Methods of producing 6-carbon chemicals via methyl-ester shielded carbon chain elongation

Patent metadata

Field	Value
Publication number	US-9580733-B2
Application number	US-201314139225-A
Country	US
Kind code	B2
Filing date	Dec 23, 2013
Priority date	Dec 31, 2012
Publication date	Feb 28, 2017
Grant date	Feb 28, 2017

Abstract

This document describes biochemical pathways for producing adipic acid, 6-aminohexanoic acid, 6-hydroxhexanoic acid, hexamethylenediamine, caprolactam, or 1,6-hexanediol by forming one or two terminal functional groups, comprised of carboxyl, amine or hydroxyl group, in a C6 aliphatic backbone substrate. These pathways, metabolic engineering and cultivation strategies described herein rely on the enzymes or homologs accepting methyl ester shielded dicarboxylic acid substrates.

First claim

What is claimed is:

1. A method for biosynthesizing a product selected from the group consisting of adipic acid, 6-aminohexanoate, 6-hydroxhexanoate, hexamethylenediamine, caprolactam and 1,6-hexanediol, said method comprising, in a recombinant host, enzymatically synthesizing a six carbon chain aliphatic backbone from oxalyl-CoA and either (i) acetyl-CoA or 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, wherein:
a polypeptide having the activity of a malonyl-[acp] O-methyltransferase converts oxalyl-CoA to oxalyl-CoA methyl ester, and
each of said two cycles of carbon chain elongation comprises using a polypeptide having the activity of (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 adipyl-CoA methyl ester or adipyl-[acp] methyl ester.

2. The method of claim 1 , wherein the six carbon chain aliphatic backbone is adipyl-[acp] or adipyl-CoA.

3. The method of claim 1 , wherein a polypeptide having the activity of a pimeloyl-[acp] methyl ester methylesterase removes the methyl group from adipyl-CoA methyl ester or adipyl-[acp] methyl ester.

4. The method of claim 1 , wherein the polypeptide having the activity of a malonyl-[acp] O-methyltransferase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16.

5. The method of claim 3 , wherein the polypeptide having the activity of a pimeloyl-[acp] methyl ester methylesterase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

6. The method of claim 1 , wherein said two terminal functional groups are the same.

7. The method of claim 6 , wherein said two terminal functional groups are amine or said two terminal functional groups are hydroxyl groups.

8. The method of claim 1 , wherein a polypeptide having the activity of a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydratase, or an alcohol dehydrogenase enzymatically forms a hydroxyl group; a polypeptide having the activity of 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; or a polypeptide having the activity of a ω-transaminase or a deacetylase enzymatically forms an amine group.

9. The method of claim 8 , 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; or said polypeptide having the activity of ω-transaminase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 8-13.

10. The method of claim 1 , wherein a polypeptide having the activity of a carboxylate reductase, enhanced by a polypeptide having the activity of a phosphopantetheinyl transferase, forms a terminal aldehyde group as an intermediate in forming the product.

11. The method of claim 10 , 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. 3-7.

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

13. The method of claim 12 , wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks.

14. The method of claim 13 , 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.

15. The method of claim 12 , wherein the host is a prokaryote.

16. The method of claim 15 , 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.

17. The method of claim 12 , wherein the host is a eukaryote.

18. The method of claim 17 , 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.

19. The method of claim 12 , wherein said host comprises one or more polypeptides having the activity of the following attenuated enzymes: polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, 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, a transhydrogenase dissipating the NADH or NADPH imbalance, an glutamate dehydrogenase dissipating the NADH or NADPH imbalance, a NADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase accepting C6 building blocks and central precursors as substrates; a glutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase; and/or
wherein said host overexpresses one or more genes encoding a polypeptide having the activity of: an oxaloacetase, a PEP carboxylase, a PEP carboxykinase, a pyruvate carboxylase, a PEP synthase, an acetyl-CoA synthetase, a 6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a formate dehydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase; a fructose 1,6 diphosphatase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase specific to the NADH or NADPH used to generate a co-factor imbalance; a methanol dehydrogenase; a formaldehyde dehydrogenase; a diamine transporter, a dicarboxylate transporter, S-adenosylmethionine synthetase; and/or a multidrug transporter.

Assignees

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

Inventors

• Botes Adriana Leonora
• Conradie Alex Van Eck

Classifications

• C12P13/005 Primary

  Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids · CPC title

• C12Y206/01

  Transaminases (2.6.1) · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12P13/02 Primary

  Amides, e.g. chloramphenicol {or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes (peptides C12P21/00 or C07K)} · CPC title

• C12N9/86

  acting on amide bonds in cyclic amides, e.g. penicillinase {(3.5.2)} · CPC title