Methods, reagents and cells for biosynthesizing compounds

Abstract

This document describes biochemical pathways for producing 7-hydroxyheptanoate methyl ester and heptanoic acid heptyl ester using one or more of a fatty acid O-methyltransferase, an alcohol O-acetyltransferase, and a monooxygenase, as well as recombinant hosts expressing one or more of such exogenous enzymes. 7-hydroxyheptanoate methyl esters and heptanoic acid heptyl esters can be enzymatically converted to pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, or 1,7-heptanediol.

1 - 83 . (canceled) 
     
     
         84 . A method of producing 7-hydroxyheptanoate methyl ester in a recombinant host via fermentation, said method comprising:
 enzymatically converting heptanoate to heptanoate methyl ester using a polypeptide having fatty acid O-methyltransferase activity; and   enzymatically converting heptanoate methyl ester to 7-hydroxyheptanoate methyl ester using a polypeptide having monooxygenase activity.   
     
     
         85 . The method of  claim 84 , wherein:
 said polypeptide having fatty acid O-methyltransferase activity has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25;   said polypeptide having monooxygenase activity has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 28, and/or SEQ ID NO: 29.   
     
     
         86 . The method of  claim 84 , wherein heptanoate is enzymatically produced from heptanoyl-CoA using:
 a polypeptide having thioesterase activity; or   a polypeptide having butanal dehydrogenase activity and a polypeptide having aldehyde dehydrogenase activity.   
     
     
         87 . The method of  claim 84 , wherein heptanoate is enzymatically produced from heptanoyl-CoA using a polypeptide having thioesterase activity, wherein said polypeptide having thioesterase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 33, or SEQ ID NO: 34. 
     
     
         88 . The method of  claim 86 , wherein heptanoyl-CoA is produced from acetyl-CoA and propanoyl-CoA via two cycles of CoA-dependent carbon chain elongation, wherein each of said two cycles of CoA-dependent carbon chain elongation comprises using:
 a polypeptide having β-ketothiolase activity or a polypeptide having acetyl-CoA carboxylase activity and a polypeptide having β-ketoacyl-[acp] synthase activity;   a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having 3-oxoacyl-CoA reductase activity;   a polypeptide having enoyl-CoA hydratase activity; and   a polypeptide having trans-2-enoyl-CoA reductase activity.   
     
     
         89 . The method of  claim 84 , said method further comprising enzymatically converting 7-hydroxyheptanoate methyl ester to 7-hydroxyheptanoate using a polypeptide having demethylase activity or a polypeptide having esterase activity. 
     
     
         90 . The method of  claim 89 , said method further comprising enzymatically converting 7-hydroxyheptanoate to at least one product chosen from pimelic acid, pimelate semialdehyde, 7-aminoheptanoate, heptamethylenediamine, and 1,7-heptanediol. 
     
     
         91 . The method of  claim 89 , said method further comprising enzymatically converting 7-hydroxyheptanoate to pimelate semialdehyde using a polypeptide having alcohol dehydrogenase activity, a polypeptide having 6-hydroxyhexanoate dehydrogenase activity, a polypeptide having 5-hydroxypentanoate dehydrogenase activity, a polypeptide having 4-hydroxybutyrate dehydrogenase activity, or a polypeptide having monooxygenase activity. 
     
     
         92 . The method of  claim 89 , said method further comprising:
 enzymatically converting 7-hydroxyheptanoate to 1,7-heptanediol using a polypeptide having carboxylate reductase activity and a polypeptide having alcohol dehydrogenase activity, wherein said polypeptide having carboxylate reductase is optionally enhanced by gene product of sfp; and   optionally further comprising enzymatically converting 1,7-heptanediol to heptamethylenediamine using a polypeptide having alcohol dehydrogenase activity and a polypeptide having ω-transaminase activity.   
     
     
         93 . The method of  claim 91 , said method further comprising:
 enzymatically converting pimelate semialdehyde to pimelic acid using a polypeptide having 5-oxopentanoate dehydrogenase activity, a polypeptide having 6-oxohexanoate dehydrogenase activity, a polypeptide having 7-oxoheptanoate dehydrogenase activity, a polypeptide having aldehyde dehydrogenase activity, or a polypeptide having monooxygenase activity;   enzymatically converting pimelate semialdehyde to 7-aminoheptanoate using a polypeptide having ω-transaminase activity; or   enzymatically converting pimelate semialdehyde to heptamethylenediamine using at least one polypeptide chosen from a polypeptide having carboxylate reductase activity and a polypeptide having ω-transaminase activity, wherein said polypeptide having carboxylate reductase is optionally enhanced by gene product of sfp.   
     
     
         94 . The method of  claim 93 , said method comprising enzymatically converting pimelate semialdehyde to 7-aminoheptanoate using a polypeptide having ω-transaminase activity, and further comprising enzymatically converting 7-aminoheptanoate to heptamethylenediamine using:
 at least one polypeptide chosen from a polypeptide having carboxylate reductase activity and a polypeptide having ω-transaminase activity, wherein the polypeptide having carboxylate reductase is optionally enhanced by gene product of sfp; and 
 optionally a polypeptide having deacetylase activity or a polypeptide having acetylputrescine deacetylase activity. 
 
     
     
         95 . The method of  claim 84 , wherein said recombinant host is:
 subjected to a cultivation strategy under aerobic, anaerobic or, micro-aerobic cultivation conditions;   cultured under conditions of nutrient limitation; and/or   retained using a ceramic membrane to maintain a high cell density during fermentation.   
     
     
         96 . The method of  claim 84 , wherein the principal carbon source fed to the recombinant host is a biological feedstock or non-biological feedstock. 
     
     
         97 . The method of  claim 84 , wherein:
 said 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; or   said 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.   
     
     
         98 . The method of  claim 84 , wherein said recombinant host is:
 a prokaryote chosen from the genera  Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus , and  Rhodococcus ; or   a eukaryote chosen from the genera  Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula , and  Kluyveromyces.      
     
     
         99 . The method of  claim 84 , wherein said recombinant host is:
 a prokaryote chosen from  Escherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans, Bacillus subtilis, Lactobacillus delbrueckii, Lactococcus lactis , and  Rhodococcus equi ; or   a eukaryote chosen from  Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans , and  Kluyveromyces lactis.      
     
     
         100 . The method of  claim 84 , wherein said recombinant host:
 comprises at least one attenuated enzyme chosen from:
 a polypeptide having polyhydroxyalkanoate synthase activity; 
 a polypeptide having acetyl-CoA thioesterase activity; 
 a polypeptide having propanoyl-CoA thioesterase activity; 
 a polypeptide having phosphotransacetylase forming acetate activity; 
 a polypeptide having acetate kinase activity; 
 a polypeptide having lactate dehydrogenase activity; 
 a polypeptide having menaquinol-fumarate oxidoreductase activity; 
 a polypeptide having 2-oxoacid decarboxylase activity producing isobutanol; 
 a polypeptide having alcohol dehydrogenase activity forming ethanol; 
 a polypeptide having triose phosphate isomerase activity; 
 a polypeptide having pyruvate decarboxylase activity; 
 a polypeptide having glucose-6-phosphate isomerase activity; 
 a polypeptide having transhydrogenase activity dissipating the cofactor gradient; 
 a polypeptide having glutamate dehydrogenase activity specific for the cofactor used to create a gradient; 
 a NADH/NADPH-utilizing polypeptide having glutamate dehydrogenase activity; 
 a polypeptide having pimeloyl-CoA dehydrogenase activity; 
 a polypeptide having acyl-CoA dehydrogenase activity accepting C7 building blocks and central precursors as substrates; 
 a polypeptide having glutaryl-CoA dehydrogenase activity; and 
 a polypeptide having pimeloyl-CoA synthetase activity; and/or 
 overexpresses at least one gene encoding a polypeptide chosen from: 
 a polypeptide having acetyl-CoA synthetase activity; 
 a polypeptide having propionate-CoA synthetase activity; 
 a polypeptide having 6-phosphogluconate dehydrogenase activity; 
 a polypeptide having transketolase activity; 
 a polypeptide having puridine nucleotide transhydrogenase activity; 
 a polypeptide having formate dehydrogenase activity; 
 a polypeptide having glyceraldehyde-3P-dehydrogenase activity; 
 a polypeptide having malic enzyme activity; 
 a polypeptide having glucose-6-phosphate dehydrogenase activity; 
 a polypeptide having fructose 1,6 diphosphatase activity; 
 a polypeptide having L-alanine dehydrogenase activity; 
 a polypeptide having L-glutamate dehydrogenase activity; 
 a polypeptide having L-glutamine synthetase activity; 
 a polypeptide having lysine transporter activity; 
 a polypeptide having dicarboxylate transporter activity; and 
 a polypeptide having multidrug transporter activity. 
   
     
     
         101 . A recombinant host comprising at least one exogenous nucleic acid encoding a polypeptide having monooxygenase activity and at least one nucleic acid encoding a polypeptide having fatty acid O-methyltransferase activity, wherein:
 said polypeptide having monooxygenase activity has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 28, or SEQ ID NO: 29; and   said polypeptide having fatty acid O-methyltransferase activity has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25,   and said recombinant host produces 7-hydroxyheptanoate methyl ester.   
     
     
         102 . The recombinant host of  claim 101 , said recombinant host further comprising an exogenous polypeptide having demethylase activity classified under EC 2.1.1.- or a polypeptide having esterase activity classified under EC 3.1.1.-,
 and said host further produces 7-hydroxyheptanoate.   
     
     
         103 . The recombinant host of  claim 102 , said recombinant host further comprising at least one exogenous polypeptide chosen from:
 a polypeptide having 4-hydroxybutyrate dehydrogenase activity;   a polypeptide having 5-hydroxypentanoate dehydrogenase activity;   a polypeptide having 5-oxopentanoate dehydrogenase activity;   a polypeptide having 6-hydroxyhexanoate dehydrogenase activity;   a polypeptide having 6-oxohexanoate dehydrogenase activity;   a polypeptide having 7-oxoheptanoate dehydrogenase activity;   a polypeptide having acetylputrescine deacetylase activity;   a polypeptide having alcohol dehydrogenase activity;   a polypeptide having aldehyde dehydrogenase activity;   a polypeptide having carboxylate reductase activity;   a polypeptide having deacetylase activity; and   a polypeptide having ω-transaminase activity.   
     
     
         104 . The recombinant host of  claim 102 , said recombinant host further comprising:
 an exogenous polypeptide having β-ketothiolase activity or an exogenous polypeptide having acetyl-CoA carboxylase activity and an exogenous polypeptide having β-ketoacyl-[acp] synthase activity;   an exogenous polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity or an exogenous polypeptide having 3-oxoacyl-CoA reductase activity;   an exogenous polypeptide having enoyl-CoA hydratase activity; and   an exogenous polypeptide having trans-2-enoyl-CoA reductase activity.   
     
     
         105 . The recombinant host of  claim 102 , said recombinant host further comprising:
 an exogenous polypeptide having thioesterase activity; or   an exogenous polypeptide having butanal dehydrogenase activity and an exogenous polypeptide having aldehyde dehydrogenase activity.   
     
     
         106 . A method of producing a bio-based polymer or resin comprising at least one bio-derived seven-carbon compound chosen from pimelic acid, pimelate semialdehyde, 7-aminoheptanoate acid, 7-hydroxyheptanoate, heptamethylenediamine, and 1,7-heptanediol, said method comprising:
 producing said at least one bio-derived seven-carbon compound using the method of  claim 90 ; and   chemically reacting said at least one bio-derived seven-carbon compound with itself or another compound in a polymer-producing reaction or resin-producing reaction.   
     
     
         107 . A method of producing a molded product comprising producing a polymer or resin using the method of  claim 106  and forming said polymer or resin into a molded product. 
     
     
         108 . A bio-derived product, bio-based product, or fermentation-derived product, wherein said product comprises:
 (i) at least one bio-derived, bio-based, or fermentation-derived compound produced using the method of  claim 90  or a composition comprising said at least one bio-derived, bio-based, or fermentation-derived compound;   (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 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 obtainable by molding the bio-derived, bio-based, or fermentation-derived polymer of (ii) or the bio-derived, bio-based, or fermentation-derived 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 or compound of (i), the bio-derived, bio-based, or fermentation-derived polymer of (ii), the bio-derived, bio-based, or fermentation-derived resin of (iii), the 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 compound or composition of (i), the bio-derived, bio-based, or fermentation-derived polymer of (ii), the bio-derived, bio-based, or fermentation-derived resin of (iii), the bio-derived, bio-based, or fermentation-derived formulation of (v), the bio-derived, bio-based, or fermentation-derived molded substance of (iv), or any combination thereof.