Methods of producing 7-carbon chemicals via aromatic compounds

Abstract

This document describes biochemical pathways for producing pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, or 1,7-heptanediol by forming two terminal functional groups, comprised of carboxyl, amine or hydroxyl group, in a C7 aliphatic backbone substrate produced from chorismate or benzoate. These pathways, metabolic engineering and cultivation strategies described herein rely on the anaerobic benzoyl-CoA degradation pathway enzymes.

1 .- 42 . (canceled) 
     
     
         43 . A method for biosynthesizing a product selected from pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, and 1,7-heptanediol, said method comprising
 (a) enzymatically synthesizing a seven carbon chain aliphatic backbone from benzoyl-CoA, and   (b) enzymatically forming two terminal functional groups selected from carboxyl, amine, and hydroxyl groups in said backbone, thereby forming the product, 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 succinate 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.   
     
     
         44 . The method of  claim 43 , wherein the seven carbon chain aliphatic backbone is pimeloyl-CoA. 
     
     
         45 . The method of  claim 44 , wherein pimeloyl-CoA is enzymatically synthesized from chorismate or benzoate via benzoyl-CoA degradation. 
     
     
         46 . The method of  claim 45 , wherein a benzoyl-CoA reductase classified under EC 1.3.7.8 converts benzoyl-CoA to cyclohexa-1,5-diene-1-carbonyl-CoA. 
     
     
         47 . The method of  claim 45 , wherein chorismate is converted to 4-hydroxybenzoate by a chorismate lyase classified under EC 4.1.3.40, or chorismate is converted to anthranilate by an anthranilate synthase classified under EC 4.1.3.27. 
     
     
         48 . The method of  claim 44 , wherein pimeloyl-CoA is enzymatically synthesized from 2-ketocyclohexane-1-carboxyl-CoA by a 2-ketocyclohexane-1-carboxyl-CoA hydrolase classified under EC 4.1.3.-. 
     
     
         49 . The method of  claim 43 , further comprising:
 converting benzoyl-CoA to cyclohexa-1,5-diene-1-carbonyl-CoA by a benzoyl-CoA reductase classified under EC 1.3.7.8;   converting cyclohexa-1,5-diene-1-carbonyl-CoA to cyclohex-1-ene-1-carboxyl-CoA by a cyclohex-1-ene-1-carboxyl-CoA dehydrogenase classified under EC 1.3.8.-;   converting cyclohex-1-ene-1-carboxyl-CoA to 2-hydroxycyclohexane-1-carboxyl-CoA by a cyclohex-1-ene-1-carboxyl-CoA hydratase classified under EC 4.2.1.-;   converting 2-hydroxycyclohexane-1-carboxyl-CoA to 2-ketocyclohexane-1-carboxyl-CoA by a 2-hydroxy cyclohexane-1-carboxyl-Co A dehydrogenase classified under EC 1.1.1.-; and/or   converting 2-ketocyclohexane-1-carboxyl-CoA to pimeloyl-CoA by a 2-ketocyclohexane-1-carboxyl-CoA hydrolase classified under EC 4.1.3.-.   
     
     
         50 . The method of  claim 43 , wherein said thioesterase has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. 
     
     
         51 . The method of  claim 43 , wherein 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. 
     
     
         52 . The method of  claim 43 , 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 7-aminoheptanoate. 
     
     
         53 . The method of  claim 52 , wherein said carboxylate reductase has at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 2-7. 
     
     
         54 . The method of  claim 43 , wherein said method is performed in a recombinant host by fermentation. 
     
     
         55 . The method of  claim 54 , wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks. 
     
     
         56 . The method of  claim 55 , wherein the biological feedstock is, or derives from, monosaccharides, disaccharides, lignocellulose, hem icellulose, 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. 
     
     
         57 . The method of  claim 54 , wherein the recombinant host is a prokaryote. 
     
     
         58 . The method of  claim 57 , wherein said prokaryote is selected  Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus,  and  Rhodococcus.    
     
     
         59 . The method of  claim 57 , wherein said prokaryote is selected from  Escherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans, Bacillus subtillis, Lactobacillus delbrueckii, Lactococcus lactis,  and  Rhodococcus equi.    
     
     
         60 . The method of  claim 54 , wherein the recombinant host is a eukaryote. 
     
     
         61 . The method of  claim 60 , wherein said eukaryote is selected from  Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula,  and  Kluyveromyces.    
     
     
         62 . The method of  claim 60 , wherein said eukaryote is selected from  Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans,  and  Kluyveromyces lactis.    
     
     
         63 . A recombinant host comprising one or more exogenous nucleic acids encoding one or more enzymes according to  claim 43 . 
     
     
         64 . A bio-derived, bio-based or fermentation-derived product, wherein said product comprises:
 i. a composition comprising at least one bio-derived, bio-based or fermentation-derived compound produced according to  claim 43 , or any combination thereof,   ii. a bio-derived, bio-based or fermentation-derived polymer comprising the bio-derived, bio-based or fermentation-derived composition or compound of i., or any combination thereof,   iii. a bio-derived, bio-based or fermentation-derived resin comprising the bio-derived, bio-based or fermentation-derived compound or bio-derived, bio-based or fermentation-derived composition of i. or any combination thereof or the bio-derived, bio-based or fermentation-derived polymer of ii. or any combination thereof,   iv. a molded substance obtained by molding the bio-derived, bio-based or fermentation-derived polymer of ii. or the bio-derived, bio-based or fermentation-derived resin of iii., or any combination thereof,   v. a bio-derived, bio-based or fermentation-derived formulation comprising the bio-derived, bio-based or fermentation-derived composition of i., bio-derived, bio-based or fermentation-derived compound of i., bio-derived, bio-based or fermentation-derived polymer of ii., bio-derived, bio-based or fermentation-derived resin of iii., or bio-derived, bio-based or fermentation-derived molded substance of iv, or any combination thereof, or   vi. a bio-derived, bio-based or fermentation-derived semi-solid or a non-semi-solid stream, comprising the bio-derived, bio-based or fermentation-derived composition of i., bio-derived, bio-based or fermentation-derived compound of i., bio-derived, bio-based or fermentation-derived polymer of ii., bio-derived, bio-based or fermentation-derived resin of iii., bio-derived, bio-based or fermentation-derived formulation of v., or bio-derived, bio-based or fermentation-derived molded substance of iv., or any combination thereof.