Methods and Materials for Producing 7-Carbon Monomers

Abstract

This document describes biochemical pathways for producing pimeloyl-CoA using a polypeptide having the enzymatic activity of a hydroperoxide lyase to form non-3-enal and 9-oxononanoate from 9-hydroxyperoxyoctadec-10,12-dienoate. Non-3-enal and 9-oxononanoate can be enzymatically converted to pimeloyl-CoA or a salt thereof using one or more polypeptides having the activity of a dehydrogenase, a CoA ligase, an isomerase, a reductase, a thioesterase, a monooxygenase, a hydratase, and/or a thiolase. Pimeloyl-CoA can be enzymatically converted to pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine, or 1,7-heptanediol, or corresponding salts thereof. This document also describes recombinant microorganisms producing pimeloyl-CoA, as well as pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine, and 1,7-heptanediol, or corresponding salts thereof.

1 . A method of producing non-3-enal and 9-oxononanoate in a recombinant microorganism, said method comprising enzymatically converting 9-hydroxyperoxyoctadec-10,12-dienoate to non-3-enal and 9-oxononanoate using an exogenous polypeptide having the activity of a hydroperoxide lyase classified under EC 4.2.99.-,
 said method optionally further comprising enzymatically converting non-3-enal to azelaic acid using one or more polypeptides comprising at least one polypeptide having the activity of a dodecenoyl-CoA isomerase classified under EC 5.3.3.8 or at least one polypeptide having the activity of an enoate reductase classified under EC 1.3.1.31.   
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1 , said method comprising enzymatically converting non-3-enal to azelaic acid using one or more polypeptides, comprising:
 at least one polypeptide having the activity of a dodecenoyl-CoA isomerase classified under EC 5.3.3.8, wherein said at least one polypeptide having the activity of a dodecenoyl-CoA isomerase classified under EC 5.3.3.8 enzymatically converts non-3-enoyl-CoA to non-2-enoyl-CoA; or   at least one polypeptide having the activity of an enoate reductase classified under EC 1.3.1.31, wherein said at least one polypeptide having the activity of an enoate reductase classified under EC 1.3.1.31 enzymatically converts non-2-enal to nonanal, and   optionally a polypeptide having the activity of a monooxygenase classified under EC 1.14.14.- or EC 1.14.15.-, wherein said polypeptide having the activity of a monooxygenase enzymatically converts nonanoic acid to 9-hydroxynonanoic acid.   
     
     
         5 - 8 . (canceled) 
     
     
         9 . The method of  claim 1 , said method further comprising producing azelaic acid in the recombinant microorganism, wherein the method comprises the steps of enzymatically converting 9-hydroxyperoxyoctadec-10,12-dienoate to non-3-enal and 9-oxononanoate using an exogenous polypeptide having the activity of a hydroperoxide lyase classified under EC 4.2.99.- and enzymatically converting non-3-enal to azelaic acid using one or more polypeptides, including at least one polypeptide having the activity of a dodecenoyl-CoA isomerase classified under EC 5.3.3.8 or at least one polypeptide having the activity of an enoate reductase classified under EC 1.3.1.31. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 9 , wherein said non-3-enal is converted to azelaic acid using one or more polypeptides having the enzymatic activities of a monooxygenase, an enal isomerase, an aldehyde dehydrogenase, a CoA ligase, a dodecenoyl-CoA isomerase, a trans-2-enoyl-CoA reductase, a thioesterase, a monooxygenase, and/or an alcohol dehydrogenase. 
     
     
         12 - 17 . (canceled) 
     
     
         18 . The method of  claim 9 , wherein said non-3-enal is converted to azelaic acid using one or more polypeptides having the enzymatic activities of a monooxygenase, an enoate reductase, an aldehyde dehydrogenase, a monooxygenase, and/or an alcohol dehydrogenase; or
 said 9-oxononanoate is converted to azelaic acid using a polypeptide having the enzymatic activity of an aldehyde dehydrogenase.   
     
     
         19 - 23 . (canceled) 
     
     
         24 . The method of  claim 9 , said method further comprising enzymatically converting azelaic acid to pimeloyl-CoA using one or more polypeptides having the enzymatic activities of a CoA ligase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, and/or a β-ketothiolase. 
     
     
         25 - 35 . (canceled) 
     
     
         36 . The method of  claim 24 , said method further comprising enzymatically converting pimeloyl-CoA to one or more of pimelate semialdehyde, pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, or 1,7-heptanediol, or corresponding salts thereof, in one or more steps. 
     
     
         37 - 45 . (canceled) 
     
     
         46 . The method of  claim 36 , wherein said pimeloyl-CoA is converted to pimelic acid using a polypeptide having the activity of a CoA ligase classified under EC 6.2.1.-., a polypeptide having the activity of a CoA transferase classified under EC 2.8.3.-, or a polypeptide having the activity of a thioesterase classified under EC 3.1.2.-. 
     
     
         47 - 49 . (canceled) 
     
     
         50 . The method of  claim 36 , wherein said pimeloyl-CoA is converted to pimelate semialdehyde using one or more polypeptides having the activity of an acetylating aldehyde dehydrogenase;
 said method further comprising optionally converting pimelate semialdehyde to 7-aminoheptanoate using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.-;   converting pimelate semialdehyde to 7-hydroxyheptanoate using one or more polypeptides having the activity of an alcohol dehydrogenase, wherein said alcohol dehydrogenase is a 4-hydroxybutanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, or a 6-hydroxyhexanoate dehydrogenase; or   converting pimelate semialdehyde to heptanedial using one or more polypeptides having the activity of a carboxylase reductase classified under EC 1.2.99.6; converting heptanedial to 7-aminoheptanal using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, EC 2.6.1.29, or EC 2.6.1.82; and converting 7-aminoheptanal to heptamethylenediamine using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.-.   
     
     
         51 - 54 . (canceled) 
     
     
         55 . The method of  claim 50 , wherein pimelate semialdehyde is converted to 7-aminoheptanoate,
 said method further comprising converting said 7-aminoheptanoate to 7-aminoheptanal using one or more polypeptides having the activity of a carboxylase reductase classified under EC 1.2.99.6, and converting said 7-aminoheptanal to heptamethylenediamine using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.-; or   converting 7-aminoheptanoate to N7-acetyl-7-aminoheptanoate using one or more polypeptides having the activity of an N-acetyltransferase classified under EC 2.3.1.32; converting N7-acetyl-7-aminoheptanoate to N7-acetyl-7-aminoheptanal using one or more polypeptides having the activity of a carboxylase reductase classified under EC 1.2.99.6; converting N7-acetyl-7-aminoheptanal is converted to N7-acetyl-1,7-diaminoheptane using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.-; and converting N7-acetyl-1,7-diaminoheptane to heptamethylenediamine using one or more polypeptides having the activity of a deacylase classified under EC 3.5.1.-.   
     
     
         56 - 59 . (canceled) 
     
     
         60 . The method of  claim 50 , wherein pimelate semialdehyde is converted to 7-hydroxyheptanoate,
 said method further comprising converting said 7-hydroxyheptanoate to 7-hydroxyheptanal using one or more polypeptides having the activity of a carboxylase reductase classified under EC 1.2.99.6; converting 7-hydroxyheptanal to 7-aminoheptanol using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.18, EC 2.6.1.19, or EC 2.6.1.48; converting 7-aminoheptanol to 7-aminoheptanal using one or more polypeptides having the activity of an alcohol dehydrogenase classified under EC 1.1.1.-; and converting 7-aminoheptanal to heptamethylenediamine using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.-; or   converting 7-hydroxyheptanoate to 7-hydroxyheptanal using a carboxylase reductase classified under EC 1.2.99.6; and converting 7-hydroxyheptanal to 1,7 heptanediol using one more polypeptides having the activity of an alcohol dehydrogenase classified under EC 1.1.1.-.   
     
     
         61 - 65 . (canceled) 
     
     
         66 . The method of claim  65 , wherein said 9-hydroxyperoxyoctadec-10,12-dienoate is enzymatically produced from octadecanoyl-CoA using one or more polypeptides having the activity of a delta9-desaturase, a delta12-desaturase, a thioesterase, and/or a 9-lipoxygenase. 
     
     
         67 - 70 . (canceled) 
     
     
         71 . The method of  claim 1 , wherein said method is performed in a recombinant microorganism. 
     
     
         72 . The method of  claim 71 , wherein said microorganism is subjected to a cultivation strategy under aerobic, anaerobic, or micro-aerobic cultivation conditions and/or said microorganism is cultured under conditions of nutrient limitation. 
     
     
         73 . (canceled) 
     
     
         74 . (canceled) 
     
     
         75 . The method of  claim 71 , wherein the principal carbon source fed to the fermentation derives from a biological feedstock. 
     
     
         76 . The method of  claim 75 , 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, plant oils, or municipal waste. 
     
     
         77 . The method of  claim 71 , wherein the principal carbon source fed to the fermentation derives from a non-biological feedstock. 
     
     
         78 . The method of  claim 77 , 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 cycloheptane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams. 
     
     
         79 . The method of  claim 71 , wherein the microorganism is a prokaryote. 
     
     
         80 . The method of  claim 79 , wherein said prokaryote is from a genus selected from  Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus , and  Rhodococcus.    
     
     
         81 . (canceled) 
     
     
         82 . The method of  claim 71 , wherein the microorganism is a eukaryote. 
     
     
         83 . The method of  claim 82 , wherein said eukaryote is from a genus selected from  Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula , and  Kluyveromyces.    
     
     
         84 - 86 . (canceled) 
     
     
         87 . The method of  claim 71 , wherein said microorganism comprises an attenuation to 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, an NADH-consuming transhydrogenase, an NADH-specific glutamate dehydrogenase, an 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. 
     
     
         88 . The method of  claim 71 , 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. 
     
     
         89 . A recombinant microorganism producing azelaic acid, said microorganism comprising one or more nucleic acids encoding a polypeptide having the enzymatic activities of any one or more of the following enzymes:
 (i) a hydroperoxide lyase, (ii) an aldehyde dehydrogenase, (iii) a CoA ligase, (iv) a dodecenoyl-CoA isomerase, (v) a trans-2-enoyl-CoA reductase, (vi) a thioesterase, (vii) a monooxygenase, and/or (viii) an alcohol dehydrogenase;   (ix) a hydroperoxide lyase, (x) an enoate reductase, (xi) an aldehyde dehydrogenase, (xii) a monooxygenase, and/or (xiii) an alcohol dehydrogenase; or   (xiv) a hydroperoxide lyase, and/or (xv) an aldehyde dehydrogenase, wherein at least one of the nucleic acids is exogenous.   
     
     
         90 . (canceled) 
     
     
         91 . (canceled) 
     
     
         92 . The recombinant microorganism of  claim 89 , said microorganism further comprising one or more exogenous nucleic acids encoding a polypeptide having one or more of the enzymatic activities of: (i) a CoA ligase, (ii) an acyl-CoA dehydrogenase, (iii) an enoyl-CoA hydratase, (iv) a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl ACP reductase, and/or (v) a β-ketothiolase, said microorganism further producing pimeloyl-CoA. 
     
     
         93 . The recombinant microorganism of  claim 92 , said microorganism further comprising one or more exogenous nucleic acids encoding a polypeptide having one or more of the enzymatic activities of a thioesterase, a CoA ligase, a CoA transferase, an acetylating aldehyde dehydrogenase, an alcohol dehydrogenase, an N-acetyltransferase, a deacylase, a ω-transaminase, a carboxylate reductase, and/or an aldehyde dehydrogenase, said microorganism further producing one or more of pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, and 1,7-heptanediol. 
     
     
         94 - 100 . (canceled) 
     
     
         101 . The recombinant microorganism of  claim 89 , said microorganism further comprising one or more nucleic acids encoding a polypeptide having one or more of the enzymatic activities of a delta9-desaturase, a delta12-desaturase, a thioesterase, or a 9-lipoxygenase. 
     
     
         102 . (canceled) 
     
     
         103 . A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having carboxylate reductase activity or a polynucleotide encoding a polypeptide having ω-transaminase activity, wherein the polynucleotide encoding a polypeptide having carboxylate reductase activity 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: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 1; (b) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 2; (c) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 3; (d) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 4, (e) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 5 and (f) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 6; and the polynucleotide encoding a polypeptide having ω-transaminase activity 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: (g) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 7; (h) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 8; (i) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 9; (i) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 10; (k) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 11 or SEQ ID NO: 48; and (l) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 12. 
     
     
         104 . (canceled) 
     
     
         105 . A composition comprising the nucleic acid construct or expression vector of  claim 103 . 
     
     
         106 . A culture medium comprising the nucleic acid construct or expression vector of  claim 103 . 
     
     
         107 - 110 . (canceled) 
     
     
         111 . Means for producing pimeloyl-CoA, comprising culturing a non-naturally occurring microorganism comprising at least one exogenous nucleic acid encoding a polypeptide having the enzymatic activity of (i) a hydroperoxide lyase, (ii) an aldehyde dehydrogenase, (iii) a CoA ligase, (iv) a dodecenoyl-CoA isomerase, (iv) a trans-2-enoyl-CoA reductase, (v) a thioesterase, (vi) an enoate reductase, (vii) a monooxygenase, (viii) an alcohol dehydrogenase, (ix) an acyl-CoA dehydrogenase, (x) an enoyl-CoA hydratase, (xi) a 3-hydroxyacyl-CoA dehydrogenase and (xii) a β-ketothiolase, expressed in a sufficient amount in said microorganism to produce pimeloyl-CoA. 
     
     
         112 . 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 produced by a method according to  claim 1  or any combination thereof,   (ii) a bio-derived, bio-based, or fermentation-derived polymer comprising the bio-derived, bio-based or fermentation-derived composition or compound of (i), or any combination thereof,   (iii) a bio-derived, bio-based, or fermentation-derived resin comprising the bio-derived, bio-based, or fermentation-derived compound or bio-derived, bio-based, or fermentation-derived composition of (i) or any combination thereof or the bio-derived, bio-based, or fermentation-derived polymer of (ii) or any combination thereof,   (iv) a molded substance obtained by molding the bio-derived, bio-based, or fermentation-derived polymer of (ii) or the bio-derived, bio-based, or fermentation-derived resin of (iii), or any combination thereof,   (v) a bio-derived, bio-based, or fermentation-derived formulation comprising the bio-derived, bio-based, or fermentation-derived composition of (i), bio-derived, bio-based, or fermentation-derived compound of (i), bio-derived, bio-based, or fermentation-derived polymer of (ii), bio-derived, bio-based, or fermentation-derived resin of (iii), or bio-derived, bio-based, or fermentation-derived molded substance of (v), 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.