgggccatg tccccatgtc acacggggta gactttcacc cgggtatgag ccgcaatgtg
420gccaaagcag ccggtatgat gggcttaact gcggagatgc tgtcccgcat gcacggcatt
480agtcgtgaaa tgcaggatgc ctttgcagcg cgcagtcatg cgcgtgcttg ggcggccact
540caatctgggg ccttcaagaa cgagattatt ccgactggcg gccatgatgc ggatggggta
600ctcaaacagt tcaactacga tgaggtgatt cggccggaaa ccaccgttga agccttgtcg
660accttacgcc cggcctttga tcctgtttct ggtaccgtta ccgcgggcac gtcgtccgcg
720ttgagcgacg gtgcggcagc catgcttgtg atgtcagaaa gccgtgctcg tgaacttggc
780ttaacaccgc gtgcgcgcat tcgtagcatg gcagtggttg gatgtgatcc gagcattatg
840gggtatggac cagtcccggc ttcgaaactc gccttgaaga aagcgggcct gagcacgtcc
900gacattgggc tgtttgaaat gaacgaagct tttgcagcgc agattctgcc gtgcatcaaa
960gacctgggtc tgatggagca gatcgacgag aaaatcaacc tgaatggcgg tgcaatcgcc
1020cttggccatc ctctgggttg cagtggtgcg cgcatttcga ccacgctgtt aaacctgatg
1080gaacgcaaag atgtccagtt cggtcttgcg accatgtgca ttggactggg tcaaggcatc
1140gcaaccgtgt tcgagcgggt ttaa
1164541197DNACupriavidus necator 54atgaaacagt tacaggatgc gtacattgtt
gcggccactc gcagtccgat tggaaaagca 60cccaagggag ccttcaagaa tacgcgcccg
gacgatctgt tagcgacgat ccttaaagcc 120gctgtggccc aggtgccgaa tctcgatccg
aaactgatcg aagatgcgat cgtcggttgc 180gcaatccctg aggctcagca aggcctgaat
gtagcccgca ttggtgcgct gctgagtgga 240ctgccaaaca cggttggcgg cattaccgtt
aatcggtttt gcgcatctgg ggtcagcgct 300gttgcgatgg cagccgatcg cattcgcgtc
ggtgaatccg acgtcatgat tgctgccggt 360gtggagtcaa tgagcatggt gcccatgatg
ggtaactctc cgagcatgag tccggaaatc 420tttacccgtg acgaaaacgt tggcattgcc
tatgggatgg gtctgacagc cgagaaagtg 480gctcagcagt ggcaagtaag ccgcgaagat
caggatgcgt tttcgctggc atcccaccag 540aaagcaattg ccgcgcaaca ggcaggcgaa
ttcaaggatg agatcacccc gattgagatt 600gtggagcgtt tccctgacct ggcaagtggg
caagtgaacg taaagacccg cacgattagc 660ctggatgaag gcccacgtcc ggaaacctct
cttgaaggct tgggtaaact ccgtccggtc 720tttgccaaca aaggctcagt aaccgcaggc
aatagctcgc agacttcgga cggtgcaggc 780gcgctgattt tggtgtcgga gaaaatcctg
aaacagttca acctggtacc gttggcgcgg 840tttgtgtcat ttgccgtccg tggtgttccg
ccggaaatca tgggtattgg tcccaaagaa 900gcgatccctg cagctctgaa agccgcgggc
ctgacccaag accaactgga ttggatcgaa 960ctcaacgaag cctttgcggc gcaatccctc
gctgtgatgc gtgaccttca gttagatcca 1020gcaaaagtga atcgcatggg tggcgcgatt
gcgctgggcc atccattggg agctaccggc 1080gcgatccgta gcgcgacagt cgttcatgcc
ttacgtcgcc acaacctgaa atatgggatg 1140gttacgatgt gtgtggggac tgggatgggt
gctgcgggca ttttcgaacg cgtttaa 1197551248DNAGordonia bronchialis
55atggcccctt gctcagtgaa agcgatgcca gaagcagtga tcgtagctca tgcacgtagt
60cccattggtc gtgctggtaa aggctcgtta aaggatgtcc gtcctgacga actgagccgc
120cagatggtag ccgctgctct cgcgaaagtg ccggaactgg caccctcgga catcgaggat
180attcactggg gtattggcca gccaggcggc caaggtgggt acaatattgc gcgtgtcatt
240gccgtggaat tgggctatga tcacatcccg ggtgtgaccg tgaatcgtta ttgctcgtcg
300agcctgcaga ccacacgcat ggcactgcat gcgattaaag caggagaagc ggatgttctc
360attagtggag gggttgagag tgttagcagc tttggcattt ccggtggggc agatggcgcc
420ccggatagta agaacccagt attcgatgac gcgcaagcac ggaccgcgaa agctgccgaa
480ggtggagcac cggcttggac tgatccacgt gagcaaggct tgattccgga tgtttacatc
540gcaatgggcc aaacggccga aaacgtagcc tcttttaccg gcatctctcg cgaagatcag
600gatcggtggt ctgtcctgag ccagaatcgc gccgaagagg ccattaacgc gggcttcttc
660gaacgcgaga tcgacccggt tacgcttccg gatggtagca cggttaacac cgatgatggc
720cctcgtgcgg gcactaccta tgagaaagtc tcacagctga aaccggtgtt tcgccctgat
780ggcaccgtga ctgcgggcaa tgcgtgtccg ctcaatgacg gtgctgccgc actggtgatt
840atgtccgaca gcaaagcgaa gcaactgggt ttaacgccct tagcgcgtgt ggtggcgaca
900gcggccactg gactgtcacc ggaaatcatg ggtctgggtc cgattgaagc catccgcaaa
960gttctgcgca tctctgggat gtccctgagc gacattgacc tggtcgaaat caacgaagcc
1020tttgcagtcc aggtactggg gagtgcgaac gagttgggga ttgaccacga caaactgaac
1080gtgtcaggtg gcgctattgc gttgggtcat ccgtttggca tgaccggggc ccgcattacc
1140acgacgcttc tgaacaatct gcagacacgc gataaaacct tcggaatcga atccatgtgt
1200gttggcggtg gccagggtat ggcgatggtc cttgaacgct tatcgtaa
1248561182DNABurkholderia sp. 56atgcgtgagg ctgtgattgt ctccaccgca
cgtacgccct tgaccaaagc gcatcgtggc 60gagttcaaca tcacaccagg cccaaccctc
gcctcgtttg ctgttcgcgc agcagtagaa 120cggagtggtg tcgaccctga tatcattgag
gatgcgatcc tgggttgcgg ctatccggaa 180ggcaccactg gccgcaatgt tgcacgccaa
agcgttattc gtgccggtct gccactgtcc 240attgcaggca caacggtcaa tcggttttgt
gcctcaggcc ttcaggcaat tgcgatggca 300gctgggcgca ttgtggtcga tggagcgccg
gcaatgattg cgggcggtgt tgagagcatt 360tcgaacatcc agacccgcga agatggagtg
tctggcctgg acccgtggat tgtcgagcac 420aaaccctcac tgtacactgc gatgatcgat
acagcggata ttgttgctcg ccgttatggg 480attagtcgcg aagctcagga tcagtttagc
gtggagagcc aacgtcgcac tgccgaggcg 540caacaagcgg gacgttatgc agacgagatt
atcccggtta ccacgaccat ggccattacg 600gacaaggaaa cccgtgcggt aagctatcgc
gaagtgacgg tgtctgccga caattgcaac 660cgtccgggga cgacctacga agcattagcg
aaacttgcgc cggtaaaagg tcctgatcag 720ttcattaccg cagggaatgc gtcccagaac
gcggatggtg cctcggcgtg cgtactgatg 780gaagcgaaag ccgccgaacg ggccaacttt
gcgccactgg gcgcgtttcg cggcttagct 840ttggctgggt gtgaaccgga tgaaatgggg
attggtccgg ttcttgctgt cccgaaactc 900ctggcacgcc atggcttaac ggtggacgat
atcggtttgt gggaactcaa cgaagccttt 960gccagtcagg ccgtatactg ccagaaacgc
ctggaaatcc cttctgaacg cctgaacgtg 1020aatggtggtg ccatttcaat cggtcatccg
ttcggcatga ctggcagccg tctggtgggc 1080cacgttctga tcgaaggacg ccgccgtggc
gtcaagtacg cggtggtgac catgtgtatg 1140gccggtggca tgggtgcggc tggtctgttc
gaaatctatt aa 1182571212DNAArthrobacter arilaitensis
57atgcagcagg catacctgta tgacgcgatt cgcaccccgt tcggtaagat cggtggtgcc
60ctgagcagtc accgtcccga tgatctggcc gcacatgtcg tacgcgaact ggttgcacgc
120agccccaaac tggacgtagc cgatatcgac gaaagcatct ttggcaatgc caacggtgct
180ggcgaagaaa accgcaatgt agcgcgtatg gctacgttac tcgcgggatt gccgacttcg
240ctcccgggaa cgaccatgaa ccgcctttgc ggttctagct tggatgcgag tattgccgcc
300tcacgccaga ttgccacagg cgatgcggac cttgttctgg tgggtggcgt ggaaagcatg
360tcccgtgcgc catgggtcct gcctaaaacc gaacgtccat ttccgatgtc gaacctggaa
420ttagcgaata cgacgcttgg atggcgtctg gtgaatccgg caatgccagg ggaatggact
480gtgtcgttag gcgaagcgac cgaacaactg cgcgaaaagc acggtatctc gcgcgaggat
540caggacgagt tcagtgctgc gtcacatcag cgtgcagcag cagcctggca agcgggcaaa
600tacgacaacc tcgtggttcc tgtcccgccg gcaaacaaac gcggcacgga agtgacacgc
660gatgaaacga ttcgcgccga tagcactgcg caaaccctgt ccaaattacg taccgtcttt
720cgcaccggcg aaaacgcgac tgtcaccgct gggaatgcct ctccaatgag tgatggtgcg
780agcgctgctt tcattgggtc agaacgtggc ggtgaactgt taggcgccgc gcctattgct
840cgcatcgcgt ctaatggcgc cgctgcgctt gatccgcagt tctttgggtt tgccccggtt
900gaggcagcga acaaagcact ggcgaaagca ggactgaagt ggtccgacat tgctgcggtg
960gagctgaacg aggcctttgc agcccagtct ctcgcgtgta tccgggcgtg ggatattgat
1020ccggcgattg tgaatgcatg gggcggtgca atctccattg gccatccgtt gggtgctagc
1080gggctgcgta ttctgggcac agttgcgcgt cgcctggcgg aatcagggga gcggtatggt
1140ctggccgcca tctgcattgg cgttggtcaa ggcttggctg ttgtagtgga gaacatcaat
1200gccaccaaat aa
1212581206DNAEscherichia coli 58atgcgtgaag cctttatttg tgacggaatt
cgtacgccaa ttggtcgcta cggcggggca 60ttatcaagtg ttcgggctga tgatctggct
gctatccctt tgcgggaact gctggtgcga 120aacccgcgtc tcgatgcgga gtgtatcgat
gatgtgatcc tcggctgtgc taatcaggcg 180ggagaagata accgtaacgt agcccggatg
gcgactttac tggcggggct gccgcagagt 240gtttccggca caaccattaa ccgcttgtgt
ggttccgggc tggacgcact ggggtttgcc 300gcacgggcga ttaaagcggg cgatggcgat
ttgctgatcg ccggtggcgt ggagtcaatg 360tcacgggcac cgtttgttat gggcaaggca
gccagtgcat tttctcgtca ggctgagatg 420ttcgatacca ctattggctg gcgatttgtg
aacccgctca tggctcagca atttggaact 480gacagcatgc cggaaacggc agagaatgta
gctgaactgt taaaaatctc acgagaagat 540caagatagtt ttgcgctacg cagtcagcaa
cgtacggcaa aagcgcaatc ctcaggcatt 600ctggctgagg agattgttcc ggttgtgttg
aaaaacaaga aaggtgttgt aacagaaata 660caacatgatg agcatctgcg cccggaaacg
acgctggaac agttacgtgg gttaaaagca 720ccatttcgtg ccaatggggt gattaccgca
ggcaatgctt ccggggtgaa tgacggagcc 780gctgcgttga ttattgccag tgaacagatg
gcagcagcgc aaggactgac accgcgggcg 840cgtatcgtag ccatggcaac cgccggggtg
gaaccgcgcc tgatggggct tggtccggtg 900cctgcaactc gccgggtgct ggaacgcgca
gggctgagta ttcacgatat ggacgtgatt 960gaactgaacg aagcgttcgc ggcccaggcg
ttgggtgtac tacgcgaatt ggggctgcct 1020gatgatgccc cacatgttaa ccccaacgga
ggcgctatcg ccttaggcca tccgttggga 1080atgagtggtg cccgcctggc actggctgcc
agccatgagc tgcatcggcg taacggtcgt 1140tacgcattgt gcaccatgtg catcggtgtc
ggtcagggca tcgccatgat tctggagcgt 1200gtttga
1206591185DNACupriavidus necator
59atgacgcgtg aagtggtagt ggtaagcggt gtccgtaccg cgatcgggac ctttggcggc
60agcctgaagg atgtggcacc ggcggagctg ggcgcactgg tggtgcgcga ggcgctggcg
120cgcgcgcagg tgtcgggcga cgatgtcggc cacgtggtat tcggcaacgt gatccagacc
180gagccgcgcg acatgtatct gggccgcgtc gcggccgtca acggcggggt gacgatcaac
240gcccccgcgc tgaccgtgaa ccgcctgtgc ggctcgggcc tgcaggccat tgtcagcgcc
300gcgcagacca tcctgctggg cgataccgac gtcgccatcg gcggcggcgc ggaaagcatg
360agccgcgcac cgtacctggc gccggcagcg cgctggggcg cacgcatggg cgacgccggc
420ctggtcgaca tgatgctggg tgcgctgcac gatcccttcc atcgcatcca catgggcgtg
480accgccgaga atgtcgccaa ggaatacgac atctcgcgcg cgcagcagga cgaggccgcg
540ctggaatcgc accgccgcgc ttcggcagcg atcaaggccg gctacttcaa ggaccagatc
600gtcccggtgg tgagcaaggg ccgcaagggc gacgtgacct tcgacaccga cgagcacgtg
660cgccatgacg ccaccatcga cgacatgacc aagctcaggc cggtcttcgt caaggaaaac
720ggcacggtca cggccggcaa tgcctcgggc ctgaacgacg ccgccgccgc ggtggtgatg
780atggagcgcg ccgaagccga gcgccgcggc ctgaagccgc tggcccgcct ggtgtcgtac
840ggccatgccg gcgtggaccc gaaggccatg ggcatcggcc cggtgccggc gacgaagatc
900gcgctggagc gcgccggcct gcaggtgtcg gacctggacg tgatcgaagc caacgaagcc
960tttgccgcac aggcgtgcgc cgtgaccaag gcgctcggtc tggacccggc caaggttaac
1020ccgaacggct cgggcatctc gctgggccac ccgatcggcg ccaccggtgc cctgatcacg
1080gtgaaggcgc tgcatgagct gaaccgcgtg cagggccgct acgcgctggt gacgatgtgc
1140atcggcggcg ggcagggcat tgccgccatc ttcgagcgta tctga
1185601284DNAClostridium viride 60atggcgcagt ttgtcactgc tcaggaagcc
gttaaacaca tcccgaatgg cagccgtgtg 60gtgcttgcgc attctacagg agaaccgcgt
actctggtga aggcaatggt tgaaaactac 120gagcagtata aagacgtcga ggtttgccac
atgctgggcc tgggacctta cgaatacacc 180aatccggaga tgaaagggca tctgtggcac
aactcgctct ttatgggccc aggtggacgg 240aaagccttta acgaaaatcg ccttgacttt
acgccagggt acttctgcga tagcatcaaa 300ttctttcgtg agggctatct gcccgttgat
gtgctgatga tgaccgtatc gcctccagat 360aaacatgggt atgtgacgtg tgggattacg
tgcgatttca ctatgccagc atttgaatgc 420gccaaaatgg tcatcgtcca ggtgaacaag
aatatgccgc gcacgttcgg tcaaaccgca 480atccacctgg acgacatcga tttcgcggta
gaagcagatg atccgctgta tggcagtgta 540ccgggtgaat tgacagacat tgatcgcaaa
attggtgaac attgtgcctc gttaatcaac 600gatggcgctt gtctgcaatt agggattggc
ggcattccga atgccgtctt gacctatctc 660accgaaaaaa acgatatggg cattcattcc
gagatgctct ctgatggcat tctgcagctg 720attaaagccg gcaacatcaa caatagcaag
aaacagattc acgtgggtaa atcagcggtt 780accttcttga acggtagtca ggaactgtac
gattatgtgg acgataatcc gagcgtagaa 840ttttatccgg tggattacat caacgacccc
tacgttattg gcaagaacga caatatggtg 900tccgttaatt cagcgttatc ggtggatctg
atggggcaaa ttgttgcaga taacctgagt 960gcgacgcgcc agatctctgg tgctggtggt
ttcgtagact ttgtccgtgg agccaccatc 1020tcaaaaggcg gcatcagcat tgtggctatg
cctagcactg cggctggtgg taaagcgagt 1080cggattgaaa tgatgtttga tgccggtcgc
ccgattaccc tgacacgctt tgagagcttc 1140tatgttgtca cggaatacgg cattgcgaaa
atgcgcggta attccttacg tacccgtgca 1200cgccaactta tcgaaattgc gcatccggat
tttcgtgacg aaatgaaaga gttctatgaa 1260aagcgctttg gcgagaaata ttaa
1284617PRTArtificial SequenceDescription
of Artificial Sequence Synthetic 7xHis tag 61His His His His His His
His 1 5
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