Methods, reagents and cells for biosynthesizing compounds

Patent metadata

Field	Value
Publication number	US-9988654-B2
Application number	US-201514740749-A
Country	US
Kind code	B2
Filing date	Jun 16, 2015
Priority date	Jun 16, 2014
Publication date	Jun 5, 2018
Grant date	Jun 5, 2018

Abstract

This document describes biochemical pathways for producing 2,3-dehydroadipyl-CoA methyl ester from precursors such as 2-oxoglutarate using one or more of a fatty acid O-methyltransferase, a thioesterase, a CoA-transferase and a CoA ligase, as well as recombinant hosts expressing one or more of such enzymes. 2,3-dehydroadipyl-CoA methyl ester can be enzymatically converted to adipyl-CoA using a trans-2-enoyl-CoA reductase, and a methylesterase, which in turn can be enzymatically converted to adipic acid, 6-aminohexanoate, 6-hydroxyhexanoate, caprolactam, hexamethylenediamine, or 1,6-hexanediol.

First claim

What is claimed is:

1. A method of shielding a carbon chain aliphatic backbone functionalized with terminal carboxyl groups in a recombinant host, said method comprising enzymatically converting 2,3-dehydroadipic acid to 2,3-dehydroadipate methyl ester in said host using a polypeptide having the activity of a fatty acid O-methyltransferase, wherein said polypeptide having the activity of a fatty acid O-methyltransferase has at least 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3.

2. The method of claim 1 , said method further comprising enzymatically converting 2,3-dehydroadipate methyl ester to adipyl-CoA using at least one polypeptide having the activity of a CoA ligase, a trans-2-enoyl-CoA reductase, or a pimelyl-[acp] methyl ester esterase, wherein said polypeptide having the activity of a CoA ligase has at least 85% sequence identity to an amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23, said polypeptide having the activity of a trans-2-enoyl-CoA reductase has at least 85% sequence identity to an amino acid sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 27, and/or said polypeptide having the activity of a pimelyl-[acp] methyl ester esterase has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6,
said method optionally further comprising enzymatically converting adipyl-CoA to adipic acid, 6 aminohexanoate, 6-hydroxyhexanoate, caprolactam, hexamethylenediamine, or 1,6 hexanediol.

3. The method of claim 1 , wherein one or more steps of said method are performed by fermentation.

4. The method of claim 1 , wherein said host is subjected to a cultivation strategy under aerobic, anaerobic, micro-aerobic, or mixed oxygen/denitrification cultivation conditions.

5. The method of claim 1 , wherein said host is cultured under conditions of phosphate, oxygen, and/or nitrogen limitation.

6. The method of claim 1 , wherein said host is retained using a ceramic membrane to maintain a high cell density during fermentation.

7. The method of claim 3 , wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks.

8. The method of claim 7 , 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.

9. The method of claim 7 , 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.

10. The method of claim 1 , wherein said host comprises one or more of the following attenuated enzymes: polyhydroxyalkanoate synthase, acetyl-CoA thioesterase, acetyl-CoA specific β-ketothiolase, phosphotransacetylase forming acetate, acetate kinase, lactate dehydrogenase, menaquinol-fumarate oxidoreductase, alcohol dehydrogenase forming ethanol, triose phosphate isomerase, pyruvate decarboxylase, glucose-6-phosphate isomerase, transhydrogenase dissipating a NADPH imbalance, glutamate dehydrogenase dissipating a NADPH imbalance, NADH/NADPH-utilizing glutamate dehydrogenase, pimeloyl-CoA dehydrogenase, acyl-CoA dehydrogenase accepting C7 building blocks and central precursors as substrates, glutaryl-CoA dehydrogenase, or adipyl-CoA synthetase.

11. The method of claim 1 , wherein said host overexpresses one or more genes encoding at least one polypeptide having acetyl-CoA synthetase, 6-phosphogluconate dehydrogenase, transketolase, puridine nucleotide transhydrogenase, formate dehydrogenase, glyceraldehyde-3P-dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase, fructose 1,6 diphosphatase, L-alanine dehydrogenase, PEP carboxylase, pyruvate carboxylase, PEP carboxykinase, PEP synthase, L-glutamate dehydrogenase specific to the NADPH used to generate a co-factor imbalance, methanol dehydrogenase, formaldehyde dehydrogenase, lysine transporter, dicarboxylate transporter, S-adenosylmethionine synthetase, 3-phosphoglycerate dehydrogenase, 3-phosphoserine aminotransferase, phosphoserine phosphatase, or a multidrug transporter activity.

12. The method of claim 1 , wherein the host is a prokaryote selected from the genus Escherichia , the genus Clostridia , the genus Corynebacteria , the genus Cupriavidus , the genus Pseudomonas , the genus Delftia , the genus Bacillus , the genus Lactobacillus , the genus Lactococcus , and the genus Rhodococcus , or a eukaryote selected from the genus Aspergillus , the genus Saccharomyces , the genus Pichia , the genus Yarrowia , the genus Issatchenkia , the genus Debaryomyces , the genus Arxula , and the genus Kluyyeromyces.

13. A method of producing 2,3-dehydroadipyl-CoA methyl ester in a recombinant host, said method comprising enzymatically converting 2,3-dehydroadipic acid to 2,3-dehydroadipate methyl ester using a polypeptide having fatty acid O-methyltransferase activity, wherein said polypeptide having fatty acid O-methyltransferase has at least 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, and further enzymatically converting 2,3-dehydroadipate methyl ester to 2,3-dehydroadipyl-CoA methyl ester using a polypeptide having the activity of a CoA ligase, wherein said polypeptide having the activity of a CoA ligase has at least 85% sequence identity to SEQ ID NO: 22 or SEQ ID NO: 23,
wherein the 2,3-dehydroadipic acid is enzymatically produced from 2,3-dehydroadipyl-CoA using a polypeptide having thioesterase activity or a protein having CoA-transferase activity, wherein said polypeptide having thioesterase activity has at least 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 4, 5, or 21 or at least 85% sequence identity to an amino acid sequence encoding a gene product of yciA or ACOT13 and said protein having CoA-transferase activity has a first subunit having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24 and a second subunit having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25,
the method optionally further comprising enzymatically converting 2,3-dehydroadipyl methyl ester to adipyl-CoA methyl ester using a polypeptide having trans-2-enoyl-CoA reductase activity, wherein said polypeptide having trans-2-enoyl-CoA reductase activity has at least 85% sequence identity to an amino acid sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 27.

14. The method of claim 13 , said method further comprising enzymatically converting 2,3-dehydroadipyl-CoA methyl ester to adipyl-CoA,
said method comprising enzymatically converting 2,3-dehydroadipyl-CoA methyl ester to adipyl-CoA methyl ester using a polypeptide having trans-2-enoyl-CoA reductase activity, wherein said polypeptide having trans-2-enoyl-CoA reductase activity has at least 85% sequence identity to an amino acid sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 27, and further enzymatically converting adipyl-CoA methyl ester to adipyl-CoA using a polypeptide having pimelyl-[acp] methyl ester esterase activity, wherein said polypeptide having pimelyl-[acp] methyl ester esterase activity has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

15. The method of claim 1 , further comprising enzymatically converting adipyl-CoA to a product selected from adipic acid, 6-aminohexanoate, 6-hydroxyhexanoate, caprolactam,

Assignees

• Invista North America Sarl
• Invista North America Sarl

Inventors

• Conradie Alex Van Eck
• Botes Adriana Leonora

Classifications

• C12Y301/01085

  Pimelyl-[acyl-carrier protein] methyl ester esterase (3.1.1.85) · CPC title

• C12N9/18

  Carboxylic ester hydrolases {(3.1.1)} · CPC title

• C12N9/0008

  acting on the aldehyde or oxo group of donors (1.2) · CPC title

• C12P7/44

  Polycarboxylic acids · CPC title

• C12P7/42

  Hydroxy-carboxylic acids · CPC title