Methods, reagents and cells for biosynthesizing compounds

Patent metadata

Field	Value
Publication number	US-9816117-B2
Application number	US-201514741228-A
Country	US
Kind code	B2
Filing date	Jun 16, 2015
Priority date	Jun 16, 2014
Publication date	Nov 14, 2017
Grant date	Nov 14, 2017

Abstract

This document describes biochemical pathways for producing 6-hydroxyhexanoate methyl ester and hexanoic acid hexyl ester using one or more of a fatty acid O-methyltransferase, an alcohol O-acetyltransferase and a monooxygenase, as well as recombinant hosts expressing one or more of such enzymes. 6-hydroxyhexanoate methyl esters and hexanoic acid hexyl ester can be enzymatically converted to adipic acid, adipate semialdehyde, 6-aminohexanoate, 6-hydroxyhexanoate, hexamethylenediamine, and 1,6-hexanediol.

First claim

What is claimed is:

1. A method of producing a terminal hydroxyl (C 3-8 hydroxyalkyl)-C(═O)OCH 3 ester in a recombinant host, said method comprising:
a) enzymatically converting a C 4-9 carboxylic acid to a (C 3-8 alkyl)-C(═O)OCH 3 ester using a polypeptide having fatty acid O-methyltransferase activity classified under EC 2.1.1.15; and
b) enzymatically converting the (C 3-8 alkyl)-C(═O)OCH 3 ester to a terminal hydroxyl (C 3-8 hydroxyalkyl)-C(═O)OCH 3 ester using a polypeptide having monooxygenase activity classified under EC 1.14.14.- or EC 1.14.15.-,
wherein the C 4-9 carboxylic acid is enzymatically produced from a C 4-9 alkanoyl-CoA using:
a polypeptide having thioesterase activity classified under EC 3.1.2.-; or
a polypeptide having butanal dehydrogenase activity classified under EC 1.2.1.10 or EC 1.2.1.57 and a polypeptide having aldehyde dehydrogenase activity classified under EC 1.2.1.3 or EC 1.2.1.4.

2. The method of claim 1 , said method further comprising enzymatically converting the terminal hydroxyl (C 3-8 hydroxyalkyl)-C(═O)OCH 3 ester to a terminal hydroxyl C 4-9 hydroxyalkanoate using a polypeptide having demethylase activity classified under EC 2.1.1.- and/or a polypeptide having esterase activity classified under EC 3.1.1.-.

3. The method of claim 1 , wherein C 4-9 carboxylic acid is hexanoate, and is enzymatically converted to hexanoate methyl ester; and the hexanoate methyl ester is enzymatically converted to 6-hydroxyhexanoate methyl ester.

4. The method of claim 1 , wherein the C 4-9 alkanoyl-CoA is hexanoyl-CoA, and hexanoate is enzymatically produced from hexanoyl-CoA.

5. The method of claim 3 , said method further comprising enzymatically converting 6-hydroxyhexanoate methyl ester to 6-hydroxyhexanoate using a polypeptide having demethylase activity classified under EC 2.1.1.- or a polypeptide having esterase activity classified under EC 3.1.1.-.

6. The method of claim 4 , wherein hexanoyl-CoA is produced from acetyl-CoA via two cycles of CoA-dependent carbon chain elongation, wherein each of said two cycles of CoA-dependent carbon chain elongation comprises using (i) a polypeptide having β-ketothiolase activity classified under EC 2.3.1.9, EC 2.3.1.16, or EC 2.3.1.174 or a polypeptide having acetyl-CoA carboxylase activity classified under EC 6.4.1.2 and a polypeptide having acetoacetyl-CoA synthase activity classified under EC 2.3.1.194, (ii) a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity classified under EC 1.1.1.35, EC 1.1.1.36, or EC 1.1.1.157 or a polypeptide having 3-oxoacyl-CoA reductase activity classified under EC 1.1.1.100, (iv) a polypeptide having enoyl-CoA hydratase activity classified under EC 4.2.1.17 or EC 4.2.1.119, and (v) a polypeptide having trans-2-enoyl-CoA reductase activity classified under EC 1.3.1.8, EC 1.3.1.38, or EC 1.3.1.44 to form hexanoyl-CoA from acetyl-CoA.

7. A method of enzymatically producing 6-hydroxyhexanoate, the method comprising:
a) enzymatically converting hexanoate to hexanoate methyl ester using a polypeptide having fatty acid O-methyltransferase activity classified under EC 2.1.1.15;
b) enzymatically converting hexanoate methyl ester to 6-hydroxyhexanoate methyl ester using a polypeptide having monooxygenase activity classified under EC 1.14.14.- or EC 1.14.15.-; and
c) enzymatically converting 6-hydroxyhexanoate methyl ester to 6-hydroxyhexanoate using a polypeptide having demethylase activity classified under EC 2.1.1.- or a polypeptide having esterase activity classified under EC 3.1.1.-.

8. The method of claim 1 , wherein said recombinant host is subjected to a cultivation strategy under aerobic, anaerobic or, micro-aerobic cultivation conditions.

9. The method of claim 8 , wherein said recombinant host is cultured under conditions of nutrient limitation.

10. The method of claim 8 , wherein said recombinant host is retained using a ceramic membrane to maintain a high cell density during fermentation.

11. The method of claim 8 , wherein the principal carbon source fed to the recombinant host derives from a biological feedstock.

12. The method of claim 11 , 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.

13. The method of claim 8 , wherein the principal carbon source fed to the recombinant host derives from a non-biological feedstock.

14. The method of claim 13 , 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 9 , wherein the recombinant host is a prokaryote selected from Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus , and Rhodococcus , or a eukaryote selected from Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula , and Kluveromyces.

16. The method of claim 3 , wherein the polypeptide having fatty acid O-methyltransferase activity classified under EC 2.1.1.15 has at least 85% sequence identity to an amino acid sequence set forth in SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24; and the polypeptide having monooxygenase activity classified under EC 1.14.14.- or EC 1.14.15.- has at least 85% sequence identity to an amino acid sequence set forth in SEQ ID NO: 13-15, SEQ ID NO:27 or SEQ ID NO:28.

17. The method of claim 4 , wherein the polypeptide having thioesterase activity classified under EC 3.1.2.- has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:32, or SEQ ID NO:33.

18. The method of claim 5 , wherein the polypeptide having demethylase activity classified under EC 2.1.1.- has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 and the polypeptide having esterase activity classified under EC 3.1.1.- has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26.

19. The method of claim 8 , wherein said recombinant host is cultured under conditions of phosphate, nitrogen, or oxygen limitation.

Assignees

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

Inventors

• Conradie Alex Van Eck
• Botes Adriana Leonora
• Haddouche Ramdane

Classifications

• C12P7/6409

  Fatty acids · CPC title

• C12Y206/01048

  5-Aminovalerate transaminase (2.6.1.48) · CPC title

• C12N9/1096 Primary

  transferring nitrogenous groups (2.6) · CPC title

• C12Y206/01018

  Beta-alanine-pyruvate transaminase (2.6.1.18) · CPC title

• C12Y102/99006

  Carboxylate reductase (1.2.99.6) · CPC title