METHODS AND MATERIALS FOR PRODUCING 7-CARBON MONOMERS

Abstract

This document describes biochemical pathways for producing 7-aminoheptanoic acid using a .beta.-ketoacyl synthase or a .beta.-ketothiolase to form an N-acetyl-5-amino-3-oxopentanoyl-CoA intermediate. 7-aminoheptanoic acid can be enzymatically converted to pimelic acid, 7-hydroxyheptanoic acid, heptamethylenediamine or 1,7-heptanediol or corresponding salts thereof. This document also describes recombinant microorganisms producing 7-aminoheptanoic acid as well as pimelic acid, 7-hydroxyheptanoic acid, heptamethylenediamine and 1,7-heptanediol or corresponding salts thereof.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/263,299, filed Dec. 4, 2015, the contents of which are incorporated herein by reference.

TECHNICAL FIELD

[0002] This invention provides methods for biosynthesizing 7-carbon monomers. For example, the present invention provides methods for making N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof using a polypeptide having the activity of a .beta.-ketoacyl synthase or a .beta.-ketothiolase and enzymatically converting N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof to 7-aminoheptanoic acid or a salt thereof using one or more polypeptides having the activity of a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase, a trans-2-enoyl-CoA reductase, a .beta.-ketothiolase, a thioesterase or a CoA-transferase and a deacetylase or methods using microorganisms expressing one or more of such enzymes. This invention also provides methods for converting 7-aminoheptanoic acid to one or more of pimelic acid, 7-hydroxyheptanoic acid, heptamethylenediamine and 1,7-heptanediol or the corresponding salts thereof using one or more polypeptides having the activity of isolated enzymes such as dehydrogenases, reductases, acetyltransferases, deacetylases, and transaminases or methods using recombinant microorganisms expressing one or more such enzymes.

BACKGROUND

[0003] Nylons are synthetic polymers composed of 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 polymerization of 7-aminoheptanoic acid, whereas Nylon 7,7 is produced by condensation polymerization of pimelic acid and heptamethylenediamine. No economically cost competitive petrochemical route exists to produce the monomers for Nylon 7 and Nylon 7,7.

[0004] Given no economically cost competitive petrochemical monomer feedstock, biotechnology offers an alternative approach via biocatalysis. Biocatalysis is the use of biological catalysts, such as enzymes, to perform biochemical transformations of, for example, bioderived feedstocks and petrochemical feedstocks which can both be viable starting materials for the biocatalysis processes.

SUMMARY

[0005] Accordingly, against this background, it is clear that there is a need for sustainable methods for producing one or more of 7-aminoheptanoate, pimelic acid, 7-hydroxyheptanoate, 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 polypeptide having the activity of a .beta.-ketoacyl synthase or a .beta.-ketothiolase to produce 7-aminoheptanoate or a salt thereof, which can be converted in one or more enzymatic steps to pimelic acid, 7-hydroxyheptanoic acid, heptamethylenediamine or 1,7-heptanediol or corresponding salts thereof. Pimelic acid and pimelate, 7hydroxyheptanoic 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 pH.

[0006] For compounds containing carboxylic acid groups such as organic monoacids, hydroxyacids, amino acids 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.

[0007] For compounds containing amine groups such as but not limited to organic amines, amino acids 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.

[0008] For compounds containing both amine groups and carboxylic acid groups such as but not limited to amino acids, 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-acid), carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-l-carboxylic 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.

[0009] It has been discovered that appropriate non-natural pathways, feedstocks, microorganisms, attenuation strategies to the microorganism's biochemical network and cultivation strategies may be combined to efficiently produce 7-aminoheptanoate as a C7 (7-carbon) building block, or convert 7-aminoheptanoate to other C7 building blocks such as pimelic acid, 7-hydroxyheptanoic acid, heptamethylenediamine or 1,7-heptanediol or the corresponding salts thereof.

[0010] In some embodiments, a terminal carboxyl group can be enzymatically formed using a thioesterase, a CoA transferase, a .omega.-transaminase, an aldehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase or a 7-oxoheptanoate dehydrogenase. See FIG. 2 and FIG. 3.

[0011] In some embodiments, a terminal amine group can be enzymatically formed using a carboxylate reductase, 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. Furthermore, 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 and be capable of transferring at least one amine group separated from a carbonyl group by at least one methylene insertion.

[0012] In some embodiments, a terminal hydroxyl group can be enzymatically formed using an alcohol dehydrogenase. See FIG. 5 and FIG. 6.

[0013] In one aspect, this document features a method of producing N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof. The method includes enzymatically converting .beta.-alanine to N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof via N-acetyl-3-aminopropanoate and N-acetyl-3-aminopropanoyl-CoA. .beta.-alanine is converted to N-acetyl-3-aminopropanoate using a polypeptide having the activity of an acetyl-transferase classified under EC 2.3.1.-. N-acetyl-3-aminopropanoate is converted to N-acetyl-3-aminopropanoyl-CoA using a polypeptide having the activity of a CoA ligase classified under EC 6.2.1.- or a CoA-transferase classified under EC 2.8.3.-.

[0014] N-acetyl-3-aminopropanoyl-CoA is converted to N-acetyl-5-amino-3-oxopentanoyl-CoA using a polypeptide having the activity of a .beta.-ketoacyl synthase classified under EC. 2.3.1.- (e.g., EC 2.3.1.180) or a .beta.-ketothiolase classified under EC. 2.3.1.- (e.g., EC 2.3.1.16 or EC 2.3.1.174) The polypeptide having the activity of a .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. Furthermore, the polypeptide having the activity of a .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 and be capable of converting N-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoA. The polypeptide having the activity of a .beta.-ketoacyl synthase can have at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. Furthermore, the polypeptide having the activity of a .beta.-ketoacyl synthase can have at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14 and be capable of converting N-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoA.

[0015] The method can include enzymatically converting N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof to 7-aminoheptanoate using a plurality of polypeptides having the activities of a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase, a trans-2-enoyl-CoA reductase, a .beta.-ketothiolase, a thioesterase or a CoA transferase and a deacetylase.

[0016] The polypeptide having the activity of a 3-hydroxyacyl-CoA dehydrogenase 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 polypeptide having the activity of an enoyl-CoA hydratase can be classified under EC 4.2.1.17 or EC 4.2.1.119. The polypeptide having the activity of a 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. The polypeptide having the activity of a .beta.-ketothiolase can be classified under EC 2.3.1.16 or EC 2.3.1.174. The polypeptide having the activity of a thioesterase or CoA transferase can be classified under EC 3.1.2.- or EC 2.8.3.- respectively. The polypeptide having the activity of a deacetylase can be classified under EC 3.5.1.-.

[0017] In one aspect, this document features a method for biosynthesizing 7-aminoheptanoate or the salt thereof. The method includes enzymatically converting N-acetyl-3-aminopropanoate to N-acetyl-5-amino-3-oxopentanoyl-CoA via N-acetyl-3-aminopropanoyl-CoA. N-acetyl-3-aminopropanoate is converted to N-acetyl-3-aminopropanoyl-CoA using a polypeptide having the activity of a CoA ligase classified under EC 6.2.1.- or a CoA-transferase classified under EC 2.8.3.-. N-acetyl-3-aminopropanoyl-CoA is converted to N-acetyl-5-amino-3-oxopentanoyl-CoA using a polypeptide having the activity of a .beta.-ketoacyl synthase classified under EC 2.3.1.- (e.g., EC 2.3.1.180) or 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. Furthermore, 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 and be capable of converting N-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoA. The .beta.-ketoacyl synthase can have at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:14. Furthermore, the .beta.-ketoacyl synthase can have at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:14 and be capable of converting N-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoA.

[0018] N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof can be converted to N-acetyl-5-amino-3-hydroxypentanoyl-CoA using a polypeptide having the activity of a 3-hydroxyacyl-CoA dehydrogenase. N-acetyl-5-amino-3-hydroxypentanoyl-CoA can be converted to N-acetyl-5-amino-pent-2-enoyl-CoA using a polypeptide having the activity of an enoyl-CoA hydratase. N-acetyl-5-amino-pent-2-enoyl-CoA can be converted to N-acetyl-5-amino-pentanoyl-CoA using a polypeptide having the activity of a trans-2-enoyl-CoA reductase. N-acetyl-5-amino-pentanoyl-CoA can be converted to N-acetyl-7-amino-3-oxoheptanoyl-CoA using a polypeptide having the activity of a .beta.-ketothiolase. N-acetyl-7-amino-3-oxoheptanoyl-CoA can be converted to N-acetyl-7-amino-3-hydroxyheptanoyl-CoA using a polypeptide having the activity of a 3-hydroxyacyl-CoA dehydrogenase. N-acetyl-7-amino-3-hydroxyheptanoyl-CoA can be converted to N-acetyl-7-amino-hept-2-enoyl-CoA using a polypeptide having the activity of an enoyl-CoA hydratase. N-acetyl-7-amino-hept-2-enoyl-CoA can be converted to N-acetyl-7-amino-heptanoyl-CoA using a polypeptide having the activity of a trans-2-enoyl-CoA reductase. N-acetyl-7-aminoheptanoyl-CoA can be converted to N-acetyl-7-amino-heptanoate using a polypeptide having the activity of a thioesterase or a CoA transferase. N-acetyl-7-amino-heptanoate can be converted to 7-aminoheptanoate using a polypeptide having the activity of a deacetylase.

[0019] Any of the methods further can include enzymatically converting 7-aminoheptanoate to pimelic acid, 7-hydroxyheptanoate, heptamethylenediamine or 1,7-heptanediol or the corresponding salts thereof in one or more steps.

[0020] For example, 7-aminoheptanoate can be enzymatically converted to pimelic acid using one or more polypeptides having the activity of a .omega.-transaminase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 5-oxopentanoate dehydrogenase or an aldehyde dehydrogenase. See FIG. 3.

[0021] For example, 7-aminoheptanoate and 7-hydroxyheptanoate can be converted to heptamethylenediamine using one or more polypeptides having the activity of a carboxylate reductase, a .omega.-transaminase, an alcohol dehydrogenase, an N-acetyltransferase, and a deacylase. See FIG. 4.

[0022] For example, 7-aminoheptanoate can be converted to 7-hydroxyheptanoate using one or more polypeptides having the activity of an alcohol dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutanoate dehydrogenase, or a .omega.-transaminase. See FIG. 5.

[0023] For example, 7-aminoheptanoate can be converted to 7-hydroxyheptanoate (see FIG. 5) and subsequently 7-hydroxyheptanoate can be converted to 1,7-heptanediol using polypeptides having the activity of a carboxylate reductase and an alcohol dehydrogenase. See FIG. 6.

[0024] The .omega.-transaminase as described in any of the figures can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO. 7-12. Furthermore, the .omega.-transaminase as described in any of the figures can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO. 7-12 and be capable of transferring at least one amine group separated from a carboxyl group by at least one methylene insertion.

[0025] The carboxylate reductase as described in any of the figures can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ D NO. 2-6 and 15. Furthermore, the carboxylate reductase as described in any of the figures can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ D NO. 2-6 and 15 and be capable of reducing a carboxyl group to a terminal aldehyde.

[0026] In any of the methods, N-acetyl-3-aminopropanoate can be enzymatically produced from .beta.-alanine, which itself can be enzymatically produced from malonyl-CoA using polypeptides having the activity of a malonyl-CoA reductase and a .beta.-alanine-pyruvate aminotransferase or from L-aspartate using a polypeptide having the activity of an aspartate .alpha.-decarboxylase.

[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 a polypeptide having the activity of (i) an aldehyde dehydrogenase classified under EC 1.2.1.3, or (ii) a 5-oxopentanoate dehydrogenase classified under EC 1.2.1.- such as encoded by CpnE, a 6-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). See FIG. 3.

[0028] In any of the methods described herein, 7-hydroxyheptanoic acid can be produced by forming the second terminal functional group in pimelate semialdehyde using a polypeptide having the activity of an alcohol dehydrogenase classified under EC 1.1.1.-, 6-hydroxyhexanoate dehydrogenase classified under EC 1.1.1.258 such as the gene product of ChnD (Iwaki et al., Appl. Environ. Microbiol., 1999, 65(11):5158-5162); a 5-hydroxypentanoate dehydrogenase classified under EC 1.1.1.- such as the gene product of cpnD, or 4-hydroxybutanoate dehydrogenase classified under EC 1.1.1.61 such as the gene product of gabD. See FIG. 5.

[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 polypeptide having the activity of a .omega.-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 3.5.1.62. See FIG. 4.

[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 a polypeptide having the activity of 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. See FIG. 6.

[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 microorganism's tolerance to high concentrations of one or more C7 (7-carbon) building blocks is improved through continuous cultivation in a selective environment.

[0034] In some embodiments, the microorganism's biochemical network is attenuated or augmented to (1) ensure the intracellular availability of acetyl-CoA and .beta.-alanine, (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 (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 microorganism containing one or more exogenous nucleic acids, using, for example, a fermentation strategy. 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 an activity of 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase, a trans-2-enoyl-CoA reductase, a .beta.-ketothiolase, a .beta.-ketoacyl synthase, a thioesterase or a CoA transferase and a deacetylase, using, for example, a fermentation strategy. In another aspect, this document features a recombinant microorganism that includes at least one exogenous nucleic acid encoding a polypeptide having the activity of (i) a .beta.-ketoacyl synthase, (ii) a .beta.-ketothiolase, (iii) a thioesterase or a CoA transferase, (iv) a deacetylase and one or more of (v) a 3-hydroxyacyl-CoA dehydrogenase, (vi) an enoyl-CoA hydratase, and (vii) a trans-2-enoyl-CoA reductase, the microorganism producing 7-aminoheptanoate or a corresponding salt thereof. See FIG. 1 and FIG. 2.

[0038] A microorganism producing 7-aminoheptanoate further can include one or more of the following exogenous polypeptides having the activity of: a .omega.-transaminase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 5-oxopentanoate dehydrogenase, or an aldehyde dehydrogenase, the microorganism further producing pimelic acid. See FIG. 3.

[0039] A microorganism producing 7-aminoheptanoate or 7-hydroxyheptanoate further can include one or more of the following exogenous polypeptides having the activity of: a carboxylate reductase, a .omega.-transaminase, a deacylase, an N-acetyl transferase, or an alcohol dehydrogenase, said microorganism further producing heptamethylenediamine. See FIG. 4.

[0040] A microorganism producing 7-aminoheptanoate further can include one or more of the following exogenous polypeptides having the activity of: a .omega.-transaminase, a 6-hydroxyhexanoate dehydrogenase, a 4-hydroxybutanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, and an alcohol dehydrogenase, the microorganism further producing 7-hydroxyheptanoate. See FIG. 5.

[0041] A microorganism producing 7-hydroxyheptanoate further can include an exogenous polypeptide having the activity of a carboxylate reductase and an exogenous polypeptide having the activity of an alcohol dehydrogenase, the microorganism further producing 1,7-heptanediol. See FIG. 6.

[0042] Any of the recombinant microorganisms described herein further can include one or more of the following exogenous polypeptides having the activity of: an aspartate-.alpha.-decarboxylase; a malonyl-CoA reductase; a .beta.-alanine-pyruvate aminotransferase; an N-acetyl transferase; a thioesterase; a CoA-transferase; and a deactylase.

[0043] Any of the recombinant microorganisms 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 cells described herein that are capable of producing C7 building blocks.

[0044] Any of the recombinant microorganisms 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 cells described herein that are capable of producing C7 building blocks.

[0045] Any of the recombinant microorganisms 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.

[0046] Any of the recombinant microorganisms 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.

[0047] In another aspect of the invention, this document features a non-naturally occurring microorganism comprising at least one exogenous nucleic acid encoding at least one polypeptide having the activity of at least one enzyme, at least one substrate and at least one product, depicted in any one of FIGS. 1 to 6.

[0048] In another aspect of the invention, this document features a plurality of nucleic acid constructs or expression vectors comprising a polynucleotide encoding a polypeptide having enzymatic activities corresponding to the polypeptides as set out in SEQ ID NO:1 to SEQ ID NO: 15 and to polypeptides having at least 70% sequence identity to the polypeptides as set out in SEQ ID NO:1 to SEQ ID NO: 15. (See FIG. 7).

[0049] In another aspect of the invention, this document features a composition comprising a nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having enzymatic activities corresponding to the polypeptides as set out in SEQ ID NO:1 to SEQ ID NO: 15 and to polypeptides having at least 70% sequence identity to the polypeptides as set out in SEQ ID NO:1 to SEQ ID NO: 15. (See FIG. 7).

[0050] In another aspect of the invention, this document features a culture medium comprising a nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having enzymatic activities corresponding to the polypeptides as set out in SEQ ID NO:1 to SEQ ID NO: 15 and to polypeptides having at least 70% sequence identity to the polypeptides as set out in SEQ ID NO:1 to SEQ ID NO: 15. (See FIG. 7).

[0051] In another aspect of the invention, this document features a non-naturally occurring biochemical network comprising N-acetyl-3-aminopropanoyl-CoA, an exogenous nucleic acid encoding a polypeptide having the activity of a .beta.-ketothiolase or a .beta.-ketoacyl synthase classified under EC. 2.3.1, and an N-acetyl-5-amino-3-oxopentanoyl-CoA.

[0052] In another aspect of the invention, this document features a non-naturally occurring biochemical network comprising at least one exogenous nucleic acid encoding a polypeptide having the enzymatic activity of (i) a .beta.-ketoacyl synthase and/or a .beta.-ketothiolase, (ii) a thioesterase or a CoA transferase, (iii) a deacetylase, and one or more of (iv) 3-hydroxyacyl-CoA dehydrogenase, (iv) an enoyl-CoA hydratase, and (v) a trans-2-enoyl-CoA reductase, said microorganism producing 7-aminoheptanoate.

[0053] In another aspect of the invention, this document features means for producing 7-aminoheptanoate, comprising culturing a non-naturally occurring microorganism comprising at least one exogenous nucleic acid encoding a polypeptide having the enzymatic activity of (i) a .beta.-ketoacyl synthase and/or a .beta.-ketothiolase, (ii) a thioesterase or a CoA transferase, (iii) a deacetylase, and one or more of (iv) a 3-hydroxyacyl-CoA dehydrogenase, (iv) an enoyl-CoA hydratase, and (v) a trans-2-enoyl-CoA reductase expressed in a sufficient amount in said microorganism to produce 7-amino-heptanoate.

[0054] In another aspect of the invention, this document features a bio-derived, bio-based or fermentation-derived product, wherein said product comprises: i. a composition comprising at least one bio-derived, bio-based or fermentation-derived compound according to any one of claims 15-28 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.

[0055] 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.

[0056] 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

[0057] FIG. 1 is a schematic of exemplary biochemical pathways leading to N-acetyl-7-aminoheptanoyl-CoA using malonyl-CoA or L-aspartate as central metabolites.

[0058] FIG. 2 is a schematic of an exemplary biochemical pathway leading to 7-aminoheptanoate using N-acetyl-7-aminoheptanoyl-CoA as a precursor.

[0059] FIG. 3 is a schematic of exemplary biochemical pathways leading to pimelic acid using 7-aminoheptanoate as a central precursor.

[0060] 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.

[0061] FIG. 5 is a schematic of exemplary biochemical pathways leading to 7-hydroxyheptanoate using 7-aminoheptanoate as a central precursor.

[0062] FIG. 6 is a schematic of an exemplary biochemical pathway leading to 1,7-heptanediol using 7-hydroxyheptanoate as a central precursor.

[0063] FIG. 7 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 massiliensecarboxylate 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: 1 CAA74523.12), an Escherichia coli .beta.-ketothiolase (see GenBank Accession No. AAC74479.1, SEQ ID NO: 13), a Bacillus subtilis .beta.-ketoacyl synthase (see GenBank Accession No. CAA74523.1, SEQ ID NO: 14), a Mycobacterium smegmatis carboxylate reductase (see GenBank Accession No. ABK75684.1, SEQ ID NO: 15), a Cupriavidus necator beta-ketothiolase (see GenBank Accession No. AAC38322.1, SEQ ID NO: 16), an Escherichia coli (see Genbank Accession No. AAC74479.1, SEQ ID NO: 17), a Clostridium propionicum acetate/propionate CoA transferase (see Genbank Accession No. CAB77207.1, SEQ ID NO: 18), a Clostridium aminobutyricum 4-hydroxybutyrate-CoA transferase (see Genbank Accession No. CAB60036.2, SEQ ID NO: 19), a Citrobacter sp. Al acetyl-CoA hydrolase/transferase transferase (see Genbank Accession No. EJF23789.1, SEQ ID NO: 20), and an Acetobacter aceti succinyl-CoA:acetate CoA-transferase (see Genbank Accesssion No. ACD85596.1, SEQ ID NO: 21).

[0064] FIG. 8 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).

[0065] 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 two carboxylate reductase preparations for converting pimelate to pimelate semialdehyde relative to the empty vector control.

[0066] 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 six carboxylate reductase preparations for converting 7-hydroxyheptanoate to 7-hydroxyheptanal relative to the empty vector control.

[0067] 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 the activity of three carboxylate reductase preparations for converting N7-acetyl-7-aminoheptanoate to N7-acetyl-7-aminoheptanal relative to the empty vector control.

[0068] FIG. 12 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.

[0069] FIG. 13 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).

[0070] FIG. 14 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.

[0071] FIG. 15 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.

[0072] 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 heptamethylenediamine to 7-aminoheptanal relative to the empty vector control.

[0073] 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 six .omega.-transaminase preparations for converting N7-acetyl-1,7-diaminoheptane to N7-acetyl-7-aminoheptanal relative to the empty vector control.

[0074] FIG. 18 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.

[0075] FIG. 19 is a schematic of the exemplary enzymatic reactions performed with 4-hydroxybutyrate-CoA transferase using either N-acetyl-.beta.-alanine (AC5) or 5-ethanamidopentanoic acid (AC7) as substrates for the formation of 5-ethanamido-3-oxopentanoyl-CoA and 7-ethanamido-3-oxoheptanoyl-CoA, respectively.

[0076] FIG. 20 is a LC-MS chromatogram of distinct peaks of chemical abundance separated by retention times as a measure of enzyme activity for of 4-hydroxybutyrate-CoA transferase for converting 5-ethanamidopentanoic acid into products, 5-ethanamidopentanoyl-CoA (g) and 7-ethanamido-3-oxopentanoyl-CoA (h).

[0077] FIG. 21 is a LC-MS ESI mass spectrum performed in positive mode that identifies the product of peak (g) (see chromatogram of FIG. 20) as ethanamidopentanoyl-CoA by comparison of the observed and expected masses for the [M+H].sup.+ and [M+2H].sup.2+ species. Expected [M+H].sup.+ for products (g): 909.2017 (1 charge) & [M+2H].sup.2+: 445.1044 (2 charges).

[0078] FIG. 22 is a LC-MS chromatogram of distinct peaks of chemical abundance separated by retention times as a measure of enzyme activity for of 4-hydroxybutyrate-CoA transferase for converting 5-ethanamidopentanoyl-CoA into the product. 7-ethanamido-3-oxopentanoyl-CoA (h).

[0079] FIG. 23 is a LC-MS ESI mass spectrum performed in positive mode that identifies the product of peak (h) (see chromatogram of FIG. 22) as ethanamido-3-oxopentanoyl-CoA by comparison of the observed and expected masses for the [M+H].sup.+ and [M+2H].sup.2+ species. Expected [M+H].sup.+ for products (h): 951.2120 (1 charge) & [M+2H]2+: 476.1097 (2 charges).

DETAILED DESCRIPTION

[0080] In general, this document provides enzymes, non-natural pathways, cultivation strategies, feedstocks, microorganisms and attenuations to the microorganism's biochemical network, for producing 7-aminoheptanoate or one or more of pimelic acid, 7-hydroxyheptanoic acid, heptamethylenediamine or 1,7-heptanediol or the corresponding salts thereof, all of which are referred to as C7 building blocks herein. The term "C7 building block" is used to denote a seven (7) carbon chain aliphatic backbone. 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] Microorganisms described herein can include endogenous pathways that can be manipulated such that 7-aminoheptanoate or one or more other C7 building blocks can be produced. In an endogenous pathway, the microorganism naturally expresses all of the enzymes catalyzing the reactions within the pathway. A 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 microorganism.

[0082] The term "exogenous" as used herein with reference to a nucleic acid (or a protein) and a microorganism 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 microorganism once in the microorganism. It is important to note that non-naturally-occurring nucleic acids can contain nucleic acid subsequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature. For example, a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a microorganism once introduced into the microorganism, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature. Thus, any vector, autonomously replicating plasmid, or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a whole does not exist in nature is considered to be non-naturally-occurring nucleic acid. It follows that genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally-occurring nucleic acid since they exist as separate molecules not found in nature. It also follows that any nucleic acid containing a promoter sequence and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally-occurring nucleic acid. A nucleic acid that is naturally-occurring can be exogenous to a particular 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 microorganism refers to a nucleic acid (or protein) that does occur in (and can be obtained from) that particular microorganism 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 microorganism of the same particular type as it is found in nature. Moreover, a microorganism "endogenously producing" or that "endogenously produces" a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound as does a microorganism of the same particular type as it is found in nature.

[0084] For example, depending on the microorganism and the compounds produced by the microorganism, one or more of the following polypeptides having the following specific enzymatic activities may be expressed in the microorganism in addition to a .beta.-ketoacyl synthase and/or a .beta.-ketothiolase:a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase, a trans-2-enoyl-CoA reductase, a thioesterase, a CoA transferase, a deacetylase, an aldehyde dehydrogenase, an alcohol dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 4-hydroxybutanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a carboxylate reductase, an N-acetyl transferase, or a .omega.-transaminase. In recombinant microorganisms expressing a polypeptide having the activity of a carboxylate reductase, a phosphopantetheinyl transferase also can be expressed as it enhances activity of the carboxylate reductase.

[0085] For example, a recombinant microorganism can include a polypeptide having the activity of an exogenous .beta.-ketoacyl synthase or a .beta.-ketothiolase and produce N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof from N-acetyl-3-aminopropanoyl-CoA. The N-acetyl-5-amino-3-oxopentanoyl-CoA or salt thereof can be converted enzymatically to N-acetyl-7-aminoheptanoyl-CoA and subsequently to 7-aminoheptanoate.

[0086] For example, a recombinant microorganism can include a polypeptide having the activity of an exogenous .beta.-ketoacyl synthase and a .beta.-ketothiolase, an exogenous thioesterase or CoA-transferase, a deacetylase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase, and a trans-2-enoyl-CoA reductase and produce 7-aminoheptanoate.

[0087] For example, a recombinant microorganism producing 7-aminoheptanoate can include one or more of the following exogenous polypeptides having the enzymatic activity of: a .omega.-transaminase, a 7-oxoheptanoate dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, or an aldehyde dehydrogenase, and further produce pimelic acid. For example, a recombinant microorganism producing 7-aminoheptanoate can include an exogenous .omega.-transaminase and an aldehyde dehydrogenase and produce pimelic acid. For example, a recombinant microorganism producing 7-aminoheptanoate can include an exogenous polypeptide having the activity of a .omega.-transaminase and one of the following exogenous polypeptides having the enzymatic activity of: a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, or a 7-oxoheptanoate dehydrogenase, and produce pimelic acid.

[0088] For example, a recombinant microorganism producing 7-aminoheptanoate can include one or more of the following exogenous polypeptides having the enzymatic activity of: a .omega.-transaminase, an alcohol dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, 4-hydroxybutanoate dehydrogenase, and further produce 7-hydroxyheptanoate. For example, a recombinant microorganism producing 7-aminoheptanoate can include an exogenous polypeptide having the activity of an alcohol dehydrogenase and an exogenous polypeptide having the activity of a .omega.-transaminase and produce 7-hydroxyheptanoate. For example, a recombinant microorganism producing 7-aminoheptanoate can include an exogenous polypeptide having the activity of a 6-hydroxyhexanoate dehydrogenase and an exogenous polypeptide having the activity of a .omega.-transaminase and produce 7-hydroxyheptanoate. For example, a recombinant microorganism producing 7-aminoheptanoate can include an exogenous polypeptide having the activity of a 5-hydroxypentanoate dehydrogenase and an exogenous polypeptide having the activity of a .omega.-transaminase and produce 7-hydroxyheptanoate. For example, a recombinant microorganism producing 7-aminoheptanoate can include an exogenous polypeptide having the activity of a 4-hydroxybutanoate dehydrogenase and an exogenous polypeptide having the activity of a .omega.-transaminase and produce 7-hydroxyheptanoate.

[0089] For example, a recombinant microorganism producing 7-aminoheptanoate can include one or more of the following exogenous polypeptides having the activity of: a carboxylate reductase, a .omega.-transaminase, a deacetylase, an N-acetyl transferase or an alcohol dehydrogenase, and produce heptamethylenediamine. For example, a recombinant microorganism producing 7-aminoheptanoate can include an exogenous polypeptide having the activity of a carboxylate reductase and one or more exogenous polypeptides having the activity of transaminases (e.g., one .omega.-transaminase or two different transaminases) and produce heptamethylenediamine. For example, a recombinant microorganism producing 7-aminoheptanoate can include an exogenous polypeptide having the activity of a carboxylate reductase, an exogenous polypeptide having the activity of a alcohol dehydrogenase, and one or more exogenous polypeptides having the activity of transaminases (e.g., one .omega.-transaminase or two different transaminases), and produce heptamethylenediamine. For example, a recombinant microorganism producing 7-aminoheptanoate can include an exogenous polypeptide having the activity of an N-acetyl transferase, a carboxylate reductase, a deacylase, and one or more exogenous transaminases (e.g., one .omega.-transaminase or two different transaminases) and produce heptamethylenediamine. For example, a recombinant microorganismproducing 7-aminoheptanoate can include one or more exogenous polypeptide having the activity of an alcohol dehydrogenase, and one or more exogenous polypeptides having the activity of transaminases (e.g., one .omega.-transaminase, or two or three different transaminases) and produce heptamethylenediamine.

[0090] For example, a recombinant microorganism producing 7-hydroxyheptanoate can include the following exogenous polypeptides having the enzymatic activity of: a carboxylate reductase and an exogenous alcohol dehydrogenase, and further produce 1,7-heptanediol.

[0091] In any of the recombinant microorganisms, the recombinant microorganism also can include one or more (e.g., one, two or three) of the following exogenous enzymes used to convert either malonyl-CoA or L-aspartate to .beta.-alanine: a malonyl-CoA reductase, an aspartate .alpha.-decarboxylase and a .beta.-alanine-pyruvate aminotransferase. For example, a recombinant microorganism can include an exogenous malonyl-CoA reductase and a .beta.-alanine-pyruvate aminotransferase and produce .beta.-alanine. For example, a recombinant microorganism can include an exogenous aspartate .alpha.-decarboxylase and produce .beta.-alanine.

[0092] In any of the recombinant microorganisms, the recombinant microorganism also can include following the exogenous enzyme used to convert .beta.-alanine to N-acetyl-3-aminopropanoate: an N-acetyl-transferase.

[0093] In any of the recombinant microorganisms, the recombinant microorganism also can include one or more (e.g., one or two) of the following exogenous enzymes used to convert N-acetyl-3-aminopropanoate to N-acetyl-3-aminopropanoyl-CoA: a CoA transferase or a CoA ligase.

[0094] In any of the recombinant microorganisms, the recombinant microorganism also can include one or more (e.g., one or two) of the following exogenous enzymes used to convert 5-ethanamidopentanoic acid to 5-ethanamidopentanoyl-CoA: a CoA transferase or a CoA ligase.

[0095] 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 EMB L.

[0096] 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.

[0097] 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 .beta.-ketothiolase (see GenBank Accession No. AAC38322.1, SEQ ID NO: 1) or an Escherichia coli .beta.-ketothiolase (see GenBank Accession No. AAC74479.1, SEQ ID NO: 13) See FIG. 7.

[0098] For example, a .beta.-ketoacyl synthase 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 Bacillus subtilis .beta.-ketoacyl synthase (see GenBank Accession No. CAA74523.1, SEQ ID NO: 14). See FIG. 7.

[0099] For example, a 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 aminobutyrium (see GenBank Accession No. CAB60036.2, SEQ ID NO: 19). See FIG. 7.

[0100] 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 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) carboxylate reductase or a Mycobacterium smegmatis carboxylate reductase (see Genbank Accession No. ABK75684.1, SEQ ID NO: 15). See, FIG. 7.

[0101] 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 .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 transaminase (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. 7.

[0102] 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 (B12seq) program from the stand-alone version of BLASTZ containing BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from Fish & Richardson's web site (e.g., www.fr.com/blast/) or the U.S. government's National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov). Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ. B12seq 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:\seql.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:\B12seq -i c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequences share homology (identity), then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology (identity), then the designated output file will not present aligned sequences. Similar procedures can be following for nucleic acid sequences except that blastn is used.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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. Enzymes with substitutions will generally have not more than 100 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, 50 or 100) 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.

[0107] Deletion variants can lack one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 50 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 microorganisms (e.g., yeast cells), expression and/or secretion of the target protein can be increased through use of a heterologous signal sequence. In some embodiments, the fusion protein can contain a carrier (e.g., KLH) useful, e.g., in eliciting an immune response for antibody generation) or ER or Golgi apparatus retention signals. Heterologous sequences can be of varying length and in some cases can be a longer sequences than the full-length target proteins to which the heterologous sequences are attached.

[0108] Engineered microorganisms 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 microorganism can include all exogenous enzymes, or can include both endogenous and exogenous enzymes. Endogenous genes of the engineered microorganisms 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 microorganisms can be referred to as recombinant microorganisms or recombinant cells. As described herein recombinant microorganisms can include nucleic acids encoding one or more of a .beta.-ketoacyl synthase, a .beta.-ketothiolase, a dehydrogenase, a reductase, a hydratase, a CoA-transferase, a CoA-ligase, a thioesterase, a deacetylase, an N-acetyltransferase and .omega.-transaminase as described herein.

[0109] 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 microorganism as a source of the enzymes, or using a plurality of lysates from different microorganisms as the source of the enzymes.

[0110] The reactions of the pathways described herein can be performed in one or more microorganisms (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, purified or extracted from of the above types of microorganism cells and used in a purified or semi-purified form. Moreover, such 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 microorganism cells, all the steps can be performed using extracted enzymes, or some of the steps can be performed in cells and others can be performed using extracted enzymes.

Enzymes

Enzymes generating N-acetyl-7-amino-heptanoyl-CoA

[0111] As depicted in FIG. 1, N-acetyl-7-amino-heptanoyl-CoA or a salt thereof can be biosynthesized from malonyl-CoA or L-aspartate through the intermediate N-acetyl-5-amino-3-oxopentanoyl-CoA, which can be produced from N-acetyl-3-aminopropanoyl-CoA using a polypeptide having the activity of a .beta.-ketoacyl synthase or a .beta.-ketothiolase. In some embodiments, a .beta.-ketothiolase may be classified under EC 2.3.1.16, such as the gene product of bktB or under EC 2.3.1.174 such as the gene product of paaJ. In some embodiments, a .beta.-ketoacyl synthase may be classified under EC 2.3.1.180 such as the gene product of fabH, under EC 2.3.1.179 such as the gene product of fabF or under EC 2.3.1.41 such as the gene product of fabB.

[0112] N-acetyl-3-aminopropanoyl-CoA or a salt thereof can be enzymatically converted from N-acetyl-3-aminopropanoate using a polypeptide having the activity of a CoA transferase classified, for example, under EC 2.8.3- or a CoA ligase classified, for example, under EC 6.2.1-. N-acetyl-3-aminopropanoate can be enzymatically produced from .beta.-alanine using a polypeptide having the activity of an N-acetyl transferase classified, for example, under EC 2.3.1.-, such as EC 2.3.1.13, EC 2.3.1.17 or EC 2.3.1.32.

[0113] .beta.-alanine itself can be enzymatically produced from malonyl-CoA using polypeptides having the activity of a malonyl-CoA-reductase and a .beta.-alanine-pyruvate aminotransferase or from L-aspartate using a polypeptide having the activity of an .alpha.-aspartate decarboxylase. In some embodiments, a malonyl-CoA-reductase may be classified under EC 1.2.1.75 and a .beta.-alanine-pyruvate aminotransferase may be classified under EC 2.6.1.18. In some embodiments, an .alpha.-aspartate decarboxylase may be classified under EC 4.1.1.11.

[0114] The intermediate N-acetyl-5-amino-3-oxopentanoyl-CoA or salt thereof can be converted to N-acetyl-7-amino-heptanoyl-CoA using polypeptides having the activity of a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase, a trans-2-enoyl-CoA reductase and a .beta.-ketothiolase. In some embodiments, a 3-hydroxyacyl-CoA dehydrogenase may be 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). In some embodiments, an enoyl-CoA hydratase may be classified under EC 4.2.1.17 such as the gene product of crt or under EC 4.2.1.119 such as the gene product of phaJ. In some embodiments, a trans-2-enoyl-CoA reductase may be classified, for example, 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). In some embodiments, a .beta.-ketothiolase may be classified under EC 2.3.1.16 such as the gene product of bktB or under EC 2.3.1.174 such as the gene product of paaJ.

Enzymes generating 7-aminoheptanoate

[0115] As depicted in FIG. 2, N-acetyl-7-amino-heptanoyl-CoA is converted to 7-aminoheptanoate using polypeptides having the activity of a thioesterase or CoA-transferase and a deacetylase.

[0116] In some embodiments, a thioesterase may be classified under EC 3.1.2.-, resulting in the production of N-acetyl-7-aminoheptanoate. 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). In some embodiments, a CoA-transferase may be 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.

[0117] In some embodiments, the first terminal amine group leading to the synthesis of 7-aminoheptanoate is enzymatically formed by a deacetylase classified, for example, under EC 3.5.1.17 such as an acyl-lysine deacetylase from Achromobacter pestifer (see, for example, Chibate et al., 1970, Methods Enzymol., 19:756-762).

Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis of Pimelic Acid

[0118] As depicted in FIG. 3, 7-aminoheptanoate can be enzymatically converted to pimelic acid. The terminal carboxyl group leading to the production of pimelic acid can be enzymatically formed using polypeptides having the activity of an aldehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, or a 7-oxoheptanoate dehydrogenase.

[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. 3.

[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-oxopentanoate 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. 3.

Enzymes Generating the Terminal Amine Groups in the Biosynthesis of Heptamethylenediamine

[0121] As depicted in FIG. 4, terminal amine groups can be enzymatically formed or removed using polypeptides having the activity of a .omega.-transaminase or a deacetylase.

[0122] In some embodiments, a terminal amine group leading to the synthesis of 7-aminoheptanoic acid is enzymatically formed in 7-aminoheptanal by a .omega.-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. 7.

[0123] An additional .omega.-transaminase that can be used in the methods and microorganisms 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).

[0124] 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 amino donor, thus forming the first terminal amine group in pimelate semialdehyde (Kaulmann et al., Enzyme and Microbial Technology, 2007, 41, 628-637).

[0125] 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).

[0126] 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).

[0127] 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. 7.

[0128] 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).

[0129] 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).

[0130] In some embodiments, the second terminal amine group leading to the synthesis of heptamethylenediamine is enzymatically formed by a deacetylase classified, for example, under EC 3.5.1.62 such as an acetylputrescine deacetylase. The acetylputrescine deacetylase from Micrococcus luteus K-11 accepts a broad range of carbon chain length substrates, such as acetylputrescine, acetylcadaverine and N8_acetylspermidine (see, for example, Suzuki et al., 1986, BBA-General Subjects, 882(1):140-142).

Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis of 1,7 Heptanediol

[0131] As depicted in FIG. 6, the terminal hydroxyl group can be enzymatically formed using a polypeptide having the activity of 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.

Enzymes generating N-acetyl-7-amino-3-oxoheptanoyl-CoA

[0132] As depicted in FIG. 1, N-acetyl-7-amino-oxoheptanoyl-CoA or a salt thereof can be biosynthesized from malonyl-CoA or L-aspartate through the intermediate N-acetyl-5-amino-3-oxopentanoyl-CoA, which can be produced from N-acetyl-5-aminopentanoyl-CoA using a polypeptide having the activity of a .beta.-ketoacyl synthase, a .beta.-ketothiolase, or a CoA-transferase. In some embodiments, a .beta.-ketothiolase may be classified under EC 2.3.1.16, such as the gene product of bktB or under EC 2.3.1.174 such as the gene product of paaJ. In some embodiments, a .beta.-ketoacyl synthase may be classified under EC 2.3.1.180 such as the gene product of fabH, under EC 2.3.1.179 such as the gene product of fabF or under EC 2.3.1.41 such as the gene product of fabB. In some embodiments, a CoA-transferase may be classified under EC 2.8.3- such as the gene product of abfT.

Biochemical Pathways

Pathways to 7-aminoheptanoate

[0133] In some embodiments, N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof is synthesized from the central metabolite, malonyl-CoA, by conversion of malonyl-CoA to malonate semialdehyde by a polypeptide having the activity of a malonyl CoA reductase classified, for example, under EC 1.2.1.75; followed by conversion of malonate semialdehyde to .beta.-alanine by a polypeptide having the activity of a .beta.-alanine-pyruvate aminotransferase classified, for example, under EC 2.6.1.18; followed by conversion of .beta.-alanine to N-acetyl-3-aminopropanoate by a polypeptide having the activity of an N-acetyl transferase classified, for example, under EC 2.3.1.13, EC 2.3.1.17 or EC 2.3.1.32; followed by conversion of N-acetyl-3-aminopropanoate to N-acetyl-3-aminopropanoyl-CoA by a polypeptide having the activity of a CoA transferase classified, for example, under EC 2.8.3.- or a polypeptide having the activity of a CoA ligase classified, for example, under EC 6.2.1.-; followed by conversion of N-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoA by a polypeptide having the activity of a .beta.-ketoacyl synthase classified under EC. 2.3.1.- (e.g., EC 2.3.1.180) or a polypeptide having the activity of a .beta.-ketothiolase classified under EC. 2.3.1.- (e.g.,EC 2.3.1.16 or EC 2.3.1.174).

[0134] In some embodiments, N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof is synthesized from the central metabolite, L-aspartate, by conversion of L-aspartate to .beta.-alanine by a polypeptide having the activity of an aspartate .alpha.-decarboxylase classified, for example, under EC 4.1.1.11; followed by conversion of .beta.-alanine to N-acetyl-3-aminopropanoate by a polypeptide having the activity of an N-acetyl transferase classified, for example, under EC 2.3.1.13, EC 2.3.1.17 or EC 2.3.1.32; followed by conversion of N-acetyl-3-aminopropanoate to N-acetyl-3-aminopropanoyl-CoA by a polypeptide having the activity of a CoA transferase classified, for example, under EC 2.8.3.- or a polypeptide having the activity of a CoA ligase classified, for example, under EC 6.2.1.-; followed by conversion of N-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoA by a polypeptide having the activity of a .beta.-ketoacyl synthase classified, for example, under EC 2.3.1.180 such as the gene product of fabH or by a polypeptide having the activity of a .beta.-ketothiolase classified, for example, under EC 2.3.1.16 such as the gene product of bktB or under EC 2.3.1.174 such as the gene product of paaJ.

[0135] The intermediate N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof is converted to N-acetyl-5-amino-3-hydroxypentanoyl-CoA by a polypeptide having the activity of 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); followed by conversion of N-acetyl-5-amino-3-hydroxypentanoyl-CoA to N-acetyl-5-amino-pent-2-enoyl-CoA using a polypeptide having the activity of an enoyl-CoA hydratase classified under, for example, EC 4.2.1.17 such as the gene product of crt or under EC 4.2.1.119 such as the gene product of phaJ; followed by conversion of N-acetyl-5-amino-pent-2-enoyl-CoA to N-acetyl-5-amino-pentanoyl-CoA by a polypeptide having the activity of a trans-2-enoyl-CoA reductase 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); followed by conversion of N-acetyl-5-amino-pentanoyl-CoA to N-acetyl-7-amino-3-oxoheptanoyl-CoA by a polypeptide having the activity of a .beta.-ketothiolase classified under, for example, EC 2.3.1.16 such as the gene product of bktB or under EC 2.3.1.174 such as the gene product of paaJ; followed by conversion of N-acetyl-7-amino-3-oxoheptanoyl-CoA to N-acetyl-7-amino-3-hydroxyheptanoyl-CoA by a polypeptide having the activity of 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); followed by conversion of N-acetyl-7-amino-3-hydroxyheptanoyl-CoA to N-acetyl-7-amino-hept-2-enoyl-CoA by a polypeptide having the activity of an enoyl-CoA-hydratase classified under, for example, EC 4.2.1.17 such as the gene product of crt or under EC 4.2.1.119 such as the gene product of phaJ; followed by conversion of N-acetyl- 7-amino-hept-2-enoyl-CoA to N-acetyl-7-aminoheptanoyl-CoA by a polypeptide having the activity of a trans-2-enoyl-CoA-reductase 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). See FIG. 1.

[0136] N-acetyl-7-aminoheptanoyl-CoA is then converted to 7-aminoheptanoate by a polypeptide having the activity of a thioesterase classified, for example, under EC 3.1.2.-or a CoA-transferase classified, for example, under EC 2.8.3.- and subsequently a polypeptide having the activity of a deacetylase classified, for example, under EC 3.5.1.17 such as an acyl-lysine deacetylase from Achromobacter pestifer (see, for example, Chibate et al., 1970, Methods Enzymol., 19:756-762).See FIG. 2.

Pathways using 7-aminoheptanoate as central precursor to pimelic acid

[0137] In some embodiments, pimelic acid is synthesized from 7-aminoheptanoate, by conversion of 7-aminoheptanoate to pimelate semialdehyde by a polypeptide having the activity of a .omega.-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.

[0138] Pimelate semialdehyde is then converted to pimelic acid by a polypeptide having the activity of a dehydrogenase classified, for example, under EC 1.2.1.- such as a 7-oxoheptanoate dehydrogenase (e.g., the gene product of ThnG), a 6-oxohexanoate dehydrogenase (e.g., the gene product of ChnE), a glutarate semialdehyde dehydrogenase classified, for example, under EC 1.2.1.20, a 5-oxopentanoate dehydrogenase such as the gene product of CpnE, or an aldehyde dehydrogenase classified under EC 1.2.1.3. See FIG. 3.

Pathway using 7-aminoheptanoate as central precursor to 7-hydroxyheptanoate

[0139] In some embodiments, 7-hydroxyheptanoate is synthesized from the central precursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoate to pimelate semialdehyde by a polypeptide having the activity of a .omega.-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; followed by conversion of pimelate semialdehyde to 7-hydroxyheptanoate by a polypeptide having the activity of 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-hydroxybutanoate dehydrogenase classified, for example, under EC 1.1.1.- such as the gene product of gabD. The alcohol dehydrogenase encoded by YMR318C has broad substrate specificity, including the oxidation of C7 alcohols. See FIG. 5.

Pathway using 7-aminoheptanoate, 7-hydroxyheptanoate, pimelate semialdehyde, or 1,7-heptanediol as a central precursor to heptamethylenediamine

[0140] In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoate to 7-aminoheptanal by a polypeptide having the activity of 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 polypeptide having the activity of 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), Segniliparus rotundus (Genbank Accession No. ADG98140.1, SEQ ID NO: 6) or Mycobacterium smegmatis (Genbank Accession No. ABK75684.1, SEQ ID NO: 15). See FIG. 4.

[0141] 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).

[0142] In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-hydroxyheptanoate (which can be produced as described in FIGS. 1, 2 and 5), 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-aminoheptanal to 7-aminoheptanol by a .omega.-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as 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 J., 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.

[0143] In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoate to N7-acetyl-7-aminoheptanoate by a polypeptide having the activity of 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 polypeptide having the activity of 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 polypeptide having the activity of 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 a polypeptide having the activity of a deacetylase classified, for example, under EC 3.5.1.62 such as an acetylputrescine deacetylase. The acetylputrescine deacetylase from Micrococcus luteus K-11 accepts a broad range of carbon chain length substrates, such as acetylputrescine, acetylcadaverine and N8_acetylspermidine (see, for example, Suzuki et al., 1986, BBA-General Subjects, 882(1):140-142).See, FIG. 4.

[0144] In some embodiments, heptamethylenediamine is synthesized from the central precursor, pimelate semialdehyde, by conversion of pimelate semialdehyde to heptanedial by a polypeptide having the activity of 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 polypeptide having the activity of 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.

[0145] In some embodiments, heptamethylenediamine is synthesized from 1,7-heptanediol by conversion of 1,7-heptanediol to 7-hydroxyheptanal using a polypeptide having the activity of 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 polypeptide having the activity of 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 a polypeptide having the activity of 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 polypeptide having the activity of 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

[0146] In some embodiments, 1,7 heptanediol is synthesized from the central precursor, 7-hydroxyheptanoate, by conversion of 7-hydroxyheptanoate to 7-hydroxyheptanal by a polypeptide having the activity of 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 a polypeptide having the activity of 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. 6.

Cultivation Strategy

[0147] In some embodiments, one or more C7 building blocks are biosynthesized in a recombinant microorganism using anaerobic, aerobic or micro-aerobic cultivation conditions. In some embodiments, the cultivation strategy entails nutrient limitation such as nitrogen, phosphate or oxygen limitation.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] The efficient catabolism of crude glycerol stemming from the production of biodiesel has been demonstrated in several microorganisms such as Escherichia coli, Cupriavidus necator, Pseudomonas oleavorans, Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem. Biotechnol., 2012, 166:1801-1813; Yang et al., Biotechnology for Biofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotechnol., 2011, 90:885-893).

[0152] 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).

[0153] The efficient catabolism of lignin-derived aromatic compounds such as benzoate analogues has been demonstrated in several microorganisms such as Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinion in Biotechnology, 2011, 22, 394-400; Perez-Pantoja et al., FEMS Microbiol. Rev., 2008, 32, 736-794).

[0154] 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).

[0155] The efficient utilization of fermentable sugars such as monosaccharides and disaccharides derived from cellulosic, hemicellulosic, cane and beet molasses, cassava, corn and other agricultural sources has been demonstrated for several microorganism such as Escherichia coli, Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcus lactis (see, e.g., Hermann et al, 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).

[0156] 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).

[0157] 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.

[0158] The efficient catabolism of methanol has been demonstrated for the methylotrophic yeast Pichia pastoris.

[0159] The efficient catabolism of ethanol has been demonstrated for Clostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008, 105(6) 2128-2133).

[0160] 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).

[0161] 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).

[0162] 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).

[0163] In some embodiments, the 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 microorganismsdescribed herein that are capable of producing one or more C7 building blocks.

[0164] In some embodiments, the 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 microorganisms described herein that are capable of producing one or more C7 building blocks.

Metabolic Engineering

[0165] 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.

[0166] Furthermore, recombinant microorganisms 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 microorganism. This document provides microorganism 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 microorganism cells can contain exogenous nucleic acids encoding enzymes catalyzing one or more of the steps of any of the pathways described herein.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] Attenuation strategies include, but are not limited to; the use of transposons, homologous recombination (double cross-over approach), mutagenesis, enzyme inhibitors and RNAi interference.

[0174] 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.

[0175] In some embodiments, the microorganism's tolerance to high concentrations of a C7 building block can be improved through continuous cultivation in a selective environment.

[0176] In some embodiments, the microorganism's endogenous biochemical network can be attenuated or augmented to (1) ensure the intracellular availability of acetyl-CoA and .beta.-alanine, (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.

[0177] 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 microorganism.

[0178] 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).

[0179] 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.

[0180] 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).

[0181] In some embodiments, enzymes that catalyze anapleurotic reactions such as PEP carboxylase and/or pyruvate carboxylase can be overexpressed in the microorganism.

[0182] In some embodiments requiring the intracellular availability of acetyl-CoA and NADH for C7 building block synthesis, endogenous genes encoding enzymes, such as menaquinol-fumarate oxidoreductase, that catalyze the degradation of phosphoenolpyruvate to succinate such as frdBC can be attenuated (see, e.g., Shen et al., 2011, supra).

[0183] 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).

[0184] 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 microorganism (Shen et al., 2011, supra).

[0185] In some embodiments, where pathways require excess NADH co-factor for C7 building block synthesis, a recombinant NADH-consuming transhydrogenase can be attenuated.

[0186] In some embodiments, an endogenous gene encoding an enzyme that catalyzes the degradation of pyruvate to ethanol such as pyruvate decarboxylase can be attenuated.

[0187] 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).

[0188] 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).

[0189] In some embodiments, carbon flux can be redirected into the pentose phosphate cycle to increase the supply of NADPH by overexpression a 6-phosphogluconate dehydrogenase and/or a transketolase (Lee et al., 2003, Biotechnology Progress, 19(5), 1444-1449).

[0190] 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 microorganisms (Brigham et al., Advanced Biofuels and Bioproducts, 2012, Chapter 39, 1065-1090).

[0191] 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 microorganisms (Brigham et al., 2012, supra).

[0192] 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 microorganisms (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 glucose-6-phosphate dehydrogenase gene such as zwf can be overexpressed in the microorganisms (Lim et al., J. Bioscience and Bioengineering, 2002, 93(6), 543-549).

[0194] 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 microorganisms(Becker et al., J. Biotechnol., 2007, 132:99-109).

[0195] 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.

[0196] 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 microorganism (Satoh et al., J. Bioscience and Bioengineering, 2003, 95(4):335-341).

[0197] 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).

[0198] In some embodiments, an endogenous glutamate dehydrogenase (EC 1.4.1.3) that utilizes both NADH and NADPH as co-factors can be attenuated.

[0199] 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).

[0200] 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).

[0201] In some embodiments using microorganisms that naturally accumulate polyhydroxyalkanoates, the endogenous polymer synthase enzymes can be attenuated in the microorganism strain.

[0202] In some embodiments, a L-alanine dehydrogenase can be overexpressed in the microorganism to regenerate L-alanine from pyruvate as an amino donor for .omega.-transaminase reactions.

[0203] In some embodiments, a L-glutamate dehydrogenase, a L-glutamine synthetase, or a alpha-aminotransaminase can be overexpressed in the microorganism to regenerate L-glutamate from 2-oxoglutarate as an amino donor for .omega.-transaminase reactions.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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).

[0208] The efflux of 7-aminoheptanoate and heptamethylenediamine can be enhanced or amplified by overexpres sing the solute transporters such as the lysE transporter from Corynebacterium glutamicum (Bellmann et al., 2001, Microbiology, 147, 1765-1774).

[0209] 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 Microorganism

[0210] Typically, one or more C7 building blocks can be produced by providing a 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.

[0211] 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.

[0212] 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

Enzyme activity of .omega.-transaminase using pimelate semialdehyde as substrate and forming 7-aminoheptanoate

[0213] A nucleotide sequence encoding a 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. 7) 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 strain. 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.

[0214] 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.

[0215] 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.

[0216] Each enzyme only control without 7-aminoheptanoate demonstrated low base line conversion of pyruvate to L-alanine. See FIG. 13. The gene product 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. 14.

[0217] 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.

[0218] The gene product 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. 15. 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

[0219] 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. 7), 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 strain 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.

[0220] 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.

[0221] 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. 8.

[0222] 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. 9), and synthesized pimelate semialdehyde.

Example 3

Enzyme activity of carboxylate reductase using 7-hydroxyheptanoate as substrate and forming 7-hydroxyheptanal

[0223] A nucleotide sequence encoding a His-tag was added to the nucleic acids from Mycobacterium marinum, Mycobacterium smegmatis, Segniliparus rugosus, Mycobacterium smegmatis, Mycobacterium massiliense, and Mycobacterium smegmatis that encode the carboxylate reductases of SEQ ID NOs: 2-6 and 15, respectively (GenBank Accession Nos. ACC40567.1, ABK71854.1, EFV11917.1, EIV11143.1, ADG98140.1, and ABK75684.1, respectively) (see FIG. 7) 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 strain 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.

[0224] 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.

[0225] 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. 8.

[0226] The gene products of SEQ ID NO 2-6 and 15, enhanced by the gene product of sfp, accepted 7-hydroxyheptanoate as substrate as confirmed against the empty vector control (see FIG. 10), and synthesized 7-hydroxyheptanal.

Example 4

Enzyme activity of .omega.-transaminase for 7-aminoheptanol, forming 7-oxoheptanol

[0227] 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. 7) 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 strain. Each resulting recombinant E. coli strain 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.

[0228] 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.

[0229] 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.

[0230] Each enzyme only control without 7-aminoheptanol had low base line conversion of pyruvate to L-alanine. See FIG. 13.

[0231] The gene products of SEQ ID NOs: 7, 9 & 10 accepted 7-aminoheptanol as substrate as confirmed against the empty vector control (see FIG. 18) 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

Enzyme activity of -.omega.transaminase using heptamethylenediamine as substrate and forming 7-aminoheptanal

[0232] 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. 7) 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 strain. Each resulting recombinant E. coli strain 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.

[0233] 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.

[0234] 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.

[0235] Each enzyme only control without heptamethylenediamine had low base line conversion of pyruvate to L-alanine. See FIG. 13.

[0236] The gene products of SEQ ID NOs: 7-12 accepted heptamethylenediamine as substrate as confirmed against the empty vector control (see FIG. 16) 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

Enzyme activity of carboxylate reductase for N7-acetyl-7-aminoheptanoate, forming N7-acetyl-7-aminoheptanal

[0237] 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. 7) 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. 8.

[0238] The gene products of SEQ ID NO 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. 11), and synthesized N7-acetyl-7-aminoheptanal.

Example 7

Enzyme activity of .omega.-transaminase using N7-acetyl-1,7-diaminoheptane, and forming N7-acetyl-7-aminoheptanal

[0239] The activity of the N-terminal His-tagged .omega.-transaminases of SEQ ID NOs: 7-12 (see Example 5, and FIG. 7) 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.

[0240] Each enzyme only control without N7-acetyl-1,7-diaminoheptane demonstrated low base line conversion of pyruvate to L-alanine. See FIG. 13.

[0241] The gene product of SEQ ID NOs: 7-12 accepted N7-acetyl-1,7-diaminoheptane as substrate as confirmed against the empty vector control (see FIG. 17) and synthesized N7-acetyl-7-aminoheptanal as reaction product.

[0242] 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

[0243] The N-terminal His-tagged carboxylate reductase of SEQ ID NO: 6 (see Example 3 and FIG. 7) 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. 8.

[0244] The gene product of SEQ ID N: 6, enhanced by the gene product of sfp, accepted pimelate semialdehyde as substrate as confirmed against the empty vector control (see FIG. 12) and synthesized heptanedial.

Example 9

Enzyme activity of 4-hydroxybuterate-CoA transferase using 5-ethanamidopentanoic acid as substrate and forming 5-ethanamidopentanoyl-CoA and 7-ethanamido-3-oxoheptanoyl-CoA

[0245] A nucleotide sequence encoding a His-tag was added to the nucleic acid sequences from Cupriavidus necator, Clostridium propionicum, Clostridium aminobutyricum, Citrobacter sp. A1, Acetobacter aceti, and E. coli K12 encoding, in sequential order, the .beta.-ketothiolase, priopionate CoA-transferase, 4-hydroxybuterate-CoA transferase. acetyl-CoA hydrolase, succinyl-CoA: acetate CoA-transferase, and thiolase of SEQ ID NOs: 16, 17, 18, 19, 20, and 21, respectively (see FIG. 7) for production of His-tagged versions of each protein. 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 strain. The resulting recombinant E. coli strains were cultivated at 37.degree. C. in a 500 mL shake flask culture containing 100 mL LB media and antibiotic selection pressure, with shaking at 230 rpm. Each culture was induced overnight at 20.degree. C. using 0.5 mM IPTG.

[0246] 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 and passage through a 0.45 .mu.m filter. Each of the His-tagged proteins was purified from the supernatant by Ni-affinity chromatography, diluted 10-fold into 50 mM HEPES buffer (pH=7.5), and concentrated by centrifugal filtration with a cut-off of 10 kD.

[0247] Enzyme assays were performed in two reactions for each of the substrates, N-acetyl-.beta.-alanine (AC5) and 5-ethanamidopentanoic acid (AC7): reaction 1 and reaction 2 (see FIG. 19 for reaction schematic).

[0248] For reaction 1, each enzyme activity assay was performed in a buffer composed of a final concentration of 25 mM HEPES buffer (pH=7.5), 50 mM N-acetyl-.beta.-alanine (AC5) or 50 mM 5-ethanamidopentanoic acic (AC7), and 2 mM acetyl CoA. Each enzyme activity assay was initiated by adding His-tag purified enzymes or the empty vector control to the assay buffer containing either the 50 mM N-acetyl-.beta.-alanine or 5-ethanamidopentanoic acid and incubated at 37.degree. C. for 2 h. The formation of 5-ethanamidopentanoyl-CoA and 7-ethanamido-3-oxoheptanoyl-CoA was monitored by LC-MS to identify products by expected masses at distinct retention times.

[0249] For reaction 2, each enzyme activity assay was performed in a buffer composed of a final concentration of 25 mM HEPES buffer (pH=7.5), 13 mM N-acetyl-.beta.-alanyl-CoA (AC5-CoA) or 2.1 mM 5-ethanamidopentanoyl-CoA (AC7-CoA), and 5 mM acetyl CoA. Each enzyme activity assay was initiated by adding His-tag purified enzymes or the empty vector control to the assay buffer containing either the 50 mM N-acetyl-.beta.-alanine or 5-ethanamidopentanoic acid (AC7-CoA) and incubated at 37.degree. C. for 2 h. The formation of 7-ethanamido-3-oxoheptanoyl-CoA was monitored by LC-MS to identify products by expected masses at distinct retention times.

[0250] The 4-hydroxybuterate-CoA transferase gene product of SEQ ID NO: 18 accepted 5-ethanamidopentanoic acid and 5-ethanamidopentanoyl-CoA as substrate and formed 5-ethanamidopentanoyl-CoA and 7-ethanamido-3-oxoheptanoyl-CoA as products, which was confirmed against the empty vector control. See row for EC 2.8.3- in Table 1, and LC-MS mass peaks confirming product identity by expected mass in FIG. 21 (5-ethanamidopentanoyl-CoA: ESI MS expected [M+H].sup.+=909.2017 and [M+2H].sup.+2=445.1044; found 909.2017 and 445.1042) and FIG. 23 (7-ethanamido-3-oxoheptanoyl-CoA: ESI MS expected [M+H].sup.+=951.2120 and [M+2H].sup.+2=476.1097; found 951.2132 and 476.1091).

[0251] Table 1 below presents the results of the enzyme assays. The enzymes are listed by EC number, gene encoding the enzyme, and name. The enzyme assays were performed with acetyl-.beta.-alanine (AC5) and 5-ethanamidopentanoic acid (AC7) substrates in a sequence of two reactions (see FIG. 19). Assays were monitored by LC-MS, and observed product (indicated by a check mark) and no product observed (indicated by x), are reported in Table 1 for the 4-hydroxybutyrate-CoA transferase.

TABLE-US-00001 AC5 AC7 Product Product Product Product Reaction Reaction Reaction Reaction E.C. Gene Name 1 2 1 2 2.3.1.16/ Q0KBP1 BktB x x x x 2.3.1.9 2.8.3.8 Q9L3F7 237 x x x x 2.8.3- Q9RM86 244 x (?*) x (?*) 2.8.3.10 J1G510 337 x x x x 2.8.3.18 B3EY95 344 x x x x 2.3.1.174 P0C7L2 PaaJ x x x x *(?): Product (f) and (e) may be present but not clear.

OTHER EMBODIMENTS

[0252] 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

221394PRTCupriavidus 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 41148PRTSegniliparus 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 abscessus 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 61186PRTSegniliparus 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 7459PRTChromobacterium 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 14325PRTBacillus subtilis 14Met Ser Lys Ala Lys Ile Thr Ala Ile Gly Thr Tyr Ala Pro Ser Arg 1 5 10 15 Arg Leu Thr Asn Ala Asp Leu Glu Lys Ile Val Asp Thr Ser Asp Glu 20 25 30 Trp Ile Val Gln Arg Thr Gly Met Arg Glu Arg Arg Ile Ala Asp Glu 35 40 45 His Gln Phe Thr Ser Asp Leu Cys Ile Glu Ala Val Lys Asn Leu Lys 50 55 60 Ser Arg Tyr Lys Gly Thr Leu Asp Asp Val Asp Met Ile Leu Val Ala 65 70 75 80 Thr Thr Thr Ser Asp Tyr Ala Phe Pro Ser Thr Ala Cys Arg Val Gln 85 90 95 Glu Tyr Phe Gly Trp Glu Ser Thr Gly Ala Leu Asp Ile Asn Ala Thr 100 105 110 Cys Ala Gly Leu Thr Tyr Gly Leu His Leu Ala Asn Gly Leu Ile Thr 115 120 125 Ser Gly Leu His Gln Lys Ile Leu Val Ile Ala Gly Glu Thr Leu Ser 130 135 140 Lys Val Thr Asp Tyr Thr Asp Arg Thr Thr Cys Val Leu Phe Gly Asp 145 150 155 160 Ala Ala Gly Ala Leu Leu Val Glu Arg Asp Glu Glu Thr Pro Gly Phe 165 170 175 Leu Ala Ser Val Gln Gly Thr Ser Gly Asn Gly Gly Asp Ile Leu Tyr 180 185 190 Arg Ala Gly Leu Arg Asn Glu Ile Asn Gly Val Gln Leu Val Gly Ser 195 200 205 Gly Lys Met Val Gln Asn Gly Arg Glu Val Tyr Lys Trp Ala Ala Arg 210 215 220 Thr Val Pro Gly Glu Phe Glu Arg Leu Leu His Lys Ala Gly Leu Ser 225 230 235 240 Ser Asp Asp Leu Asp Trp Phe Val Pro His Ser Ala Asn Leu Arg Met 245 250 255 Ile Glu Ser Ile Cys Glu Lys Thr Pro Phe Pro Ile Glu Lys Thr Leu 260 265 270 Thr Ser Val Glu His Tyr Gly Asn Thr Ser Ser Val Ser Ile Val Leu 275 280 285 Ala Leu Asp Leu Ala Val Lys Ala Gly Lys Leu Lys Lys Asp Gln Ile 290 295 300 Val Leu Leu Phe Gly Phe Gly Gly Gly Leu Thr Tyr Thr Gly Leu Leu 305 310 315 320 Ile Lys Trp Gly Met 325 151167PRTMycobacterium smegmatis 15Met 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 Gly 755 760 765 Thr Glu Ile Arg Thr Val Leu Leu Thr Gly Ala Thr Gly Phe Leu Gly 770 775 780 Arg Tyr Leu Ala Leu Glu Trp Leu Glu Arg Met Asp Leu Val Asp Gly 785 790 795 800 Lys Val Ile Cys Leu Val Arg Ala Arg Ser Asp Asp Glu Ala Arg Ala 805 810 815 Arg Leu Asp Ala Thr Phe Asp Thr Gly Asp Ala Thr Leu Leu Glu His 820 825 830 Tyr Arg Ala Leu Ala Ala Asp His Leu Glu Val Ile Ala Gly Asp Lys 835 840 845 Gly Glu Ala Asp Leu Gly Leu Asp His Asp Thr Trp Gln Arg Leu Ala 850 855 860 Asp Thr Val Asp Leu Ile Val Asp Pro Ala Ala Leu Val Asn His Val 865 870 875 880 Leu Pro Tyr Ser Gln Met Phe Gly Pro Asn Ala Leu Gly Thr Ala Glu 885 890 895 Leu Ile Arg Ile Ala Leu Thr Thr Thr Ile Lys Pro Tyr Val Tyr Val 900 905 910 Ser Thr Ile Gly Val Gly Gln Gly Ile Ser Pro Glu Ala Phe Val Glu 915 920 925 Asp Ala Asp Ile Arg Glu Ile Ser Ala Thr Arg Arg Val Asp Asp Ser 930 935 940 Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu Leu 945 950 955 960 Arg Glu Ala His Asp Trp Cys Gly Leu Pro Val Ser Val Phe Arg Cys 965 970 975 Asp Met Ile Leu Ala Asp Thr Thr Tyr Ser Gly Gln Leu Asn Leu Pro 980 985 990 Asp Met Phe Thr Arg Leu Met Leu Ser Leu Val Ala Thr Gly Ile Ala 995 1000 1005 Pro Gly Ser Phe Tyr Glu Leu Asp Ala Asp Gly Asn Arg Gln Arg 1010 1015 1020 Ala His Tyr Asp Gly Leu Pro Val Glu Phe Ile Ala Glu Ala Ile 1025 1030 1035 Ser Thr Ile Gly Ser Gln Val Thr Asp Gly Phe Glu Thr Phe His 1040 1045 1050 Val Met Asn Pro Tyr Asp Asp Gly Ile Gly Leu Asp Glu Tyr Val 1055 1060 1065 Asp Trp Leu Ile Glu Ala Gly Tyr Pro Val His Arg Val Asp Asp 1070 1075 1080 Tyr Ala Thr Trp Leu Ser Arg Phe Glu Thr Ala Leu Arg Ala Leu 1085 1090 1095 Pro Glu Arg Gln Arg Gln Ala Ser Leu Leu Pro Leu Leu His Asn 1100 1105 1110 Tyr Gln Gln Pro Ser Pro Pro Val Cys Gly Ala Met Ala Pro Thr 1115 1120 1125 Asp Arg Phe Arg Ala Ala Val Gln Asp Ala Lys Ile Gly Pro Asp 1130 1135 1140 Lys Asp Ile Pro His Val Thr Ala Asp Val Ile Val Lys Tyr Ile 1145 1150 1155 Ser Asn Leu Gln Met Leu Gly Leu Leu 1160 1165 16393PRTCupriavidus necator 16Met 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 His Ala Gly Val Asp Pro Lys Ala 275 280 285 Met Gly Ile Gly Pro Val Pro Ala Thr Lys Ile Ala Leu Glu Arg Ala 290 295 300 Gly Leu Gln Val Ser Asp Leu Asp Val Ile Glu Ala Asn Glu Ala Phe 305 310 315 320 Ala Ala Gln Ala Cys Ala Val Thr Lys Ala Leu Gly Leu Asp Pro Ala 325 330 335 Lys Val Asn Pro Asn Gly Ser Gly Ile Ser Leu Gly His Pro Ile Gly 340 345 350 Ala Thr Gly Ala Leu Ile Thr Val Lys Ala Leu His Glu Leu Asn Arg 355 360 365 Val Gln Gly Arg Tyr Ala Leu Val Thr Met Cys Ile Gly Gly Gly Gln 370 375 380 Gly Ile Ala Ala Ile Phe Glu Arg Ile 385 390 17401PRTEscherichia coli 17Met 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 18524PRTClostridium propionicum 18Met Arg Lys Val Pro Ile Ile Thr Ala Asp Glu Ala Ala Lys Leu Ile 1 5 10 15 Lys Asp Gly Asp Thr Val Thr Thr Ser Gly Phe Val Gly Asn Ala Ile 20 25 30 Pro Glu Ala Leu Asp Arg Ala Val Glu Lys Arg Phe Leu Glu Thr Gly 35 40 45 Glu Pro Lys Asn Ile Thr Tyr Val Tyr Cys Gly Ser Gln Gly Asn Arg 50 55 60 Asp Gly Arg Gly Ala Glu His Phe Ala His Glu Gly Leu Leu Lys Arg 65 70 75 80 Tyr Ile Ala Gly His Trp Ala Thr Val Pro Ala Leu Gly Lys Met Ala 85 90 95 Met Glu Asn Lys Met Glu Ala Tyr Asn Val Ser Gln Gly Ala Leu Cys 100 105 110 His Leu Phe Arg Asp Ile Ala Ser His Lys Pro Gly Val Phe Thr Lys 115 120 125 Val Gly Ile Gly Thr Phe Ile Asp Pro Arg Asn Gly Gly Gly Lys Val 130 135 140 Asn Asp Ile Thr Lys Glu Asp Ile Val Glu Leu Val Glu Ile Lys Gly 145 150 155 160 Gln Glu Tyr Leu Phe Tyr Pro Ala Phe Pro Ile His Val Ala Leu Ile 165 170 175 Arg Gly Thr Tyr Ala Asp Glu Ser Gly Asn Ile Thr Phe Glu Lys Glu 180 185 190 Val Ala Pro Leu Glu Gly Thr Ser Val Cys Gln Ala Val Lys Asn Ser 195 200 205 Gly Gly Ile Val Val Val Gln Val Glu Arg Val Val Lys Ala Gly Thr 210 215 220 Leu Asp Pro Arg His Val Lys Val Pro Gly Ile Tyr Val Asp Tyr Val 225 230 235 240 Val Val Ala Asp Pro Glu Asp His Gln Gln Ser Leu Asp Cys Glu Tyr 245 250 255 Asp Pro Ala Leu Ser Gly Glu His Arg Arg Pro Glu Val Val Gly Glu 260 265 270 Pro Leu Pro Leu Ser Ala Lys Lys Val Ile Gly Arg Arg Gly Ala Ile 275 280 285 Glu Leu Glu Lys Asp Val Ala Val Asn Leu Gly Val Gly Ala Pro Glu 290 295 300 Tyr Val Ala Ser Val Ala Asp Glu Glu Gly Ile Val Asp Phe Met Thr 305 310 315 320 Leu Thr Ala Glu Ser Gly Ala Ile Gly Gly Val Pro Ala Gly Gly Val 325 330 335 Arg Phe Gly Ala Ser Tyr Asn Ala Asp Ala Leu Ile Asp Gln Gly Tyr 340 345 350 Gln Phe Asp Tyr Tyr Asp Gly Gly Gly Leu Asp Leu Cys Tyr Leu Gly 355 360 365 Leu Ala Glu Cys Asp Glu Lys Gly Asn Ile Asn Val Ser Arg Phe Gly 370 375 380 Pro Arg Ile Ala Gly Cys Gly Gly Phe Ile Asn Ile Thr Gln Asn Thr 385 390 395 400 Pro Lys Val Phe Phe Cys Gly Thr Phe Thr Ala Gly Gly Leu Lys Val 405 410 415 Lys Ile Glu Asp Gly Lys Val Ile Ile Val Gln Glu Gly Lys Gln Lys 420 425 430 Lys Phe Leu Lys Ala Val Glu Gln Ile Thr Phe Asn Gly Asp Val Ala 435 440 445 Leu Ala Asn Lys Gln Gln Val Thr Tyr Ile Thr Glu Arg Cys Val Phe 450 455 460 Leu Leu Lys Glu Asp Gly Leu His Leu Ser Glu Ile Ala Pro Gly Ile 465 470 475 480 Asp Leu Gln Thr Gln Ile Leu Asp Val Met Asp Phe Ala Pro Ile Ile 485 490 495 Asp Arg Asp Ala Asn Gly Gln Ile Lys Leu Met Asp Ala Ala Leu Phe 500 505 510 Ala Glu Gly Leu Met Gly Leu Lys Glu Met Lys Ser 515 520 19438PRTClostridium aminobutyricum 19Met Asp Trp Lys Lys Ile Tyr Glu Asp Arg Thr Cys Thr Ala Asp Glu 1 5 10 15 Ala Val Lys Ser Ile Lys Ser Gly Asp Arg Val Leu Phe Ala His Cys 20 25 30 Val Ala Glu Pro Pro Val Leu Val Glu Ala Met Val Ala Asn Ala Ala 35 40 45 Ala Tyr Lys Asn Val Thr Val Ser His Met Val Thr Leu Gly Lys Gly 50 55 60 Glu Tyr Ser Lys Pro Glu Tyr Lys Glu Asn Phe Thr Phe Glu Gly Trp 65 70 75 80 Phe Thr Ser Pro Ser Thr Arg Gly Ser Ile Ala Glu Gly His Gly Gln 85 90 95 Phe Val Pro Val Phe Phe His Glu Val Pro Ser Leu Ile Arg Lys Asp 100 105 110 Ile Phe His Val Asp Val Phe Met Val Met Val Ser Pro Pro Asp His 115 120 125 Asn Gly Phe Cys Cys Val Gly Val Ser Ser Asp Tyr Thr Met Gln Ala 130 135 140 Ile Lys Ser Ala Lys Ile Val Leu Ala Glu Val Asn Asp Gln Val Pro 145 150 155 160 Val Val Tyr Gly Asp Thr Phe Val His Val Ser Glu Ile Asp Lys Phe 165 170 175 Val Glu Thr Ser His Pro Leu Pro Glu Ile Gly Leu Pro Lys Ile Gly 180 185 190 Glu Val Glu Ala Ala Ile Gly Lys His Cys Ala Ser Leu Ile Glu Asp 195 200 205 Gly Ser Thr Leu Gln Leu Gly Ile Gly Ala Ile Pro Asp Ala Val Leu 210 215 220 Ser Gln Leu Lys Asp Lys Lys His Leu Gly Ile His Ser Glu Met Ile 225 230 235 240 Ser Asp Gly Val Val Asp Leu Tyr Glu Ala Gly Val Ile Asp Cys Ser 245 250 255 Gln Lys Ser Ile Asp Lys Gly Lys Met Ala Ile Thr Phe Leu Met Gly 260 265 270 Thr Lys Arg Leu Tyr Asp Phe Ala Ala Asn Asn Pro Lys Val Glu Leu 275 280 285 Lys Pro Val Asp Tyr Ile Asn His Pro Ser Val Val Ala Gln Cys Ser 290 295 300 Lys Met Val Cys Ile Asn Ala Cys Leu Gln Val Asp Phe Met Gly Gln 305 310 315 320 Ile Val Ser Asp Ser Ile Gly Thr Lys Gln Phe Ser Gly Val Gly Gly 325 330 335 Gln Val Asp Phe Val Arg Gly Ala Ser Met Ser Ile Asp Gly Lys Gly 340 345 350 Lys Ala Ile Ile Ala Met Pro Ser Val Ala Lys Lys Lys Asp Gly Ser 355 360 365 Met Ile Ser Lys Ile Val Pro Phe Ile Asp His Gly Ala Ala Val Thr 370 375 380 Thr Ser Arg Asn Asp Ala Asp Tyr Val Val Thr Glu Tyr Gly Ile Ala 385 390 395 400 Glu Met Lys Gly Lys Ser Leu Gln Asp Arg Ala Arg Ala Leu Ile Asn 405 410 415 Ile Ala His Pro Asp Phe Lys Asp Glu Leu Lys Ala Glu Phe Glu Lys 420 425 430 Arg Phe Asn Ala Ala Phe 435 20488PRTCitrobacter sp. 20Met Lys Arg Met Ser Ala Glu Gln Ala Ala Glu Ile Ile Gln His Asp 1 5 10 15 Asp Met Val Ala Phe Ser Gly Phe Thr Pro Ala Gly Ser Pro Lys Ala 20 25 30 Leu Pro Thr Ala Ile Ala Gln Arg Ala Cys Glu Gln His Gln Asn Gly 35 40 45 Gln Pro Phe Gln Ile Arg Leu Leu Thr Gly Ala Ser Ile Gly Ala Ala 50 55 60 Ala Asp Asp Val Leu Ser Ala Ala Asp Ala Val Ser Trp Arg Ala Pro 65 70 75 80 Tyr Gln Thr Ser Ser Gly Leu Arg Asp Lys Ile Asn Gln Gly Gln Val 85 90 95 Arg Phe Val Asp Leu His Leu Ser Glu Val Ala Gln Met Val Asn Tyr 100 105 110 Gly Phe Phe Gly Glu Ile Asp Val Ala Val Ile Glu Ala Ser Ala Ile 115 120 125 Ala Pro Asp Gly Arg Ile Trp Leu Ser Ser Gly Ile Gly Asn Ala Pro 130 135 140 Thr Trp Leu Leu Arg Ala Lys Lys Val Ile Ile Glu Leu Asn His Tyr 145 150 155 160 His Asn Pro Arg Val Ala Glu Phe Ala Asp Ile Val Ile Pro Gly Ala 165 170 175 Pro Pro Arg Arg Asn Ser Val Pro Ile Phe His Thr Met Asp Arg Val 180 185 190 Gly Ser Gln Cys Val Gln Ile Asp Pro Lys Lys Val Val Ala Val Val 195 200 205 Asp Thr Glu Leu Pro Asp Ala Gly Asn Ala Ser Asp Lys Thr Asn Pro 210 215 220 Val Ser Gln Gln Ile Ala Asp Asn Val Val Ser Phe Leu Leu Ala Glu 225 230 235 240 Met Ala His Lys Arg Ile Pro Ala Glu Phe Leu Pro Leu Gln Ser Gly 245 250 255 Val Gly Asn Ile Asn Asn Ala Val Met Ala Arg Leu Gly Glu Asn Pro 260 265 270 Asp Ile Pro Pro Phe Met Met Tyr Ser Glu Val Leu Gln Glu Ser Val 275 280 285 Val His Leu Leu Glu Thr Gly Lys Ile Ser Gly Ala Ser Ala Ser Ser 290 295 300 Leu Thr Ile Ser Ala Pro Ser Leu Gln Lys Ile Tyr Asp Asn Met Asp 305 310 315 320 Phe Phe Ala Ser Arg Ile Val Leu Arg Pro Gln Glu Ile Ser Asn Asn 325 330 335 Pro Glu Ile Ile Arg Arg Leu Gly Val Ile Ala Leu Asn Val Gly Leu 340 345 350 Glu Phe Asp Ile Tyr Gly His Ala Asn Ser Thr His Val Ala Gly Val 355 360 365 Asn Leu Met Asn Gly Ile Gly Gly Ser Gly Asp Phe Glu Arg Asn Ala 370 375 380 Tyr Leu Ser Ile Phe Met Ala Pro Ser Ile Ala Lys Gly Gly Lys Ile 385 390 395 400 Ser Thr Ile Val Pro Met Cys Ser His Val Asp His Ser Glu His Ser 405 410 415 Val Lys Val Ile Val Thr Glu Gln Gly Ile Ala Asp Leu Arg Gly Leu 420 425 430 Ser Pro Met Gln Arg Ala His Thr Ile Ile Asn Asn Cys Ala His Pro 435 440 445 Leu Tyr Arg Asp Tyr Leu His Arg Tyr Leu Glu Lys Ala Pro Gly Gly 450 455 460 His Ile His His Asp Leu Ser His Ala Phe Asp Leu His Arg Asn Leu 465 470 475 480 Leu Glu Thr Gly Ser Met Leu Gly 485 21505PRTAcetobacter aceti 21Met Thr Glu Arg Ile Arg Asn Val Ala Leu Arg Ser Lys Val Cys Pro 1 5 10 15 Ala Glu Thr Ala Ser Glu Leu Ile Lys His Gly Asp Val Val Gly Thr 20 25 30 Ser Gly Phe Thr Gly Ala Gly Tyr Pro Lys Glu Val Pro Lys Ala Leu 35 40 45 Ala Gln Arg Met Glu Ala Ala His Asp Arg Gly Glu Lys Tyr Gln Ile 50 55 60 Ser Leu Ile Thr Gly Ala Ser Thr Gly Pro Gln Leu Asp Gly Glu Leu 65 70 75 80 Ala Lys Ala Asn Gly Val Tyr Phe Arg Ser Pro Phe Asn Thr Asp Ala 85 90 95 Thr Met Arg Asn Arg Ile Asn Ala Gly Glu Thr Glu Tyr Phe Asp Asn 100 105 110 His Leu Gly Gln Val Ala Gly Arg Ala Val Gln Gly Asn Tyr Gly Lys 115 120 125 Phe Asn Ile Ala Leu Val Glu Ala Thr Ala Ile Thr Glu Asp Gly Gly 130 135 140 Ile Val Pro Thr Ser Ser Val Gly Asn Ser Gln Thr Phe Leu Asn Leu 145 150 155 160 Ala Glu Lys Val Ile Ile Glu Val Asn Glu Trp Gln Asn Pro Met Leu 165 170 175 Glu Gly Ile His Asp Ile Trp Asp Gly Asn Val Ser Gly Val Pro Thr 180 185 190 Arg Asp Ile Val Pro Ile Val Arg Ala Asp Gln Arg Val Gly Gly Pro 195 200 205 Val Leu Arg Val Asn Pro Asp Lys Ile Ala Ala Ile Val Arg Thr Asn 210 215 220 Asp Arg Asp Arg Asn Ala Pro Phe Ala Ala Pro Asp Glu Thr Ala Lys 225 230 235 240 Ala Ile Ala Gly Tyr Leu Leu Asp Phe Phe Gly His Glu Val Lys Gln 245 250 255 Asn Arg Leu Pro Pro Ser Leu Leu Pro Leu Gln Ser Gly Val Gly Asn 260 265 270 Val Ala Asn Ala Val Leu Glu Gly Leu Lys Glu Gly Pro Phe Glu Asn 275 280 285 Leu Val Gly Tyr Ser Glu Val Ile Gln Asp Gly Met Leu Ala Met Leu 290 295 300 Asp Ser Gly Arg Met Arg Ile Ala Ser Ala Ser Ser Phe Ser Leu Ser 305 310 315 320 Pro Glu Ala Ala Glu Glu Ile Asn Asn Arg Met Asp Phe Phe Arg Ser 325 330 335 Lys Ile Ile Leu Arg Gln Gln Asp Val Ser Asn Ser Pro Gly Ile Ile 340 345 350 Arg Arg Leu Gly Cys Ile Ala Met Asn Gly Met Ile Glu Ala Asp Ile 355 360 365 Tyr Gly Asn Val Asn Ser Thr Arg Val Met Gly Ser Lys Met Met Asn 370 375 380 Gly Ile Gly Gly Ser Gly Asp Phe Ala Arg Ser Ser Tyr Leu Ser Ile 385 390 395 400 Phe Leu Ser Pro Ser Thr Ala Lys Gly Gly Lys Ile Ser Ala Ile Val 405 410 415 Pro Met Ala Ala His Val Asp His Ile Met Gln Asp Ala Gln Ile Phe 420 425 430 Val Thr Glu Gln Gly Leu Ala Asp Leu Arg Gly Leu Ser Pro Val Gln 435 440 445 Arg Ala Arg Glu Ile Ile Ser Lys Cys Ala His Pro Asp Tyr Arg Pro 450 455 460 Met Leu Gln Asp Tyr Phe Asp Arg Ala Leu Lys Asn Ser Phe Gly Lys 465 470 475 480 His Thr Pro His Leu Leu Thr Glu Ala Leu Ser Trp His Gln Arg Phe 485 490 495 Ile Asp Thr Gly Thr Met Leu Pro Ser 500 505 227PRTArtificial SequenceDescription of Artificial Sequence Synthetic 7xHis tag 22His His His His His His His 1 5

Claims

1. A method of producing N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof, said method comprising enzymatically converting N-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof using a polypeptide having the activity of a .beta.-ketoacyl synthase or a .beta.-ketothiolase classified under EC. 2.3.1.- and/or a CoA transferase classified under EC 2.8.3.-.

2. The method of claim 1, wherein said polypeptide having the activity of a .beta.-ketoacyl synthase is classified under EC 2.3.1.41, EC 2.3.1.179 or EC 2.3.1.180 and wherein said polypeptide having the activity of a .beta.-ketothiolase is classified under EC 2.3.1.16 or EC 2.3.1.174.

3. The method of claim 1 any one of claims 1 2, wherein said polypeptide having the activity of a .beta.-ketothiolase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1 or 13 and said polypeptide having the activity of a .beta.-ketoacyl synthase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14 and said polypeptide having the activity of a CoA transferase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19.

4. The method of claim 3, wherein said polypeptide having the activity of a .beta.-ketothiolase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1 or 13 and is capable of converting N-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoA and said polypeptide having the activity of a .beta.-ketoacyl synthase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14 and is capable of converting N-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoA and said polypeptide having the activity of a CoA transferase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19 and is capable of converting N-acetyl-3-aminopropanoyl-CoA to N-acetyl-5-amino-3-oxopentanoyl-CoA.

5. The method of claim 1, further comprising enzymatically converting N-acetyl-5-amino-3-oxopentanoyl-CoA or the salt thereof to 7-aminoheptanoate using polypeptides having the enzymatic activities of a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase, a trans-2-enoyl-CoA reductase, a .beta.-ketothiolase, a thioesterase or a CoA transferase and a deacetylase.

6.-12. (canceled)

13. A method for biosynthesizing 7-aminoheptanoate, said method comprising enzymatically synthesizing N-acetyl-5-amino-3-oxopentanoy/-CoA or the salt thereof from N-acetyl-3-aminopropanoyl-CoA using a polypeptide having the activity of a .beta.-ketoacyl synthase or a .beta.-ketothiolase classified under EC. 2.3.1.- and/or a CoA transferase classified under EC 2.8.3-, and enzymatically converting N-acetyl-5-amino-3-oxopentanoyl-CoA or the salt thereof to 7-aminoheptanoate.

14. The method of claim 13, wherein N-acetyl-5-amino-3-oxopentanoyl-CoA or the salt thereof is converted to N-acetyl-5-amino-3-hydroxypentanoyl-CoA using a polypeptide having the activity of a 3-hydroxyacyl-CoA dehydrogenase; N-acetyl-5-amino-3-hydroxypentanoyl-CoA is converted to 5-amino-pent-2-enoyl-CoA using polypeptide having the activity of an enoyl-CoA hydratase; N-acetyl-5-amino-pent-2-enoyl-CoA is converted to N-acetyl-5-amino-pentanoyl-CoA using a polypeptide having the activity of a trans-2-enoyl-CoA-reductase; N-acetyl-5-amino-pentanoyl-CoA is converted to N-acetyl-7-amino-3-oxoheptanoyl-CoA using a polypeptide having the activity of a 13-ketothiolase; N-acetyl-7-amino-3-oxoheptanoyl-CoA is converted to N-acetyl-7-amino-3-hydroxyheptanoyl-CoA using a polypeptide having the activity of a 3-hydroxyacyl-CoA-dehydrogenase; N-acetyl-7-amino-3-hydroxyheptanoyl-CoA is converted to N-acetyl-7-amino-hept-2-enoyl-CoA using a polypeptide having the activity of an enoyl-CoA hydratase; N-acetyl-7-amino-hept-2-enoyl-CoA is converted to N-acetyl-7-aminoheptanoyl-CoA using a polypeptide having the activity of a trans-2-enoyl-CoA reductase; N-acetyl-7-aminoheptanoyl-CoA is converted to N-acetyl-7-aminoheptanoate using a polypeptide having the activity of a thioesterase or a CoA transferase; and N-acetyl-7-aminoheptanoate is converted to 7-aminoheptanoate using a polypeptide having the activity of a deacetylase.

15. The method of claim 5, said method further comprising enzymatically converting 7-am inoheptanoate to pimelic acid, 7-hydroxyheptanoate, heptamethylenediamine or 1,7-heptanediol or a corresponding salt thereof in one or more steps.

16. The method of claim 15, wherein 7-aminoheptanoate is converted to pimelic acid using one or more polypeptides having the enzymatic activity of a .omega.-transaminase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 5-oxopentanoate dehydrogenase, or an aldehyde dehydrogenase.

17. The method of claim 15, wherein 7-aminoheptanoate is converted to 7-hydroxyheptanoate using one or more polypeptides having the enzymatic activity of an alcohol dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutanoate dehydrogenase, and a .omega.-transaminase.

18. The method of claim 15, wherein 7-aminoheptanoate is converted to heptamethylenediamine using polypeptides having the enzymatic activity of a carboxylate reductase and a .omega.-transaminase.

19. The method of claim 15, wherein 7-aminoheptanoate is converted to heptamethylenediamine using polypeptides having the enzymatic activity of a carboxylate reductase, a .omega.-transaminase and an alcohol dehydrogenase.

20. The method of claim 15, wherein 7-aminoheptanoate is converted to heptamethylenediamine using polypeptides having the enzymatic activity of an N-acetyltransferase, a carboxylate reductase, a .omega.-transaminase, and a deacetylase.

21. The method of claim 15, wherein 7-aminoheptanoate is converted to heptamethylenediamine using polypeptides having the enzymatic activity of an alcohol dehydrogenase and a .omega.-transaminase.

22. (canceled)

23. (canceled)

24. The method of claim 15, wherein 7-hydroxyheptanoate is converted to 1,7-heptanediol using a polypeptide having the activity of a carboxylate reductase and a polypeptide having the activity of an alcohol dehydrogenase.

25. (canceled)

26. (canceled)

27. The method of claim 1, wherein said N-acetyl-3-aminopropanoyl-CoA is enzymatically produced from malonyl-CoA or L-aspartate.

28. The method of claim 27, wherein said N-acetyl-3-aminopropanoyl-CoA is enzymatically produced from malonyl-CoA or L-aspartate using one or more polypeptides having the enzymatic activity of a malonyl-CoA-reductase, a .beta.-alanine-pyruvate aminotransferase, an .alpha.-aspartate decarboxylase, an N-acetyl transferase, a CoA transferase and a CoA Ligase.

29. The method of claim 1, wherein said method is performed in a recombinant microorganism.

30. The method of claim 29, wherein said microorganism is subjected to a cultivation strategy under aerobic, anaerobic or, micro-aerobic cultivation conditions.

31. The method of claim 29, wherein said microorganism is cultured under conditions of nutrient limitation.

32. (canceled)

33. The method of claim 29, wherein the principal carbon source fed to the fermentation derives from a biological feedstock.

34. (canceled)

35. The method of claim 29, wherein the principal carbon source fed to the fermentation derives from a non-biological feedstock.

36. (canceled)

37. The method of claim 29, wherein the microorganism is a prokaryote.

38. (canceled)

39. (canceled)

40. The method of claim 29, wherein the microorganism is a eukaryote.

41. (canceled)

42. (canceled)

43. The method of claim 29, wherein the microorganism's tolerance to high concentrations of a C7 building block is improved relative to a wild type organism.

44. (canceled)

45. The method of claim 29 , wherein said microorganism 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.

46. The method of claim 29, wherein said microorganism 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.

47. A recombinant microorganism comprising at least one exogenous nucleic acid encoding a polypeptide having the enzymatic activity of (i) a .beta.-ketoacyl synthase and/or a .beta.-ketothiolase, (ii) a thioesterase or a CoA transferase, (iii) a deacetylase, and one or more of (iv) 3-hydroxyacyl-CoA dehydrogenase, (iv) an enoyl-CoA hydratase, and (v) a trans-2-enoyl-CoA reductase, said microorganism producing 7-am inoheptanoate or a corresponding salt thereof.

48. The recombinant microorganism of claim 47, said microorganism further comprising one or more of the following exogenous enzymes: .omega.-transaminase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 5-pentanoate dehydrogenase, or an aldehyde dehydrogenase, said microorganism further producing pimelic acid or a corresponding salt thereof.

49. The recombinant microorganism of claim 47, said microorganism further comprising one or more of the following exogenous enzymes: a .omega.-transaminase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutanoate dehydrogenase, and an alcohol dehydrogenase, said microorganism further producing 7-hydroxyheptanoate or a corresponding salt thereof.

50. The recombinant microorganism of claim 47, said microorganism further comprising 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 microorganism further producing heptamethylenediamine or a corresponding salt thereof.

51.-54. (canceled)

55. The recombinant microorganism of claim 47, said microorganism further comprising an exogenous carboxylate reductase and an exogenous alcohol dehydrogenase, said microorganism further producing 1,7-heptanediol or a corresponding salt thereof.

56. The recombinant microorganism of claim 47, said microorganism further comprising one or more of the following exogenous enzymes: an aspartate-.alpha.-decarboxylase; a malonyl-CoA reductase; a .beta.-alanine-pyruvate-aminotransferase; an N-acetyl transferase; a thioesterase; a CoA-transferase; a CoA ligase and a deacetylase.

57. A non-naturally occurring microorganism comprising at least one exogenous nucleic acid encoding at least one polypeptide having the activity of at least one enzyme, at least one substrate and at least one product, depicted in any one of FIGS. 1 to 6.

58. A nucleic acid construct or expression vector 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 NO: 1; and (b) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 13.

59. A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having .beta.-ketoacyl synthase 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.-ketoacyl synthase activity has at least 70% sequence identity to the polypeptide of SEQ ID NO: 14.

60. A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having carboxylate reductase 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 carboxylate reductase activity is selected from the group consisting of: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 2; (b) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 3; (c) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 4; (d) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 5; (e) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 6; and (f) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 15.

61. A nucleic acid construct or expression vector comprising 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 NO: 7; (b) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 8; (c) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 9; (d) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 10; (e) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO:11 and (f) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO:12.

62. (canceled)

63. (canceled)

64. A non-naturally occurring biochemical network comprising an N-acetyl-3-aminopropanoyl-CoA, an exogenous nucleic acid encoding a polypeptide having the activity of a .beta.-ketothiolase or a .beta.-ketoacyl synthase classified under EC. 2.3.1, and an N-acetyl-5-amino-3-oxopentanoyl-CoA.

65. A non-naturally occurring biochemical network comprising at least one exogenous nucleic acid encoding a polypeptide having the enzymatic activity of (i) a .beta.-ketoacyl synthase and/or a .beta.-ketothiolase, (ii) a thioesterase or a CoA transferase, (iii) a deacetylase, and one or more of (iv) 3-hydroxyacyl-CoA dehydrogenase, (iv) an enoyl-CoA hydratase, and (v) a trans-2-enoyl-CoA reductase, said microorganism producing 7-aminoheptanoate.

66. (canceled)

67. A bio-derived, bio-based or fermentation-derived product, wherein said product comprises: (i) a composition comprising at least one bio-derived, bio-based or fermentation-derived compound according to claim 15 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.

68. A method of producing 7-ethanamido-3-oxoheptanoyl-CoA or a salt thereof, said method comprising enzymatically converting 5-ethanamidopentanoic acid to 7-ethanamido-3-oxoheptanoyl-CoA or a salt thereof using a polypeptide having the activity of a .beta.-ketoacyl synthase or a .beta.-ketothiolase classified under EC. 2.3.1.-, further comprising enzymatically converting 5-ethanamidopentanoic acid or the salt thereof to 7-ethanamido-3-oxoheptanoyl-CoA using polypeptides having the enzymatic activities of a .beta.-ketothiolase, CoA transferase, acetyl-CoA hydrolase, and thiolase.

69. The method of claim 68, wherein said CoA-transferase is classified under EC 2.8.3.-.

70. A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having CoA transferase 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 CoA transferase activity has at least 70% sequence identity to the polypeptide of SEQ ID NO: 19.

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