Methods, reagents and cells for biosynthesizing compounds

Patent metadata

Field	Value
Publication number	US-9896702-B2
Application number	US-201514740554-A
Country	US
Kind code	B2
Filing date	Jun 16, 2015
Priority date	Jun 16, 2014
Publication date	Feb 20, 2018
Grant date	Feb 20, 2018

Abstract

This document describes biochemical pathways for producing 2(E)-heptenedioyl-CoA methyl ester from precursors such as 2-oxo-glutarate, acetyl-CoA, or succinyl-CoA using one or more of a fatty acid O-methyltransferase, a thioesterase, a CoA-transferase, a CoA ligase, as well as recombinant hosts expressing one or more of such enzymes. 2(E)-heptenedioyl-CoA methyl ester can be enzymatically converted to pimeloyl-CoA using a trans-2-enoyl-CoA reductase, and a methylesterase. Pimeloyl-CoA can be enzymatically converted to pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, or 1,7-heptanediol.

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 using at least one exogenous polypeptide expressed by said host, said method comprising:
enzymatically converting a n-carboxy-2-enoic acid to a n-carboxy-2-enoate methyl ester in said host by contacting said n-carboxy-2-enoic acid with a polypeptide having fatty acid O-methyltransferase activity, wherein n+1 reflects the length of the carbon chain aliphatic backbone and the polypeptide having fatty acid O-methyltransferase activity has at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 1-3.

2. A method of producing 2(E)-heptenedioyl-CoA methyl ester in a recombinant host using at least one exogenous polypeptide expressed by said host, said method comprising:
enzymatically converting 2(E)-heptenedioate to 2(E)-heptenedioate methyl ester by contacting 2(E)-heptenedioate with a polypeptide having fatty acid O-methyltransferase activity, wherein said polypeptide having fatty acid O-methyltransferase activity has at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 1-3;
enzymatically converting 2(E)-heptenedioate methyl ester to 2(E)-heptenedioyl-CoA methyl ester by contacting 2(E)-heptenedioate methyl ester with a polypeptide having the activity of a CoA ligase, wherein said polvpeptide having the activity of a CoA ligase has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23;
enzymatically converting 2(E)-heptenedioyl-CoA methyl ester to pimeloyl-CoA methyl ester by contacting 2(E)-heptenedioyl-CoA methyl ester with a polypeptide having the activity of a trans-2-enoyl-CoA reductase, wherein said polvpeptide having the activity of a trans-2-enoyl-CoA reductase has at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27; and
enzymatically converting pimeloyl-CoA methyl ester to pimeloyl-CoA by contacting pimeloyl-CoA methyl ester with a polypeptide having the activity of a pimelyl-[acp]methyl ester esterase, wherein 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.

3. The method of claim 2 , further comprising enzymatically converting pimeloyl-CoA to a product selected from pimelic acid, pimelate semialdehyde, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, and 1.7-heptanediol, wherein pimeloyl-CoA is converted to said product using at least one exogenous polypeptide expressed by said host, wherein said exogenous polypeptide has thioesterase, reversible CoA-ligase, glutaconate CoA-transferase, ω-transaminase, 6-hydroxyhexanoate dehydrogenase, 5-hydroxypentanoate dehydrogenase, 4-hydroxybutyrate dehydrogenase, alcohol dehydrogenase, carboxylate reductase, or phosphopantetheine transferase enhancer activity, wherein:
said polypeptide having the activity of a thioesterase has at least 85% sequence similarity to the amino acid sequence set forth in any one of SEQ ID NOs: 4, 5, or 21;
said polypeptide having the activity of a reversible CoA-ligase is classified under EC 6.2.1.5;
said polypeptide having the activity of a glutaconate CoA-transferase has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24 and SEQ ID NO: 25;
said polypeptide having the activity of a ω-transaminase has at least 85% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 13-18;
said polypeptide having the activity of a 6-hydroxyhexanoate dehydrogenase has at least 85% sequence identity to the amino acid sequence of a gene product of chnD;
said polypeptide having the activity of a 5-hydroxypentanoate dehydrogenase has at least 85% sequence identity to the amino acid sequence of a gene product of cpnD;
said polypeptide having the activity of a 4-hydroxybutyrate dehydrogenase has at least 85% sequence identity to the amino acid sequence of a gene product of gbd;
said polypeptide having the activity of an alcohol dehydrogenase has at least 85% sequence identity to the amino acid seauence of a gene product of any one of hadH, IdhA, yghD, or YMR318C or the amino acid sequence of the protein having GenBank Accession No. CAA81612.1;
said polypeptide having the activity of a carboxylate reductase has at least 85% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 7-12; and
said polypeptide having the activity of a phosphopantetheine transferase enhancer has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 20 or at least 85% sequence identity to the amino acid sequence of the gene products of griC and griD.

4. The method of claim 3 , wherein said product is pimelate semialdehyde and said method comprises:
contacting pimeloyl-CoA with a polypeptide having acetylating aldehyde dehydrogenase activity, wherein said polypeptide having acetylating aldehyde dehydrogenase activity has at least 85% seauence identity to the amino acid sequence of the gene product of pduB or pduP; or
contacting pimeloyl-CoA with a polypeptide having thioesterase, reversible CoA-ligase, or glutaconate CoA-transferase activity to produce pimelic acid and contacting pimelic acid with a polypeptide having carboxylate reductase activity optionally in combination with a polypeptide having the activity of a phosphopantetheine transferase enhancer to produce pimelate semialdehyde, wherein
said polypeptide having thioesterase activity has at least 85% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 4, 5, or 21;
said polypeptide having reversible CoA-ligase activity is classified under EC 6.2.1.5;
said polypeptide having glutaconate CoA-transferase activity has at least 85% sequence identity to SEQ ID NOs: 24 and 25;
said polypeptide having the activity of a carboxylate reductase has at least 85% seauence identity to the amino acid seauence set forth in any one of SEQ ID NOs: 7-12; and
said polypeptide having the activity of a phosphopantetheine transferase enhancer has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 20 or at least 85% sequence identity to the amino acid sequence of the gene products of griC and griD.

5. The method of claim 4 , said method further comprising
enzymatically converting pimelate semialdehyde to 7-aminoheptanoate by contacting pimelate semialdehyde with a polypeptide having ω-transaminase activity, wherein said polypeptide having ω-transaminase activity has at least 85% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 13-18.

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

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

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

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

10. The method of claim 6 , wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks, 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

Assignees

• Invista North America Sarl
• Invista North America Sarl

Inventors

• Conradie Alex Van Eck
• Botes Adriana Leonora

Classifications

• C12N9/1029

  transferring groups other than amino-acyl groups (2.3.1) · CPC title

• C07C69/533

  Monocarboxylic acid esters having only one carbon-to-carbon double bond · CPC title

• C12P7/24

  containing a carbonyl group · CPC title

• C12Y206/01018

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

• C12Y206/01029

  Diamine transaminase (2.6.1.29) · CPC title