Methods of producing 7-carbon chemicals via pyruvate and succinate semialdehyde aldol condensation

Abstract

This document describes biochemical pathways for producing one or more of pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine and 1,7-heptanediol by forming one or two terminal functional groups, comprised of carboxyl, amine or hydroxyl groups, in a C7 aliphatic backbone substrate produced from succinate semialdehyde or pyruvate. These pathways, metabolic engineering and cultivation strategies described herein rely on the aldol condensation of succinate semialdehyde and pyruvate.

1 - 40 . (canceled) 
     
     
         41 . A method for biosynthesizing at least one product chosen from pimelic acid, 7-hydroxyheptanoate, heptamethylenediamine, and 1,7-heptanediol, said method comprising:
 enzymatically synthesizing a seven carbon chain aliphatic backbone from succinate semialdehyde and pyruvate via aldol condensation, wherein the aldol condensation comprises an aldol reaction catalyzed by a 4-hydroxy-2-oxopimelate aldolase and a dehydration catalyzed by a 2-hydroxyhepta-2,4-dienedioate hydratase; and   enzymatically forming two terminal functional groups chosen from carboxyl, amine, and hydroxyl groups in said seven carbon chain aliphatic backbone, thereby forming said at least one product.   
     
     
         42 . The method of  claim 41 , wherein said seven carbon chain aliphatic backbone is pimeloyl-CoA or pimelate semialdehyde. 
     
     
         43 . The method of  claim 41 , wherein the product of the aldol condensation, 2,4-dihydroxyhept-2-enedioate or its tautomer 4-hydroxy-2-oxo-pimelate, is converted to:
 pimeloyl-CoA using said 2-hydroxyhepta-2,4-dienedioate hydratase and at least one enzyme chosen from: an enoate reductase; a 2-hydroxyglutarate dehydrogenase; a glutaconate CoA-transferase; a 2-hydroxyglutaryl-CoA dehydratase; and an enoyl-CoA reductase or an enoyl-[acp] reductase; or   pimelate semialdehyde using said 2-hydroxyhepta-2,4-dienedioate hydratase and at least one enzyme chosen from: an enoate reductase; a 2-hydroxyglutarate dehydrogenase; a glutaconate CoA-transferase; a 2-hydroxyglutaryl-CoA dehydratase; and a carboxylate reductase.   
     
     
         44 . The method of  claim 43 , wherein said enoate reductase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16 or SEQ ID NO: 17. 
     
     
         45 . The method of  claim 41 , wherein said at least one product comprises two terminal amine groups, two terminal carboxyl groups, two terminal hydroxyl groups, or a terminal hydroxyl group and a terminal carboxyl group. 
     
     
         46 . The method of  claim 45 , wherein a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydratase, or an alcohol dehydrogenase enzymatically forms said two terminal hydroxyl groups. 
     
     
         47 . The method of  claim 41 , wherein a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a CoA-transferase, or a reversible CoA-ligase enzymatically forms a terminal carboxyl group. 
     
     
         48 . The method of  claim 47 , wherein said thioesterase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. 
     
     
         49 . The method of  claim 41 , wherein a ω-transaminase or a deacetylase enzymatically forms a terminal amine group. 
     
     
         50 . The method of  claim 49 , wherein said ω-transaminase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 8-13. 
     
     
         51 . The method of  claim 41 , wherein a carboxylate reductase, enhanced by a phosphopantetheinyl transferase, forms a terminal aldehyde group as an intermediate in forming the product. 
     
     
         52 . The method of  claim 51 , 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. 
     
     
         53 . The method of  claim 41 , wherein said method is performed in a recombinant host by fermentation. 
     
     
         54 . The method of  claim 53 , wherein the principal carbon source fed to said fermentation derives from a biological or non-biological feedstock. 
     
     
         55 . The method of  claim 54 , wherein:
 said 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   said 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.   
     
     
         56 . The method of  claim 53 , wherein said recombinant host is a prokaryote or a eukaryote. 
     
     
         57 . The method of  claim 53 , wherein said recombinant host's tolerance to high concentrations of a C7 building block is improved through continuous cultivation in a selective environment. 
     
     
         58 . The method of  claim 53 , wherein said recombinant host comprises at least one attenuated enzyme chosen from: a lactate dehydrogenase; a menaquinol-fumarate oxidoreductase; an alcohol dehydrogenase producing ethanol; a pyruvate decarboxylase; a 2-oxoacid decarboxylase generating isobutanol; a polymer synthase; a NADH-specific L-glutamate dehydrogenase; a NADH-consuming transhydrogenase; a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase that degrades C7 building blocks and their precursors; a glutaryl-CoA dehydrogenase; and a pimeloyl-CoA synthetase. 
     
     
         59 . The method of  claim 53 , wherein said recombinant host overexpresses at least one gene encoding a gene product chosen from: a PEP carboxykinase; a PEP carboxylase; a pyruvate carboxylase; a PEP synthase; a formate dehydrogenase; a L-alanine dehydrogenase; a NADPH-specific L-glutamate dehydrogenase; a diamine transporter, a dicarboxylate transporter; and a multidrug transporter. 
     
     
         60 . A recombinant host comprising at least one exogenous nucleic acid encoding a 4-hydroxy-2-oxopimelate aldolase, a 2-hydroxyhepta-2,4-dienedioate hydratase, an enoate reductase, a 2-hydroxyglutarate dehydrogenase, a glutaconate CoA-transferase, a 2-hydroxyglutaryl-CoA dehydratase, and an enoyl-CoA reductase or an enoyl-[acp] reductase, said host producing pimeloyl-CoA. 
     
     
         61 . A recombinant host comprising at least one exogenous nucleic acid encoding a 4-hydroxy-2-oxopimelate aldolase, a 2-hydroxyhepta-2,4-dienedioate hydratase, an enoate reductase, a 2-hydroxyglutarate dehydrogenase, a glutaconate CoA-transferase, a 2-hydroxyglutaryl-CoA dehydratase, and a carboxylate reductase, said host producing pimelate semialdehyde.