Methods of producing 7-carbon chemicals via carbon chain elongation associated with cyclohexane carboxylate synthesis

Patent metadata

Field	Value
Publication number	US-9637764-B2
Application number	US-201314138904-A
Country	US
Kind code	B2
Filing date	Dec 23, 2013
Priority date	Dec 31, 2012
Publication date	May 2, 2017
Grant date	May 2, 2017

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 carbon chain elongation enzymes or homologs thereof associated with the cyclohexane carboxylate biosynthesis from Syntrophus aciditrophicus or 2-aminoadipate lysine biosynthesis.

First claim

What is claimed is:

1. A method for biosynthesizing a product selected from the group consisting of pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine, and 1,7-heptanediol, said method comprising:
(i) contacting 2-oxoglutarate with enzymes comprising a homocitrate synthase classified under EC 2.3.3.14 or EC 2.3.3.13, a homocitrate dehydratase and a homoaconitate hydratase classified under EC 4.2.1.114, EC 4.2.1.36 or EC 4.2.1.33 an iso(homo) n citrate dehydrogenase classified under EC 1.1.1.85, EC 1.1.1.87, or EC 1.1.1.286, an indolepyruvate decarboxylase classified under EC 4.1.1.43 or EC 4.1.1.74, a glutarate semialdehyde dehydrogenase classified under EC 1.2.1.20, 1.2.1.16, or EC 1.2.1.79, and a glutarate:CoA ligase classified under EC 6.2.1.6 to produce glutaryl-CoA, or
contacting acetyl-CoA with enzymes comprising a β-ketothiolase classified under EC 2.3.1.9, an acetyl-CoA carboxylase classified under EC 6.4.1.2, an acetoacetyl-CoA synthase classified under EC 2.3.1.194, a 3-hydroxyacyl-CoA dehydrogenase classified under EC 1.1.1.-, an enoyl-CoA reductase classified under 4.2.1.-, a glutaconyl-CoA decarboxylase classified under EC 4.1.1.70, and an enoyl-[acp] reductase classified under EC 1.3.1.9 or a trans-2-enoyl-CoA reductase classified under EC 1.3.1.44 or a glutaryl-CoA dehydrogenase to produce glutaryl-CoA;
(ii) contacting said glutaryl-CoA with enzymes comprising a β-ketothiolase classified under EC 2.3.1.- or a β-ketoacyl-[acp] synthase classified under EC 2.3.1.41, EC 2.3.1.179, EC 2.3.1.180, a 3-hydroxyadipyl-CoA dehydrogenase classified under EC 1.1.1.157, an enoyl-CoA hydratase, and an enoyl-[acp] reductase classified under EC 1.3.1.9 or a trans-2-enoyl-CoA reductase classified under EC 1.3.1.44 to produce pimeloyl-CoA, or
contacting said glutaryl-CoA with enzymes comprising a β-ketothiolase classified under EC 2.3.1.- or a β-ketoacyl-[acp] synthase classified under EC 2.3.1.41, EC 2.3.1.179, EC 2.3.1.180, a 6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase classified under EC 3.7.1.-, a 6-hydroxycyclohex-1-ene-1-carboxyl-CoA dehydrogenase classified under EC 1.1.1.-, a cyclohexa-1,5-dienecarbonyl-CoA hydratase classified under EC 4.2.1.100, a cyclohex-1-ene-1-carboxyl-CoA dehydrogenase classified under EC 1.3.8.-, a cyclohex-1-ene-1-carboxyl-CoA hydratase classified under EC 4.2.1-, a 2-hydroxycyclohexanecarboxyl-CoA dehydrogenase classified under EC 1.1.1.-, and a 2-ketocyclohexanecarboxyl-CoA hydrolase classified under EC 4.1.3.36 to produce pimeloyl-CoA;
(iii) contacting said pimeloyl-CoA with enzymes comprising a thioesterase classified under EC 3.1.2., an aldehyde dehydrogenase classified under EC 1.2.1.3, a 6-oxohexanoate dehydrogenase classified under EC 1.2.1.63, a 7-oxoheptanoate dehydrogenase classified under EC 1.2.1.-, a glutaconate-CoA transferase classified under EC 2.8.3.12, or a reversible CoA-ligase classified under EC 6.2.1.5 to produce pimelic acid or 7-hydroxyheptanoate, and/or contacting said pimeloyl-CoA with enzymes comprising an ω-transaminase classified under EC 2.6.1.- or a deacetylase classified under 3.5.1.62 to produce 7-aminoheptanoate or heptamethylenediamine; and
(iv) optionally, contacting said 7-hydroxyheptanoate from step (iii) with an alcohol dehydrogenase classified under EC 1.1.1.- to produce 1,7-heptanediol;
wherein at least one of said enzymes is a heterologous enzyme.

2. The method of claim 1 , wherein said thioesterase comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 and wherein said ω-transaminase comprises an amino acid sequence that is at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 8-13.

3. The method of claim 1 , further comprising contacting said pimelic acid with a carboxylate reductase to form a terminal aldehyde group as an intermediate in forming said 7-aminoheptanoate.

4. The method of claim 3 , wherein said carboxylate reductase comprises an amino acid sequence that is at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 2-7.

5. The method of claim 1 , wherein said method is performed in a recombinant host cell by fermentation of said recombinant host cell.

6. The method of claim 5 , wherein said fermentation comprises a principal carbon source obtained from biological or non-biological feedstocks.

7. The method of claim 6 , wherein said biological feedstock is, or obtained 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 said non-biological feedstock is, or obtained from, natural gas, syngas, CO 2 /H 2 , methanol, ethanol, benzoate, or terephthalic acid/isophthalic acid mixture waste streams.

8. The method of claim 5 , wherein said recombinant host cell is a prokaryote.

9. The method of claim 8 , wherein said prokaryote is a genus selected from the group consisting of Escherichia, Clostridia, Corynebacteria Cupriavidus, Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus , and Rhodococcus.

10. The method of claim 5 , wherein said recombinant host cell is a eukaryote.

11. The method of claim 10 , wherein said eukaryote is a genus selected from the group consisting of Aspergillus Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula , and Kluyveromyces.

12. The method of claim 5 , wherein said recombinant host cell comprises one or more of the following attenuated enzymes: a lactate dehydrogenase, a menaquinol-fumarate oxidoreductase, an alcohol dehydrogenase producing ethanol, a pyruvate decarboxylase, a 2-oxoacid decarboxylase generating isobutanol, a formate dehydrogenase, a polymer synthase, a NADPH-specific L-glutamate dehydrogenase, a NADP H-consuming transhydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase that degrades C7 building blocks and their precursors; a glutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase; and/or wherein said recombinant host cell overexpresses one or more genes encoding: a PEP carboxykinase, a PEP carboxylase, a pyruvate carboxylase, a PEP synthase, a L-alanine dehydrogenase; a NADH-specific L-glutamate dehydrogenase; a L-glutamine synthetase; a diamine transporter; a dicarboxylate transporter; and/or a multidrug transporter.

13. The method of claim 8 , wherein said prokaryote is selected from the group consisting of Escherichia coli, Clostridium ljungdhalii, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans, Pseudomaonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans, Bacillus subtilis, Lactobacillus delbrueckii, Lactobacillus lactis , and Rhodococcus equi.

14. The method of claim 10 , wherein said eukaryote is selected from the group consisting of Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans , and Kluyveromyces lactis.

Assignees

• Invista North America S Á R L
• Invista North America Sarl

Inventors

• Botes Adriana Leonora
• Conradie Alex Van Eck
• Chen Changlin
• Pearlman Paul S

Classifications

• C12P13/001 Primary

  Amines; Imines · CPC title

• C12P13/002

  Nitriles (-CN) · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12Y301/02002

  Palmitoyl-CoA hydrolase (3.1.2.2) · CPC title

• C12P7/18

  polyhydric · CPC title