Methods for producing 6-carbon chemicals via methyl-ester shielded carbon chain elongation

Abstract

This document describes biochemical pathways for producing adipic acid, 6-aminohexanoic acid, 6-hydroxhexanoic acid, hexamethylenediamine, caprolactam, or 1,6-hexanediol by forming one or two terminal functional groups, comprised of carboxyl, amine or hydroxyl group, in a C6 aliphatic backbone substrate. These pathways, metabolic engineering and cultivation strategies described herein rely on the enzymes or homologs accepting methyl ester shielded dicarboxylic acid substrates.

1 .- 41 . (canceled) 
     
     
         42 . A method for biosynthesizing a product selected from the group consisting of adipic acid, 6-aminohexanoate, 6-hydroxhexanoate, hexamethylenediamine, caprolactam and 1,6-hexanediol, said method comprising
 (a) enzymatically synthesizing a six carbon chain aliphatic backbone from oxalyl-CoA and either (i) acetyl-CoA or malonyl-CoA via two cycles of methyl ester shielded carbon chain elongation or (ii) malonyl-[acp] via two cycles of methyl-ester shielded carbon chain elongation, and   (b) enzymatically forming two terminal functional groups selected from the group consisting of carboxyl, amine, and hydroxyl groups in said backbone, thereby forming the product, wherein   the six carbon chain aliphatic backbone is adipyl-[acp] or adipyl-CoA, and wherein   a thioesterase classified under EC 3.1.2.-, an aldehyde dehydrogenase classified under EC 1.2.1.-, a 7-oxoheptanoate dehydrogenase classified under EC 1.2.1.-, a 6-oxohexanoate dehydrogenase classified under EC 1.2.1.63, a glutaconate CoA-transferase classified under EC 2.8.3.12, or a reversible succinyl-CoA ligase classified under EC 6.2.1.5 enzymatically forms a terminal carboxyl group,   a ω-transaminase classified under EC 2.6.1.-, or a deacetylase classified under EC 3.5.1.62 enzymatically forms an amine group, and/or   a 6-hydroxyhexanoate dehydrogenase classified under EC 1.1.1.258, a 5-hydroxypentanoate dehydrogenase classified under EC 1.1.1.-, a 4-hydroxybutyrate dehydratase classified under EC 1.1.1.-, or an alcohol dehydrogenase classified under EC 1.1.1.- enzymatically forms a hydroxyl group.   
     
     
         43 . The method of  claim 42 , wherein a malonyl-[acp] O-methyltransferase classified under EC 2.1.1.197 converts oxalyl-CoA to oxalyl-CoA methyl ester. 
     
     
         44 . The method of  claim 42 , wherein each of said two cycles of carbon chain elongation comprises using (i) a β-ketoacyl-[acp] synthase classified under EC 2.3.1.- or a β-ketothiolase classified under EC 2.3.1.16, (ii) a 3-oxoacyl-[acp] reductase classified under EC 1.1.1.100, an acetoacetyl-CoA reductase classified under EC 1.1.1.36, a 3-hydroxyacyl-CoA dehydrogenase classified under EC 1.1.1.35 or EC 1.1.1.157, or a 3-hydroxybutyryl-CoA dehydrogenase classified under EC 1.1.1.157, (iii) an enoyl-CoA hydratase classified under EC 4.2.1.17 or EC 4.2.1.119, or a 3-hydroxyacyl-[acp] dehydratase classified under EC 4.2.1.59, and (iv) an enoyl-[acp] reductase classified under EC 1.3.1.10 or a trans-2-enoyl-CoA reductase classified under EC 1.3.1.38, EC 1.3.1.8, or EC 1.3.1.44 to produce adipyl-CoA methyl ester or adipyl-[acp] methyl ester. 
     
     
         45 . The method of  claim 44 , wherein a pimeloyl-[acp] methyl ester methylesterase classified under EC 3.1.1.85 removes the methyl group from adipyl-CoA methyl ester or adipyl-[acp] methyl ester. 
     
     
         46 . The method of  claim 43 , wherein the malonyl-[acp] O-methyltransferase has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. 
     
     
         47 . The method of  claim 45 , wherein the pimeloyl-[acp] methyl ester methylesterase has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. 
     
     
         48 . The method of  claim 42 , wherein said thioesterase has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and/or said ω-transaminase has at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 8-13. 
     
     
         49 . The method of  claim 42 , wherein a carboxylate reductase classified under EC 1.2.99.6, enhanced by a phosphopantetheinyl transferase classified under EC 2.7.8.-, forms a terminal aldehyde group as an intermediate in forming the 6-aminohexanoate. 
     
     
         50 . The method of  claim 49 , wherein said carboxylate reductase has at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 3-7. 
     
     
         51 . The method of  claim 42 , wherein said method is performed in a recombinant host by fermentation. 
     
     
         52 . The method of  claim 51 , wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks. 
     
     
         53 . The method of  claim 52 , 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, or municipal waste; or wherein the non-biological feedstock is, or derives from, natural gas, syngas, CO 2 /H 2 , methanol, ethanol, benzoate, or terephthalic acid/isophthalic acid mixture waste streams. 
     
     
         54 . The method of  claim 51 , wherein the recombinant host is a prokaryote. 
     
     
         55 . The method of  claim 54 , wherein said prokaryote is from the genus  Escherichia  such as  Escherichia coli , from the genus  Clostridia  such as  Clostridium ljungdahlii, Clostridium autoethanogenum  or  Clostridium kluyveri , from the genus  Corynebacteria  such as  Corynebacterium glutamicum , from the genus  Cupriavidus  such as  Cupriavidus necator  or  Cupriavidus metallidurans , from the genus  Pseudomonas  such as  Pseudomonas fluorescens, Pseudomonas putida  or  Pseudomonas oleavorans , from the genus  Delftia  such as  Delftia acidovorans , from the genus  Bacillus  such as  Bacillus subtillis , from the genus  Lactobacillus  such as  Lactobacillus delbrueckii , from the genus  Lactococcus  such as  Lactococcus lactis , or from the genus  Rhodococcus  such as  Rhodococcus equi.    
     
     
         56 . The method of  claim 51 , wherein the recombinant host is a eukaryote. 
     
     
         57 . The method of  claim 56 , wherein said eukaryote is from the genus  Aspergillus  such as  Aspergillus niger , from the genus  Saccharomyces  such as  Saccharomyces cerevisiae , from the genus  Pichia  such as  Pichia pastoris , from the genus  Yarrowia  such as  Yarrowia lipolytica , from the genus  Issatchenkia  such as  Issathenkia orientalis , from the genus  Debaryomyces  such as  Debaryomyces hansenii , from the genus  Arxula  such as  Arxula adenoinivorans , or from the genus  Kluyveromyces  such as  Kluyveromyces lactis.    
     
     
         58 . A recombinant host comprising at least one exogenous nucleic acid encoding (i) a malonyl-[acp] O-methyltransferase classified under EC 2.1.1.197, (ii) a β-ketoacyl-[acp] synthase classified under EC 2.3.1.- or a β-ketothiolase classified under EC 2.3.1.16, (iii) a 3-oxoacyl-[acp] reductase classified under EC 1.1.1.100, an acetoacetyl-CoA reductase classified under EC 1.1.1.36, a 3-hydroxyacyl-CoA dehydrogenase classified under EC 1.1.1.35 or EC 1.1.1.157, or a 3-hydroxybutyryl-CoA dehydrogenase classified under EC 1.1.1.157, (iv) an enoyl-CoA hydratase classified under EC 4.2.1.17 or EC 4.2.1.119, or 3-hydroxyacyl-[acp] dehydratase classified under EC 4.2.1.59, (v) an enoyl-[acp] reductase classified under EC 1.3.1.10 or a trans-2-enoyl-CoA reductase classified under EC 1.3.1.38, EC 1.3.1.8, or EC 1.3.1.44, and (vi) a pimeloyl-[acp] methyl ester methylesterase classified under EC 3.1.1.85, said recombinant host producing adipyl-[acp] or adipyl-CoA. 
     
     
         59 . The recombinant host of  claim 58 , further comprising at least one exogenous nucleic acid encoding one or more of a thioesterase classified under EC 3.1.2.-, an aldehyde dehydrogenase classified under EC 1.2.1.-, a 7-oxoheptanoate dehydrogenase classified under EC 1.2.1.-, a 6-oxohexanoate dehydrogenase classified under EC 1.2.1.63, a glutaconate CoA-transferase classified under EC 2.8.3.12, a reversible succinyl-CoA ligase classified under EC 6.2.1.5, an acetylating aldehyde dehydrogenase classified under EC 1.2.1.10, or a carboxylate reductase classified under EC 1.2.99.6, said recombinant host producing adipic acid or adipate semialdehyde. 
     
     
         60 . The recombinant host of  claim 59 , further comprising at least one exogenous nucleic acid encoding a ω-transaminase classified under EC 2.6.1.-, said recombinant host producing 6-aminohexanoate. 
     
     
         61 . The recombinant host of  claim 59 , further comprising at least one exogenous nucleic acid encoding one or more of a 4-hydroxybutyrate dehydrogenase classified under EC 1.1.1.-, a 5-hydroxypentanoate dehydrogenase classified under EC 1.1.1.-, or a 6-hydroxyhexanoate dehydrogenase classified under EC 1.1.1.258, said recombinant host producing 6-hydroxyhexanoic acid. 
     
     
         62 . The recombinant host of  claim 59 , further comprising at least one exogenous nucleic acid encoding one or more of a ω-transaminase classified under EC 2.6.1.-, a deacetylase classified under EC 3.5.1.62, an N-acetyl transferase classified under EC 2.3.1.32, or an alcohol dehydrogenase classified under EC 1.1.1.-, said recombinant host producing hexamethylenediamine. 
     
     
         63 . The recombinant host of  claim 61 , further comprising at least one exogenous nucleic acid encoding a carboxylate reductase classified under EC 1.2.99.6 or an alcohol dehydrogenase classified under EC 1.1.1.-, said recombinant host producing 1,6-hexanediol. 
     
     
         64 . The recombinant host of  claim 60 , further comprising at least one exogenous nucleic acid encoding a lactamase classified under EC 3.5.2.-, said recombinant host producing caprolactam.