Methods and materials for producing 7-carbon monomers

Abstract

This document describes biochemical pathways for producing 7-aminoheptanoic acid using a β-ketoacyl synthase or a β-ketothiolase to form either a 5-amino-3-oxopentanoyl-[ACP] or 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 the 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 the corresponding salts thereof.

1 . A method of producing 5-amino-3-oxopentanoyl-[ACP] or 5-amino-3-oxopentanoyl-CoA or corresponding salts thereof, said method comprising enzymatically converting β-alanyl-[ACP] or β-alanyl-CoA respectively to 5-amino-3-oxopentanoyl-[ACP] or 5-amino-3-oxopentanoyl-CoA or corresponding salts thereof, using a polypeptide having the activity of a β-ketoacyl synthase or a β-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 β-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 β-ketothiolase is classified under EC 2.3.1.16 or EC 2.3.1.174. 
     
     
         3 . The method of  claim 1 , wherein said polypeptide having the activity of a β-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 β-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 β-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 β-alanyl-CoA to 5-amino-3-oxopentanoyl-CoA and said polypeptide having the activity of a β-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 β-alanyl-CoA and β-alanyl-[ACP] to 5-amino-3-oxopentanoyl-CoA and 5-amino-3-oxopentanoyl-[ACP] respectively 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 β-alanyl-CoA to 5-amino-3-oxopentanoyl-CoA. 
     
     
         5 . The method of  claim 1 , further comprising enzymatically converting 5-amino-3-oxopentanoyl-[ACP] or 5-amino-3-oxopentanoyl-CoA or the corresponding salts thereof to 7-aminoheptanoate or the salt thereof using polypeptides having the enzymatic activities of a 3-oxoacyl-[ACP] reductase or a 3-hydroxyacyl-CoA dehydrogenase, a 3-hydroxyacyl-[ACP] dehydratase or an enoyl-CoA hydratase, an enoyl-[ACP] reductase or a trans-2-enoyl-CoA reductase, a β-ketoacyl synthase or a β-ketothiolase and a thioesterase or a CoA transferase. 
     
     
         6 - 14 . (canceled) 
     
     
         15 . A method for biosynthesizing 7-aminoheptanoate, said method comprising enzymatically synthesizing 5-amino-3-oxopentanoyl-[ACP] or 5-amino-3-oxopentanoyl-CoA or the salts thereof from β-alanyl-[ACP] or β-alanyl-CoA using a polypeptide having the activity of a β-ketoacyl synthase or a β-ketothiolase classified under EC. 2.3.1.- and/or a CoA transferase classified under EC 2.8.3-, and enzymatically converting 5-amino-3-oxopentanoyl-[ACP] or 5-amino-3-oxopentanoyl-CoA or the salts thereof to 7-aminoheptanoate. 
     
     
         16 . The method of  claim 15 , wherein 5-amino-3-oxopentanoyl-[ACP] is converted to 5-amino-3-hydroxypentanoyl-[ACP] using a polypeptide having the activity of 3-oxoacyl-[ACP] reductase and 5-amino-3-oxopentanoyl-CoA is converted to 5-amino-3-hydroxypentanoyl-CoA using a polypeptide having the activity of a 3-hydroxyacyl-CoA dehydrogenase; 5-amino-3-hydroxypentanoyl-[ACP] is converted to 5-amino-pent-2-enoyl-[ACP] using a polypeptide having the activity of a 3-hydroxyacyl-[ACP] dehydratase and 5-amino-3-hydroxypentanoyl-CoA is converted to 5-amino-pent-2-enoyl-CoA using a polypeptide having the activity of an enoyl-CoA hydratase; 5-amino-pent-2-enoyl-[ACP] is converted to 5-amino-pentanoyl-[ACP] using a polypeptide having the activity of an enoyl-[ACP] reductase and 5-amino-pent-2-enoyl-CoA is converted to 5-amino-pentanoyl-CoA using a polypeptide having the activity of a trans-2-enoyl-CoA-reductase; 5-amino-pentanoyl[ACP] is converted to 7-amino-3-oxoheptanoyl-[ACP] using a polypeptide having the activity of a β-ketoacyl synthase and 5-amino-pentanoyl-CoA is converted to 7-amino-3-oxoheptanoyl-CoA using a polypeptide having the activity of a β-ketothiolase; 7-amino-3-oxoheptanoyl-[ACP] is converted to 7-amino-3-hydroxyheptanoyl-[ACP] using a polypeptide having the activity of a 3-oxoacyl-[ACP]reductase and 7-amino-3-oxoheptanoyl-CoA is converted to 7-amino-3-hydroxyheptanoyl-CoA using a polypeptide having the activity of a 3-hydroxyacyl-CoA-dehydrogenase; 7-amino-3-hydroxyheptanoyl-[ACP] is converted to 7-amino-hept-2-enoyl-[ACP] using a polypeptide having the activity of a 3-hydroxyacyl-[ACP] dehydratase and 7-amino-3-hydroxyheptanoyl-CoA is converted to 7-amino-hept-2-enoyl-CoA using a polypeptide having the activity of an enoyl-CoA hydratase; 7-amino-hept-2-enoyl-[ACP] is converted to 7-aminoheptanoyl-[ACP] using a polypeptide having the activity of an enoyl-[ACP] reductase and 7-amino-hept-2-enoyl-CoA is converted to 7-aminoheptanoyl-CoA using a polypeptide having the activity of a trans-2-enoyl-CoA reductase; and 7-aminoheptanoyl-[ACP] is converted to 7-aminoheptanoate using a polypeptide having the activity of a thioesterase and 7-aminoheptanoyl-CoA is converted to 7-aminoheptanoate using a polypeptide having the activity of a thioesterase or a CoA transferase. 
     
     
         17 . The method of  claim 5 , said method further comprising enzymatically converting 7-aminoheptanoate to pimelic acid, 7-hydroxyheptanoate, heptamethylenediamine or 1,7-heptanediol or corresponding salts thereof in one or more steps. 
     
     
         18 . The method of  claim 17 , wherein 7-aminoheptanoate is converted to pimelic acid using one or more polypeptides having the enzymatic activity of a ω-transaminase a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 5-oxopentanoate dehydrogenase, or an aldehyde dehydrogenase. 
     
     
         19 . The method of  claim 17 , 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 ω-transaminase. 
     
     
         20 . The method of  claim 17 , wherein 7-aminoheptanoate is converted to heptamethylenediamine using polypeptides having the enzymatic activity of a carboxylate reductase and one or more ω-transaminases. 
     
     
         21 . The method of  claim 17 , wherein 7-aminoheptanoate is converted to heptamethylenediamine using polypeptides having the enzymatic activity of a carboxylate reductase, a ω-transaminase and an alcohol dehydrogenase. 
     
     
         22 . The method of  claim 17 , wherein 7-aminoheptanoate is converted to heptamethylenediamine using polypeptides having the enzymatic activity of an N-acetyltransferase, a carboxylate reductase, a ω-transaminase, and a deacylase. 
     
     
         23 . The method of  claim 17 , wherein 7-aminoheptanoate is converted to heptamethylenediamine using polypeptides having the enzymatic activity of one or more alcohol dehydrogenases and one or more ω-transaminases. 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 17 , wherein 7-hydroxyheptanoate is converted to 1,7-heptanediol using a polypeptide having the enzymatic activity of a carboxylate reductase and a polypeptide having the enzymatic activity of an alcohol dehydrogenase. 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 1 , wherein said β-alanyl-[ACP] or said β-alanyl-CoA is enzymatically produced from malonyl-CoA or L-aspartate. 
     
     
         28 . The method of  claim 27 , wherein β-alanyl-[ACP] or said β-alanyl-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 β-alanine-pyruvate aminotransferase, an α-aspartate decarboxylase, a CoA transferase and an [ACP]-acetyltransferase. 
     
     
         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 β-ketoacyl synthase and/or a β-ketothiolase, (ii) a thioesterase or a CoA transferase, and one or more of (iii) a 3-oxoacyl-ACP reductase or 3-hydroxyacyl-CoA dehydrogenase, (iv) a 3-hydroxyacyl-[ACP]-dehydratase or an enoyl-CoA hydratase, and (v) an enoyl-[ACP]-reductase or a trans-2-enoyl-CoA reductase, said microorganism producing 7-aminoheptanoate or a corresponding salt thereof. 
     
     
         48 . The recombinant microorganism of  claim 47 , said microorganism further comprising one or more of the following exogenous polypeptides having the enzymatic activity of: ω-transaminase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 5-oxopentanoate 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 polypeptides having the enzymatic activity of: a ω-transaminase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutanoate dehydrogenase, and an alcohol dehydrogenase, said microorganism further producing 7-hydroxyheptanoate. 
     
     
         50 . The recombinant microorganism of  claim 47 , said microorganism further comprising one or more of the following exogenous polypeptides having the enzymatic activity of: a carboxylate reductase, a ω-transaminase, a deacylase, an N-acetyl transferase, or an alcohol dehydrogenase, said microorganism further producing heptamethylenediamine. 
     
     
         51 - 54 . (canceled) 
     
     
         55 . The recombinant microorganism of  claim 47 , said microorganism further comprising an exogenous polypeptide having the enzymatic activity of a carboxylate reductase and an exogenous alcohol dehydrogenase, said microorganism further producing 1,7-heptanediol. 
     
     
         56 . The recombinant microorganism of  claim 47 , said microorganism further comprising one or more of the following exogenous polypeptides having the enzymatic activity of: an aspartate-α-decarboxylase; a malonyl-CoA reductase; a β-alanine-pyruvate aminotransferase; a CoA-transferase; or an [ACP]-acetyl transferase. 
     
     
         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 7 . 
     
     
         58 . A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having β-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 β-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 β-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 β-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 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 ω-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 ω-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 β-alanyl-[ACP] or β-alanyl-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 a 5-amino-3-oxopentanoyl-[ACP] or 5-amino-3-oxopentanoyl-CoA respectively. 
     
     
         65 . 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, and one or more of (iii) a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-[ACP] reductase, (iv) an enoyl-CoA hydratase or 3-hydroxyacyl-[ACP] dehydratase, and (v) a trans-2-enoyl-CoA reductase or enoyl-[ACP] reductase, said microorganism producing 7-aminoheptanoate or the corresponding salt thereof. 
     
     
         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 5-amino-3-oxopentanoyl-CoA or a salt thereof, said method comprising enzymatically converting β-alanine to 5-amino-3-oxopentanoyl-CoA or a salt thereof using a polypeptide having the activity of a β-ketoacyl synthase or a β-ketothiolase classified under EC. 2.3.1.-, further comprising enzymatically converting β-alanine or the salt thereof to 5-amino-3-oxopentanoyl-CoA using polypeptides having the enzymatic activities of a β-ketothiolase, CoA transferase, acetyl-CoA hydrolase, succinyl-CoA: acetate CoA-transferase, 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.