Methods of producing 6-carbon chemicals using 2,6-diaminopimelate as precursor to 2-aminopimelate

Abstract

This document describes biochemical pathways for producing 2-aminopimelate from 2,6-diaminopimelate, and methods for converting 2-aminopimelate to one or more of adipic acid, adipate semialdehyde, caprolactam, 6-aminohexanoic acid, 6-hexanoic acid, hexamethylenediamine, or 1,6-hexanediol by decarboxylating 2-aminopimelate into a six carbon chain aliphatic backbone and enzymatically forming one or two terminal functional groups, comprised of carboxyl, amine or hydroxyl group, in the backbone.

1 - 76 . (canceled) 
     
     
         77 . A method of biosynthesizing at least one product chosen from adipic acid, adipate semialdehyde, 6-aminohexanoic acid, 6-hydroxyhexanoic acid, caprolactam, hexamethylenediamine, and 1,6-hexanediol in a recombinant host via fermentation, said method comprising:
 enzymatically converting 2,6-diaminopimelate to 2-aminopimelate using at least one polypeptide having an activity chosen from 2-hydroxyacyl-CoA dehydratase activity, mutase activity, ammonia lyase activity, and enoate reductase activity; and   enzymatically converting 2-aminopimelate to said at least one product.   
     
     
         78 . The method of  claim 77 , wherein 2,6-diaminopimelate is enzymatically converted to (S) 2-aminopinelate or (R) 2-aminopimelate. 
     
     
         79 . The method of  claim 77 , said method comprising:
 using said polypeptide having 2-hydroxyacyl-CoA dehydratase activity and said polypeptide having enoate reductase activity to enzymatically convert 2,6-diaminopimelate to 2-aminopimelate; and/or   further using at least one polypeptide having an activity chosen from diaminopimelate dehydrogenase activity, 2-hydroxycarboxylate dehydrogenase activity, CoA-transferase activity, 2-hydroxyacid dehydratase activity, and carboxylate reductase activity to enzymatically convert 2,6-diaminopimelate to 2-aminopimelate.   
     
     
         80 . The method of  claim 77 , said method comprising:
 using said polypeptide having mutase activity, said polypeptide having ammonia lyase activity, and said polypeptide having enoate reductase activity to enzymatically convert 2,6-diaminopimelate to 2-aminopimelate; and/or   further using at least one polypeptide having an activity chosen from CoA ligase activity, CoA-transferase activity, carboxylate reductase activity, and aldehyde dehydrogenase activity to enzymatically convert 2,6-diaminopimelate to 2-aminopimelate.   
     
     
         81 . The method of  claim 77 , wherein:
 said polypeptide having enoate reductase activity has at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 16-22;   said polypeptide having 2-hydroxyacyl-CoA dehydratase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 28;   said polypeptide having mutase activity has at east 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26; or   said polypeptide having ammonia lyase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23.   
     
     
         82 . The method of  claim 77 , wherein 2-aminopimelate is enzymatically converted to said at least one product using:
 at least one polypeptide having an activity chosen from α-oxoacid decarboxylase activity classified under EC 4.1.1.-, α-aminoacid decarboxylase activity classified under EC 4.1.1.-, synthase activity, and activity of a dehydrogenase complex; and   optionally one or more polypeptides having an activity chosen from aldehyde dehydrogenase activity, alcohol dehydrogenase activity, CoA-transferase activity, carboxylate reductase activity, α-aminotransferase activity, thioesterase activity, hydrolase activity, ω-transaminase activity, N-acetyltransferase activity, and deacylase activity.   
     
     
         83 . The method of  claim 82 , wherein:
 said polypeptide having α-oxoacid decarboxylase activity is classified under EC 4.1.1.43, EC 4.1.1.71, EC 4.1.1.72, or EC 4.1.1.74;   said polypeptide having α-aminoacid decarboxylase activity is classified under EC 4.1.1.15, EC 4.1.1.1 EC 4.1.1.18, or EC 4.1.1.19;   said polypeptide having synthase activity is classified under EC 2.2.1.6; or   said polypeptide having the activity of a dehydrogenase complex comprises activities classified under EC 1.2.4.2, EC 1.8.1.4, or EC 2.3.1.61.   
     
     
         84 . The method of  claim 77 , wherein:
 said at least one product is adipic acid and is biosynthesized from 2-aminopimelate using at least one polypeptide having an activity chosen from α-aminotransferase activity, 2-oxoacid decarboxylase activity, synthase activity, dehydrogenase complex activity, thioesterase activity, CoA-transferase activity, CoA-ligase activity, and aldehyde dehydrogenase activity;   said at least one product is adipate semialdehyde and is biosynthesized from 2-aminopimelate using at least one polypeptide having an activity chosen from α-aminotransferase activity, 2-oxoacid decarboxylase activity, and synthase activity;   said at least one product is 6-aminohexanoic acid and is biosynthesized from 2-aminopimelate using a polypeptide having α-aminoacid decarboxylase activity; or   said at least one product is 6-hydroxyhexanoic acid and is biosynthesized from 2-aminopimelate using at least one polypeptide having an activity chosen from α-aminotransferase activity, 2-oxoacid decarboxylase activity, synthase activity, and alcohol dehydrogenase activity.   
     
     
         85 . The method of  claim 84 , wherein:
 said polypeptide having 2-oxoacid decarboxylase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 34;   said polypeptide having a-amino acid decarboxylase activity has at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 29-34; and/or   said polypeptide having thioesterase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.   
     
     
         86 . The method of  claim 84 , wherein said at least one product is adipate semialdehyde and is biosynthesized from 2-aminopimelate using at least one polypeptide having an activity chosen from a-aminotransferase activity, 2-oxoacid decarboxylase activity, and synthase activity, and further wherein said method comprises:
 biosynthesizing 6-aminohexanoic acid from adipate semialdehyde using a polypeptide having ω-transaminase activity;   biosynthesizing 6-aminohexanoic acid from adipate semialdehyde using a polypeptide having ω-transaminase activity and further biosynthesizing hexamethylenediamine from 6-aminohexanoic acid using at least one polypeptide having an activity chosen from carboxylate reductase activity, N-acetyltransferase activity, ω-transaminase activity, and deacylase activity;   biosynthesizing 6-aminohexanoic acid from adipate semialdehyde using a polypeptide having ω-transminase activity and further biosynthesizing caprolactam from 6-aminohexanoic acid using a polypeptide having the activity of a hydrolase; or   biosynthesizing hexamethylenediamine from adipate semialdehyde using at least one polypeptide having carboxylate reductase activity or ω-transaminase activity.   
     
     
         87 . The method of  claim 86 , wherein:
 said polypeptide having carboxylate reductase activity has at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 3-7; and/or   said polypeptide having ω-transaminase activity has at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 8-13.   
     
     
         88 . The method of  claim 84 , wherein said at least one product is 6-hydroxyhexanoic acid and is biosynthesized from 2-aminopimelate using at least one polypeptide having an activity chosen from α-aminotransferase activity, 2-oxoacid decarboxylase activity, synthase activity, and alcohol dehydrogenase activity, and further wherein said method comprises:
 biosynthesizing hexamethylenediamine from 6-hydroxyhexanoic acid using at least one polypeptide having an activity chosen from carboxylate reductase activity, ω-transaminase activity, and alcohol dehydrogenase activity; and/or 
 biosynthesizing 1,6-hexanediol from 6-hydroxyhexanoic acid using a polypeptide having carboxylate reductase activity and a polypeptide having alcohol dehydrogenase activity. 
 
     
     
         89 . The method of  claim 77 , wherein said recombinant host is:
 subjected to a cultivation strategy under aerobic or micro-aerobic cultivation conditions;   cultured under conditions of nitrogen, phosphate, or oxygen limitation; and/or   retained using a ceramic membrane to maintain a high cell density during said fermentation.   
     
     
         90 . The method of  claim 77 , further comprising feeding a principal feedstock to the fermentation derived from a biological feedstock or a non-biological feedstock. 
     
     
         91 . The method of  claim 90 , wherein:
 said biological feedstock 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   said non-biological feedstock 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 I isophthalic acid mixture waste streams.   
     
     
         92 . The method of  claim 77 , 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.      
     
     
         93 . The method of  claim 77 , wherein said recombinant host is:
 a prokaryote chosen from  Escherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans, Pseudomonasfluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans, Bacillus subtillis, 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.      
     
     
         94 . The method of  claim 77 , wherein:
 said recombinant host exhibits tolerance to high concentrations of a C6 building block, and wherein the tolerance to high concentrations of a C6 building block is improved through continuous cultivation in a selective environment; and/or   said recombinant host comprises at least one modification chosen from:
 increased intracellular concentration of oxaloacetate for biosynthesis of a C6 building block, wherein said intracellular concentration is increased in the host by overexpressing recombinant genes forming oxaloacetate; 
 an imbalance in NADPH that can be balanced via the formation of a C6 building block; 
 an exogenous lysine biosynthesis pathway synthesizing lysine from 2-oxoglutarate via 2-oxoadipate; 
 an exogenous lysine biosynthesis pathway synthesizing lysine from oxaloacetate to meso 2,6-diaminopimelate; 
 attenuated endogenous degradation pathways of central metabolites and central precursors leading to and including C6 building blocks; and 
 the efflux of a C6 building block across the cell membrane to the extracellular media is enhanced or amplified by genetically engineering structural modifications to the cell membrane or increasing any associated transporter activity for a C6 building block. 
   
     
     
         95 . A composition comprising:
 at least one bioderived 6-carbon compound chosen from adipic acid, adipate semialdehyde, 6-aminohexanoic acid, 6-hydroxyhexanoic acid, caprolactam, hexamethylenediamine, and 1,6-hexanediol, wherein said at least one bioderived 6-carbon compound is biosynthesized using the method of  claim 77 ; and   a compound other than the bioderived 6-carbon compound.   
     
     
         96 . A biobased polymer or resin comprising at least one bioderived 6-carbon compound chosen from adipic acid, adipate semialdehyde, 6-aminohexanoic acid, 6-hydroxyhexanoic acid, caprolactam, hexamethylenediamine, and 1,6-hexanediol, wherein said at least one bioderived compound is biosynthesized using the method of  claim 77 . 
     
     
         97 . A molded product obtainable by molding a biobased polymer or resin of  claim 93 . 
     
     
         98 . A process for producing a biobased polymer or resin comprising biosynthezing at least one bioderived 6-carbon compound chosen from adipic acid, adipate semialdehyde, 6-aminohexanoic acid, 6-hydroxyhexanoic acid, caprolactam, hexamethylenediamine, and 1,6-hexanediol using the method of  claim 77 , wherein said method further comprises:
 culturing or growing a host cell under conditions and for a sufficient period of time to produce said at least one bioderived 6-carbon compound; and   chemically reacting said at least one bioderived 6-carbon compound with itself or another compound in a polymer-producing or resin-producing reaction.   
     
     
         99 . A biochemical network comprising meso-2,6-diaminopimelate and at least one enzyme chosen from a dehydrogenase, a CoA-transferase, a dehydratase, a reductase, a mutase, a CoA-ligase, an ammonia lyase, and a thioesterase, wherein said at least one enzyme is capable of enzymatically converting the meso-2,6-diaminopimelate to 2-aminopimelate, and further wherein said biochemical network optionally comprises:
 an α-aminotransferase, wherein said α-aminotransferase is capable of enzymatically converting 2-aminopimelate to 2-oxo-pimelate; 
 an α-aminotransferase and at least one enzyme chosen from a synthase, a dehydrogenase complex, and a decarboxylase, wherein:
 said α-aminotransferase is capable of enzymatically converting 2-aminopimelate to 2-oxo-pimelate; and 
 said at least one enzyme is capable of enzymatically converting 2-oxo-pimelate to adipyl-CoA or adipate semialdehyde; 
 
 an α-aminotransferase, at least one first enzyme chosen from decarboxylase, a synthase, and a dehydrogenase complex, and at least one second enzyme chosen from a dehydrogenase, a CoA-transferase, a CoA-ligase, and a thioesterase, wherein:
 said α-aminotransferase is capable of enzymatically converting 2-aminopimelate to 2-oxo-pimelate; 
 said at least one first enzyme is capable of enzymatically converting 2-oxo-pimelate to adipyl-CoA or adipate semialdehyde; and 
 said at least one second enzyme is capable of enzymatically converting adipyl-CoA or adipate semialdehyde to adipic acid; 
 
 a decarboxylase, wherein said decarboxylase is capable of enzymatically converting 2-aminopimelate to 6-aminohexanoic acid; 
 a decarboxylase and at least one enzyme chosen from a hydrolase, a reductase, a transaminase, an N-acetyltransferase, and a deacetylase, wherein:
 said decarboxylase is capable of enzymatically converting 2-aminopimelate to 6-aminohexanoic acid; and 
 said at least one enzyme is capable of enzymatically converting 6-aminohexanoic acid into at least one of caprolactam or hexamethylenediamine; 
 
 at least one enzyme chosen from an aminotransferase, a synthase, a decarboxylase, and a dehydrogenase, wherein said at least one enzyme is capable of enzymatically converting 2-aminopimelate to 6-hydroxyhexanoic acid; or 
 at least one first enzyme chosen from an aminotransferase, a synthase, a decarboxylase, and a dehydrogenase and at least one second enzyme chosen from a reductase, a transaminase, or an alcohol dehydrogenase, wherein:
 said at least one first enzyme is capable of enzymatically converting 2-aminopimelate to 6-hydroxyhexanoic acid; and 
 said at least one second enzyme is capable of enzymatically converting 6-hydroxyhexanoic acid into at least one of hexamethylenediamine and 1,6-hexanediol.