SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 29, 2016, is named 12444_0620-00000_SL.txt and is 118,685 bytes in size.

TECHNICAL FIELD

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 β-ketoacyl synthase or a β-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 β-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

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.

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

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 β-ketoacyl synthase or a β-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, 7-hydroxyheptanoic acid and 7-hydroxyheptanoate, and 7-aminoheptanoic acid and 7-aminoheptanoate are used interchangeably herein to refer to the compound in any of its neutral or ionized forms, including any salt forms thereof. It is understood by those skilled in the art that the specific form will depend on pH.

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.

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.

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-carboxylic acid, glucoheptonic acid, 4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like or 2) when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases are known in the art and include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases are known in the art and include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. The salt can be isolated as is from the system or converted to the free acid by reducing the pH to below the pKa through addition of acid or treatment with an acidic ion exchange resin.

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.

In some embodiments, a terminal carboxyl group can be enzymatically formed using a thioesterase, a CoA transferase, a co-transaminase, an aldehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase or a 7-oxoheptanoate dehydrogenase. SeeFIG. 2andFIG. 3.

In some embodiments, a terminal amine group can be enzymatically formed using a carboxylate reductase, a ω-transaminase or a deacylase. SeeFIG. 4A-E. The co-transaminase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs. 7-12. Furthermore, the co-transaminase can have at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs. 7-12 and be capable of transferring at least one amine group separated from a carbonyl group by at least one methylene insertion.

In some embodiments, a terminal hydroxyl group can be enzymatically formed using an alcohol dehydrogenase. SeeFIG. 5andFIG. 6.

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 β-alanine to N-acetyl-5-amino-3-oxopentanoyl-CoA or a salt thereof via N-acetyl-3-aminopropanoate and N-acetyl-3-aminopropanoyl-CoA. β-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.-.

N-acetyl-3-aminopropanoyl-CoA is converted to N-acetyl-5-amino-3-oxopentanoyl-CoA using a polypeptide having the activity of a β-ketoacyl synthase classified under EC. 2.3.1.- (e.g., EC 2.3.1.180) or a β-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 β-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 β-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 β-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 β-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.

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 β-ketothiolase, a thioesterase or a CoA transferase and a deacetylase.

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 β-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.-.

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 β-ketoacyl synthase classified under EC 2.3.1.- (e.g., EC 2.3.1.180) or a β-ketothiolase classified under EC. 2.3.1.- (e.g., EC 2.3.1.16 or EC 2.3.1.174). The β-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 β-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 β-ketoacyl synthase can have at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:14. Furthermore, the β-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.

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 β-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.

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.

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

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 co-transaminase, an alcohol dehydrogenase, an N-acetyltransferase, and a deacylase. SeeFIG. 4A-B.

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 co-transaminase. SeeFIG. 5.

For example, 7-aminoheptanoate can be converted to 7-hydroxyheptanoate (seeFIG. 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. SeeFIG. 6.

The ω-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 ω-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.

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 ID 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.

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

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). SeeFIG. 3.

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. SeeFIG. 5.

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 ω-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. SeeFIG. 4B-C.

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. SeeFIG. 6.

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.

In some embodiments, the non-biological feedstock can be or can derive from natural gas, syngas, CO2/H2, 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.

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.

In some embodiments, the microorganism's biochemical network is attenuated or augmented to (1) ensure the intracellular availability of acetyl-CoA and β-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.

In some embodiments, a cultivation strategy is used to achieve anaerobic, micro-aerobic, or aerobic cultivation conditions.

In some embodiments, the cultivation strategy includes limiting nutrients, such as limiting nitrogen, phosphate or oxygen.

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 β-ketothiolase, a β-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 β-ketoacyl synthase, (ii) a β-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. SeeFIG. 1andFIG. 2.

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

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 ω-transaminase, a deacylase, an N-acetyl transferase, or an alcohol dehydrogenase, said microorganism further producing heptamethylenediamine. SeeFIG. 4A-B.

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

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. SeeFIG. 6.

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

Any of the recombinant microorganisms can be a prokaryote such as a prokaryote from a genus selected from the group consisting ofEscherichia; Clostridia; Corynebacteria;Cupriavidus; Pseudomonas; Delftia; Bacilluss;Lactobacillus; Lactococcus; andRhodococcus. For example, the prokaryote can be selected from the group consisting ofEscherichia coli, Clostridium ljungdahlii, Clostridiumautoethanogenum,Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonasoleavorans,Delftia acidovorans, Bacillus subtillis, Lactobacillus delbrueckii, Lactococcus lactis, andRhodococcus equi. Such prokaryotes also can be sources of genes for constructing recombinant cells described herein that are capable of producing C7 building blocks.

Any of the recombinant microorganisms can be a eukaryote such as a eukaryote from a genus selected from the group consisting ofAspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula, andKluyveromyces. For example, the eukaryote can be selected from the group consisting ofAspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowiahpolytica, Issathenkiaorientalis, Debaryomyces hansenii, Arxulaadenoinivorans, andKluyveromyces lactis. Such eukaryotes also can be sources of genes for constructing recombinant cells described herein that are capable of producing C7 building blocks.

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.

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.

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 ofFIGS. 1 to 6.

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. (SeeFIG. 7A-E).

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. (SeeFIG. 7A-E).

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. (SeeFIG. 7A-E).

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 β-ketothiolase or a β-ketoacyl synthase classified under EC. 2.3.1, and an N-acetyl-5-amino-3-oxopentanoyl-CoA.

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 β-ketoacyl synthase and/or a β-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.

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 β-ketoacyl synthase and/or a β-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.

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 original claims 15-28 (numbered according to the as-filed U.S. application Ser. No. 15/368,092 filed Dec. 2, 2016, and published as U.S. 2018/0066296 on Mar. 8, 2018, incorporated herein by reference) 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.

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.

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.

