Methods Of Producing 7-Carbon Chemicals Via Methyl-Ester Shielded Carbon Chain Elongation

Abstract

This document describes biochemical pathways for producing pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine or 1,7-heptanediol by forming two terminal functional groups, comprised of carboxyl, amine or hydroxyl group, in a C7 aliphatic backbone substrate. These pathways, metabolic engineering and cultivation strategies described herein rely on enzymes or homologs accepting methyl ester shielded dicarboxylic acid substrates.

1 . A method for biosynthesizing a product selected from the group consisting of pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine and 1,7-heptanediol, said method comprising enzymatically synthesizing a seven carbon chain aliphatic backbone from (i) acetyl-CoA and 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 enzymatically forming two terminal functional groups selected from the group consisting of carboxyl, amine, and hydroxyl groups in said backbone, thereby forming the product. 
     
     
         2 . The method of  claim 1 , wherein the seven carbon chain aliphatic backbone is pimeloyl-[acp] or pimeloyl-CoA. 
     
     
         3 . The method of  claim 1 , wherein a malonyl-[acp] O-methyltransferase converts malonyl-CoA to malonyl-CoA methyl ester or converts malonyl-[acp] to malonyl-[acp] methyl ester. 
     
     
         4 . The method of  claim 3 , wherein each of said two cycles of carbon chain elongation comprises using (i) a β-ketoacyl-[acp] synthase or a β-ketothiolase, (ii) a 3-oxoacyl-[acp] reductase, an acetoacetyl-CoA reductase, a 3-hydroxyacyl-CoA dehydrogenase or a 3-hydroxybutyryl-CoA dehydrogenase, (iii) an enoyl-CoA hydratase or a 3-hydroxyacyl-[acp] dehydratase, and (iv) an enoyl-[acp] reductase or a trans-2-enoyl-CoA reductase to produce pimeloyl-[acp] methyl ester from malonyl-[acp] methyl ester or produce pimeloyl-CoA methyl ester from malonyl-CoA methyl ester. 
     
     
         5 . The method of  claim 4 , wherein a pimeloyl-[acp] methyl ester methylesterase removes the methyl group from pimeloyl-CoA methyl ester or pimeloyl-[acp] methyl ester. 
     
     
         6 . The method of  claim 3 , wherein the malonyl-[acp]O-methyltransferase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. 
     
     
         7 . The method of  claim 5 , wherein the pimeloyl-[acp] methyl ester methylesterase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. 
     
     
         8 .- 13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydratase, or an alcohol dehydrogenase enzymatically forms a hydroxyl group. 
     
     
         15 . The method of  claim 1 , wherein a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a glutaconate CoA-transferase, or a reversible succinyl-CoA ligase enzymatically forms a terminal carboxyl group. 
     
     
         16 . The method of  claim 15 , wherein said thioesterase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. 
     
     
         17 . The method of  claim 1 , wherein a ω-transaminase or a deacetylase enzymatically forms the amine group. 
     
     
         18 . The method of  claim 17 , wherein said w-transaminase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 8-13. 
     
     
         19 . The method of  claim 1 , wherein a carboxylate reductase, enhanced by a phosphopantetheinyl transferase, forms a terminal aldehyde group as an intermediate in forming the product. 
     
     
         20 . The method of  claim 19 , wherein said carboxylate reductase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 2-7. 
     
     
         21 . The method of  claim 1 , wherein said method is performed in a recombinant host by fermentation. 
     
     
         22 . The method of  claim 21 , wherein:
 (1) said recombinant host is subjected to a cultivation strategy under aerobic, anaerobic, micro-aerobic or mixed oxygen/denitrification cultivation conditions,   (2) said recombinant host is cultured under conditions of nutrient limitation,   (3) said recombinant host is retained using a ceramic hollow fiber membrane to maintain a high cell density during fermentation, and/or   (4) the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks.   
     
     
         23 .- 25 . (canceled) 
     
     
         26 . The method of  claim 22 , 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, and/or 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 cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams. 
     
     
         27 . (canceled) 
     
     
         28 . The method of  claim 21 , wherein the recombinant host is a prokaryote or a eukaryote. 
     
     
         29 . The method of  claim 28 , 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 acidovorans , from the genus  Bacillus  such as  Bacillus subtillis ; from the genes  Lactobacillus  such as  Lactobacillus delbrueckii ; from the genus  Lactococcus  such as  Lactococcus lactis  or from the genus  Rhodococcus  such as  Rhodococcus equi , or
 wherein said eukaryote is from the genus  Aspergillus  such as  Asperqillus niqer , 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.      
     
     
         30 .- 31 . (canceled) 
     
     
         32 . The method of  claim 21 , wherein the recombinant host's tolerance to high concentrations of a C7 building block is improved through continuous cultivation in a selective environment. 
     
     
         33 .- 34 . (canceled) 
     
     
         35 . A recombinant host comprising at least one exogenous nucleic acid encoding (i) a malonyl-[acp] O-methyltransferase, (ii) a β-ketoacyl-[acp] synthase or a β-ketothiolase, (iii) a 3-oxoacyl-[acp] reductase, acetoacetyl-CoA reductase, a 3-hydroxyacyl-CoA dehydrogenase or a 3-hydroxybutyryl-CoA dehydrogenase, (iv) an enoyl-CoA hydratase or 3-hydroxyacyl-[acp] dehydratase, (v) an enoyl-[acp] reductase or a trans-2-enoyl-CoA reductase, and (vi) a pimeloyl-[acp] methyl ester methylesterase, said host producing pimeloyl-[acp] or pimeloyl-CoA. 
     
     
         36 . The recombinant host of  claim 35 , said host comprising at least one exogenous nucleic acid encoding one or more of a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a glutaconate CoA-transferase, a reversible succinyl-CoA ligase, an acetylating aldehyde dehydrogenase, or a carboxylate reductase, said host producing pimelic acid or pimelate semialdehyde. 
     
     
         37 . The recombinant host of  claim 36 , said host comprising at least one exogenous nucleic acid encoding a ω-transaminase, said host producing 7-aminoheptanoate. 
     
     
         38 . The recombinant host of  claim 36 , said host further comprising one or more of a 4-hydroxybutyrate dehydrogenase, a 5-hydroxypentanoate dehydrogenase or a 6-hydroxyhexanoate dehydrogenase, said host producing 7-hydroxyheptanoic acid. 
     
     
         39 . The recombinant host of  claim 36 , said host further comprising at least one exogenous nucleic acid encoding a ω-transaminase, a deacetylase, an N-acetyl transferase or an alcohol dehydrogenase, said host producing heptamethylenediamine. 
     
     
         40 . The recombinant host of  claim 38 , said host further comprising at least one exogenous nucleic acid encoding a (i) carboxylate reductase enhanced by a phosphopantetheinyl transferase or (ii) an alcohol dehydrogenase, said host producing 1,7-heptanediol.