Methods of producing 7-carbon chemicals via carbon chain elongation associated with cyclohexane carboxylate synthesis

Abstract

This document describes biochemical pathways for producing pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine or 1,7-heptanediol by forming two terminal functional groups, comprised of carboxyl, amine or hydroxyl group, in a C7 aliphatic backbone substrate. These pathways, metabolic engineering and cultivation strategies described herein rely on the carbon chain elongation enzymes or homologs thereof associated with the cyclohexane carboxylate biosynthesis from Syntrophus aciditrophicus or 2-aminoadipate lysine biosynthesis.

What is claimed is: 
     
         1 . A method for biosynthesizing a product selected from the group consisting of pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine, and 1,7-heptanediol, said method comprising enzymatically synthesizing a seven carbon chain aliphatic backbone using enzymes associated with cyclohexane carboxylate biosynthesis or the 2-aminoadipate lysine biosynthesis pathway, and enzymatically forming one or two terminal functional groups selected from the group consisting of carboxyl, amine, and hydroxyl groups in said backbone, thereby forming the product. 
     
     
         2 . The method of  claim 1 , wherein the seven carbon chain aliphatic backbone is pimeloyl-CoA. 
     
     
         3 . The method of  claim 1  or  2 , wherein pimeloyl-CoA is enzymatically synthesized from acetyl-CoA or 2-oxoglutarate via 3-ketopimeloyl-CoA. 
     
     
         4 . The method of  claim 3 , wherein a β-ketothiolase or a β-ketoacyl [acp] synthase converts glutaryl-CoA to 3-ketopimeloyl-CoA. 
     
     
         5 . The method of  claim 4 , wherein glutaryl-CoA is produced from crotonyl-CoA using a glutaconyl-CoA decarboxylase or a glutaryl-CoA dehydrogenase. 
     
     
         6 . The method of any one of  claims 3 - 5 , wherein 3-ketopimeloyl-CoA is converted to 3-hydroxypimeloyl-CoA using a 3-hydroxyadipyl-CoA dehydrogenase. 
     
     
         7 . The method of  claim 6 , wherein 3-hydroxypimeloyl-CoA is converted to 6-ketocyclohex-1-ene-1-carboxyl-CoA by a 6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase. 
     
     
         8 . The method of  claim 7 , wherein 3-hydroxypimeloyl-CoA is converted to 2,3-dehydropimeloyl-CoA by an enoyl-CoA hydratase. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein said two terminal functional groups are the same. 
     
     
         10 . The method of any one of  claims 1 - 8 , wherein said two terminal functional groups are different. 
     
     
         11 . The method of  claim 10 , wherein said product comprises a terminal amine and a terminal carboxyl group. 
     
     
         12 . The method of  claim 10 , wherein said product comprises a terminal hydroxyl group and a terminal carboxyl group. 
     
     
         13 . The method of any one of  claims 1 - 9 , wherein said two terminal functional groups are amine. 
     
     
         14 . The method of any one of  claims 1 - 9 , wherein said two terminal functional groups are hydroxyl groups. 
     
     
         15 . The method of  claim 12  or  claim 14 , wherein a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydratase, or an alcohol dehydrogenase enzymatically forms the two hydroxyl groups. 
     
     
         16 . The method of any one of  claims 1 - 14 , wherein a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a CoA-transferase, or a reversible CoA-ligase enzymatically forms a terminal carboxyl group. 
     
     
         17 . The method of  claim 16 , wherein said thioesterase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. 
     
     
         18 . The method of  claim 11  or  claim 13 , wherein a ω-transaminase or a deacetylase enzymatically forms the amine group. 
     
     
         19 . The method of  claim 18 , wherein said ω-transaminase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO. 8-13. 
     
     
         20 . The method of any one of  claims 1 - 14 , wherein a carboxylate reductase in combination with a phosphopantetheine transferase enhancer form a terminal aldehyde group as an intermediate in forming the product. 
     
     
         21 . The method of  claim 20 , wherein said carboxylate reductase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO. 2-7. 
     
     
         22 . The method of any of the preceding claims, wherein said method is performed in a recombinant host by fermentation. 
     
     
         23 . The method of  claim 22 , wherein said host is subjected to a cultivation strategy under anaerobic, micro-aerobic or mixed oxygen/denitrification cultivation conditions. 
     
     
         24 . The method of  claim 22  or  claim 23 , wherein said host is cultured under conditions of nutrient limitation. 
     
     
         25 . The method according to any one of  claims 22 - 24 , wherein said host is retained using a ceramic hollow fiber membrane to maintain a high cell density during fermentation. 
     
     
         26 . The method of any one of  claims 22 - 25 , wherein the final electron acceptor is nitrate. 
     
     
         27 . The method of any one of  claims 22 - 26 , wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks. 
     
     
         28 . The method of  claim 27 , wherein the biological feedstock is, or derives from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers'solubles, or municipal waste. 
     
     
         29 . The method of  claim 27 , wherein the non-biological feedstock is, or derives from, natural gas, syngas, CO 2 /H 2 , methanol, ethanol, benzoate, non-volatile residue (NVR) caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams. 
     
     
         30 . The method of any one of  claims 22 - 29 , wherein the host is a prokaryote. 
     
     
         31 . The method of  claim 30 , wherein said prokaryote is from the genus  Escherichia  such as  Escherichia coli ; from the genus  Clostridia  such as  Clostridium ljungdahlii, Clostridium autoethanogenum  or  Clostridium kluyveri ; from the genus  Corynebacteria  such as  Corynebacterium glutamicum ; from the genus  Cupriavidus  such as  Cupriavidus necator  or  Cupriavidus metallidurans ; from the genus  Pseudomonas  such as  Pseudomonas fluorescens, Pseudomonas putida  or  Pseudomonas oleavorans ; from the genus  Delftia acidovorans , from the genus  Bacillus  such as  Bacillus subtillis ; from the genes  Lactobacillus  such as  Lactobacillus delbrueckii ; from the genus  Lactococcus  such as  Lactococcus lactis  or from the genus  Rhodococcus  such as  Rhodococcus equi.    
     
     
         32 . The method of any one of  claims 22 - 29 , wherein the host is a eukaryote. 
     
     
         33 . The method of  claim 32 , wherein said eukaryote is from the genus  Aspergillus  such as  Aspergillus niger ; from the genus  Saccharomyces  such as  Saccharomyces cerevisiae ; from the genus  Pichia  such as  Pichia pastoris ; from the genus  Yarrowia  such as  Yarrowia lipolytica , from the genus  Issatchenkia  such as  Issathenkia orientalis , from the genus  Debaryomyce s such as  Debaryomyces hansenii , from the genus  Arxula  such as  Arxula adenoinivorans , or from the genus  Kluyveromyces  such as  Kluyveromyces lactis .    
     
     
         34 . The method of any one of  claims 22 - 33 , wherein the host's tolerance to high concentrations of a C7 building block is improved through continuous cultivation in a selective environment. 
     
     
         35 . The method of any one of  claims 22 - 34 , wherein said host comprises one or more of the following attenuated enzymes: a phosphotransacetylase generating acetate, an acetate kinase; a lactate dehydrogenase; a menaquinol-fumarate oxidoreductase; an alcohol dehydrogenase producing ethanol; a pyruvate decarboxylase; a 2-oxoacid decarboxylase generating isobutanol; a polymer synthase; a NADPH-specific L-glutamate dehydrogenase; a NADPH/NADH L-glutamate dehydrogenase; a NADH-consuming transhydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase that degrades C7 building blocks and their precursors; a glutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase. 
     
     
         36 . The method of any one of  claims 22 - 35 , wherein said host overexpresses one or more genes encoding: an acetyl-CoA synthetase, a formate dehydrogenase, a PEP carboxykinase, a PEP carboxylase, a pyruvate carboxylase, a PEP synthase, a L-alanine dehydrogenase; a NADH-specific L-glutamate dehydrogenase; a diamine transporter; a dicarboxylate transporter; and/or a multidrug transporter. 
     
     
         37 . A recombinant host comprising at least one exogenous nucleic acid encoding i) a β-ketothiolase or a) β-ketoacyl [acp] synthase, (ii) a 6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase or an enoyl-CoA hydratase, and (iii) a trans-2-enoyl-CoA reductase, an enoyl-[acp] reductase, or a 2-ketocyclohexanecarboxyl-CoA hydrolase, said host producing pimeloyl-CoA. 
     
     
         38 . The recombinant host of  claim 37 , further comprising a glutaconyl-CoA decarboxylase or a glutaryl-CoA dehydrogenase. 
     
     
         39 . The recombinant host of  claim 37  or  claim 38 , said host further comprising one or more of an aldehyde dehydrogenase, a 6-oxohexanoate dehydrogenase, a thioesterase, a 7-oxoheptanoate dehydrogenase, a reversible succinyl-CoA-ligase, a glutaconate CoA transferase, a carboxylate reductase, or an acetylating aldehyde dehydrogenase said host producing pimelate or pimelate semialdehyde. 
     
     
         40 . The recombinant host of  claim 39 , said host further comprising a ω-transaminase, said host producing 7-aminoheptanoate. 
     
     
         41 . The recombinant host of  claim 39 , said host further comprising one or more of a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, or a 4-hydroxybutyrate dehydrogenase, said host producing 7-hydroxyheptanoic acid. 
     
     
         42 . The recombinant host of any one of  claims 39 - 41 , said host further comprising one or more of a deacetylase, an N-acetyl transferase, or an alcohol dehydrogenase, said host producing heptamethylenediamine. 
     
     
         43 . The recombinant host of  claim 41 , said host further comprising a carboxylate reductase or an alcohol dehydrogenase, said host producing 1,7-heptanediol.