Methods of producing 7-carbon chemicals from long chain fatty acids via oxidative cleavage

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 the fatty acid synthesis pathway and oxidative cleavage of long chain acyl-[acp] intermediates by a monooxgenase (e.g., cytochrome P450) such as that encoded by BioI from microorganisms such as Bacillus subtillis.

1 . A method for biosynthesizing a product selected from the group consisting of pimelic acid, 7-hydroxyheptanoic acid, heptamethylenediamine, and 1,7-heptanediol, said method comprising enzymatically synthesizing two seven carbon chain aliphatic backbones from a long chain acyl-[acp] intermediate 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 two seven carbon chain aliphatic backbones are pimeloyl-[acp] and heptanoate. 
     
     
         3 . The method of  claim 2 , wherein pimeloyl-[acp] and heptanoate are enzymatically synthesized from acetyl-CoA and malonyl-CoA via fatty acid synthesis and the oxidative cleavage of a long chain acyl-[acp] intermediate by a polypeptide having the activity of a monooxgenase, wherein the polypeptide having the activity of a monooxgenase is encoded by BioI. 
     
     
         4 .- 9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein (i) a polypeptide having the activity of a monooxygenase, a polypeptide having the activity of an oxidoreductase and ferredoxin, (ii) a polypeptide having the activity of a 6-hydroxyhexanoate dehydrogenase, a polypeptide having the activity of a 5-hydroxypentanoate dehydrogenase, or a polypeptide having the activity of a 4-hydroxybutyrate dehydrogenase, or (iii) a polypeptide having the activity of an alcohol dehydrogenase enzymatically forms the two hydroxyl groups. 
     
     
         11 . The method of  claim 10 , wherein said polypeptide having the activity of a monooxygenase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:14-16. 
     
     
         12 . The method of  claim 1 , wherein a polypeptide having the activity of a thioesterase, a polypeptide having the activity of an aldehyde dehydrogenase, a polypeptide having the activity of a 7-oxoheptanoate dehydrogenase, or a polypeptide having the activity of a 6 oxohexanoate dehydrogenase enzymatically forms a terminal carboxyl group. 
     
     
         13 . The method of  claim 12 , wherein said polypeptide having the activity of a thioesterase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:23, or SEQ ID NO:24. 
     
     
         14 . The method of  claim 1 , wherein a polypeptide having the activity of a ω-transaminase or a polypeptide having the activity of a deacetylase enzymatically forms an amine group. 
     
     
         15 . The method of  claim 14 , wherein said polypeptide having the activity of a ω-transaminase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:8-13. 
     
     
         16 . The method of  claim 1 , wherein a polypeptide having the activity of a carboxylate reductase and a polypeptide having the activity of a phosphopantetheinyl transferase form a terminal aldehyde group as an intermediate in forming the product. 
     
     
         17 . The method of  claim 16 , wherein said polypeptide having the activity of a carboxylate reductase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:2-7. 
     
     
         18 . The method of  claim 1 , wherein said method is performed in a recombinant host by fermentation. 
     
     
         19 .- 21 . (canceled) 
     
     
         22 . The method of  claim 18 , wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks. 
     
     
         23 . 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; 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. 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 18 , wherein the recombinant host is a prokaryote or a eukaryote. 
     
     
         26 . The method of  claim 25 , 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 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  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.      
     
     
         27 .- 29 . (canceled) 
     
     
         30 . The method of  claim 18 ,
 wherein said recombinant host comprises one or more polypeptides having the activity of the following attenuated enzymes: a polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, an acetyl-CoA specific β-ketothiolase, 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, an NADPH-consuming transhydrogenase, an NADPH-specific glutamate dehydrogenase, an NADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoA dehydrogenase, an acyl-CoA dehydrogenase accepting C7 building blocks and central precursors as substrates, a glutaryl-CoA dehydrogenase or a pimeloyl-CoA synthetase; and/or   wherein said recombinant host overexpresses one or more genes encoding polypeptides having the activity of the following enzymes: an acetyl-CoA carboxylase, an acetyl-CoA synthetase, a 6-phosphogluconate dehydrogenase, a transketolase, a puridine nucleotide transhydrogenase, a glyceraldehyde-3P-dehydrogenase, a malic enzyme, a glucose-6-phosphate dehydrogenase, a fructose 1,6 diphosphatase, a L-alanine dehydrogenase, an NADPH L-glutamate dehydrogenase, a diamine transporter, a dicarboxylate transporter, and/or a multidrug transporter.   
     
     
         31 . (canceled) 
     
     
         32 . A recombinant host comprising at least one exogenous nucleic acid encoding a polypeptide having the activity of a monooxygenase and a polypeptide having the activity of an aldehyde dehydrogenase, said recombinant host producing pimeloyl-[acp] and heptanoate. 
     
     
         33 . The recombinant host of  claim 32 , further comprising one or more polypeptides having the activity of a monooxygenase, a thioesterase, an aldehyde dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, or a carboxylate reductase, said recombinant host producing pimelic acid, pimelate semialdehyde, or 7-hydroxyheptanoate. 
     
     
         34 . The recombinant host of  claim 33 , further comprising:
 a) a polypeptide having the activity of a ω-transaminase, said recombinant host producing 7-aminoheptanoate,   b) one or more polypeptides having the activity of a carboxylate reductase, a ω-transaminase, a deacetylase, an N-acetyl transferase, or an alcohol dehydrogenase, said recombinant host producing heptamethylenediamine, and/or   c) a polypeptide having the activity of a carboxylate reductase or an alcohol dehydrogenase, said recombinant host producing 1,7-heptanediol.   
     
     
         35 .- 36 . (canceled)