Methods, reagents and cells for biosynthesizing compounds

Abstract

This document describes biochemical pathways for producing 2,4-pentadienoyl-CoA by forming one or two terminal functional groups, comprised of carboxyl or hydroxyl group, in a C5 backbone substrate such as glutaryl-CoA, glutaryl-[acp] or glutarate methyl ester. 2,4-pentadienoyl-CoA can be enzymatically converted to 1,3-butadiene.

1 - 22 . (canceled) 
     
     
         23 . A method of making glutarate, said method comprising (i) enzymatically converting glutaryl-[acp] methyl ester to glutaryl-[acp] or glutaryl-CoA methyl ester to glutaryl-CoA using a polypeptide having pimeloyl-[acp] methyl ester methylesterase activity, and (ii) enzymatically converting glutaryl-[acp] or glutaryl-CoA to glutarate using at least one polypeptide having thioesterase activity, reversible CoA-ligase activity, a CoA-transferase activity, an acylating dehydrogenase activity, an aldehyde dehydrogenase activity, a glutarate semialdehyde dehydrogenase activity, or a succinate-semialdehyde dehydrogenase activity. 
     
     
         24 . The method of  claim 23 , wherein said polypeptide having pimeloyl-[acp] methyl ester methylesterase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. 
     
     
         25 . The method of  claim 23 , wherein glutaryl-[acp] or glutaryl-CoA is enzymatically converted to glutaric acid using a polypeptide having thioesterase activity. 
     
     
         26 . The method of  claim 23 , wherein glutaryl-[acp] or glutaryl-CoA is enzymatically converted to glutaric acid using a polypeptide having reversible CoA-ligase activity or a CoA-transferase activity. 
     
     
         27 . The method of  claim 23 , wherein glutaryl-[acp] or glutaryl-CoA is enzymatically converted to glutaric acid using a polypeptide having an acylating dehydrogenase activity, an aldehyde dehydrogenase activity, a glutarate semialdehyde dehydrogenase activity, or a succinate-semialdehyde dehydrogenase activity. 
     
     
         28 . The method of  claim 23 , wherein the method is performed in a host subjected to a cultivation strategy under aerobic or micro-aerobic cultivation conditions, a host cultured under conditions of nutrient limitation either via nitrogen, phosphate or oxygen limitation, and/or a host retained using a ceramic membrane to maintain a high cell density during fermentation. 
     
     
         29 - 30 . (canceled) 
     
     
         31 . The method of  claim 23 , wherein a principal carbon source fed to the fermentation is derived from a biological feedstock which is, or derives from monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid and formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste or a non-biological feedstock which is, or derives from, natural gas, syngas, CO 2 /H 2 , methanol, ethanol, benzoate, non-volatile residue (NVR) or a caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams. 
     
     
         32 - 34 . (canceled) 
     
     
         35 . The method of  claim 23 , wherein the host is method is performed in a prokaryote selected from the group consisting of  Escherichia; Clostridia; Corynebacteria; Cupriavidus; Pseudomonas; Delftia; Bacilluss; Lactobacillus; Lactococcus;  and  Rhodococcus  or a eukaryote is selected from the group consisting of  Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula,  and  Kluyveromyces.    
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 35 , wherein the prokaryote is selected from the group consisting of  Escherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans, Bacillus subtillis, Lactobacillus delbrueckii, Lactococcus lactis,  and  Rhodococcus equi.    
     
     
         38 - 39 . (canceled) 
     
     
         40 . The method of  claim 35 , wherein the eukaryote is selected from the group consisting of  Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans,  and  Kluyveromyces lactis.    
     
     
         41 . The method of  claim 23 , wherein the method is performed in a host exhibiting tolerance to high concentrations of a C5 building block, and wherein the tolerance to high concentrations of a C5 building block is improved through continuous cultivation in a selective environment. 
     
     
         42 . The method of  claim 23 , wherein the method is performed in a host expressing one or more of the following exogenous polypeptides having an acetyl-CoA synthetase, a 6-phosphogluconate dehydrogenase; a transketolase; a feedback resistant threonine deaminase; a puridine nucleotide transhydrogenase; a formate dehydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase; a fructose 1,6 diphosphatase; a propionyl-CoA synthetase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase; a L-glutamine synthetase; a lysine transporter; a dicarboxylate transporter; and/or a multidrug transporter activity. 
     
     
         43 . The method of  claim 23 , wherein the method is performed in a host comprises comprising an attenuation of one or more polypeptides having an activity selected from the group consisting of: polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, an acetyl-CoA specific β-ketothiolase, an acetoacetyl-CoA reductase, a phosphotransacetylase forming acetate, an acetate kinase, a lactate dehydrogenase, a menaquinol-fumarate oxidoreductase, a 2-oxoacid decarboxylase producing isobutanol, an alcohol dehydrogenase forming ethanol, a triose phosphate isomerase, a pyruvate decarboxylase, a glucose-6-phosphate isomerase, a transhydrogenase dissipating the cofactor imbalance, aglutamate dehydrogenase specific for the co-factor for which an imbalance is created, a NADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase accepting C5 building blocks and central precursors as substrates; a glutaryl-CoA dehydrogenase; and a pimeloyl-CoA synthetase. 
     
     
         44 . A recombinant host cell comprising at least one exogenous nucleic acid encoding a polypeptide having malonyl-CoA O-methyltransferase activity; and a polypeptide having thioesterase activity, the host producing glutarate methyl ester. 
     
     
         45 . The host of  claim 44 , the host further comprising an exogenous polypeptide having carboxylate reductase activity, said host further producing glutarate semialdehyde methyl ester. 
     
     
         46 . The host of  claim 44 , the host further comprising one or more exogenous polypeptides having an activity selected from the group consisting of synthase activity, dehydrogenase activity, dehydratase activity, and reductase activity. 
     
     
         47 . The host of  claim 44 , the host further comprising one or more exogenous polypeptides having an activity selected from the group consisting of synthase activity, β-ketothiolase activity, dehydrogenase activity, hydratase activity, and reductase activity. 
     
     
         48 . The host of  claim 44 , wherein the polypeptide having malonyl-CoA O-methyltransferase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:13. 
     
     
         49 . The host of  claim 44 , wherein the polypeptide having thioesterase activity has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 14 or 15. 
     
     
         50 . The host of  claim 45 , wherein the polypeptide having reductase activity has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 11 or 12. 
     
     
         51 . The host of  claim 44  further comprising an exogenous polypeptide having esterase activity, the host further producing glutaric acid or 5-oxopentanoic acid. 
     
     
         52 . The host of  claim 44  further comprising one or more exogenous polypeptides having an activity selected from the group consisting of esterase activity, 6-hydroxyhexanoate dehydrogenase activity, 4-hydroxybutyrate dehydrogenase activity, 5-hydroxypentanoate dehydrogenase activity, and alcohol dehydrogenase activity, the host producing 5-hydroxypentanoic acid. 
     
     
         53 . The host of  claim 52 , further comprising one or more exogenous polypeptides having an activity selected from the group consisting of CoA-transferase activity, a synthase activity, and dehydratase activity, the host producing 2,4-pentadienoyl-CoA from 5-hydroxypentanoic acid. 
     
     
         54 . The host of  claim 53 , further comprising one or more exogenous polypeptides having an activity selected from the group consisting of hydratase activity, thioesterase activity, decarboxylase activity, dehydrogenase activity, CoA-transferase activity, and dehydratase activity, the host producing 1,3-butadiene from 2,4-pentadienoyl-CoA. 
     
     
         55 . The host of  claim 54 , wherein said polypeptide having thioesterase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15 
     
     
         56 . The host of  claim 45 , wherein the polypeptide having carboxylate reductase activity has at least 70% sequence identity to any one of the amino acid sequences set forth in any one of SEQ ID NOs: 2-7. 
     
     
         57 . A recombinant host comprising at least one exogenous nucleic acid encoding a polypeptide having pimeloyl-[acp] methyl ester methylesterase activity, and at least one polypeptide having an activity selected from the group consisting of thioesterase activity, reversible CoA-ligase activity, a CoA-transferase activity, an acylating dehydrogenase activity, an aldehyde dehydrogenase activity, a glutarate semialdehyde dehydrogenase activity, and a succinate-semialdehyde dehydrogenase activity. 
     
     
         58 . The recombinant host of  claim 57 , wherein said polypeptide having pimeloyl-[acp] methyl ester methylesterase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. 
     
     
         59 . A bio-derived product, bio-based product 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 23 , or any one of  FIGS. 1-7 , 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.   
     
     
         60 . A method of increasing the activity of a polypeptide having carboxylate reductase activity on a substituted or unsubstituted C 4 -C 8  dicarboxylic acid, the method comprising enzymatically converting said C 4 -C 8  dicarboxylic acid to a HOC(═O)(C 2 -C 6  alkyl)-C(═O)OCH 3  ester using a polypeptide having malonyl-CoA methyltransferase activity before enzymatically converting the HOC(═O)(C 2 -C 6  alkyl)-C(═O)OCH 3  ester to a HC(═O)(C 2 -C 6  alkyl)-C(═O)OCH 3  using a polypeptide having carboxylate reductase activity. 
     
     
         61 . The method of  claim 60 , further comprising converting said HC(═O)(C 2 -C 6  alkyl)-C(═O)OCH 3  to a HOCH 2 (C2-C6 alkyl)-C(═O)OCH 3  using a polypeptide having dehydrogenase activity. 
     
     
         62 . The method of  claim 61 , further comprising enzymatically converting the HOCH 2 (C 2 -C 6  alkyl)-C(═O)OCH 3  to a HOCH 2 (C 2 -C 6  alkyl)-C(═O)OH using a polypeptide having esterase activity.