Methods of producing 6-carbon chemicals using 2,6-diaminopimelate as precursor to 2-aminopimelate

Patent metadata

Field	Value
Publication number	US-9745607-B2
Application number	US-201514714164-A
Country	US
Kind code	B2
Filing date	May 15, 2015
Priority date	May 15, 2014
Publication date	Aug 29, 2017
Grant date	Aug 29, 2017

Abstract

This document describes biochemical pathways for producing 2-aminopimelate from 2,6-diaminopimelate, and methods for converting 2-aminopimelate to one or more of adipic acid, adipate semialdehyde, caprolactam, 6-aminohexanoic acid, 6-hexanoic acid, hexamethylenediamine, or 1,6-hexanediol by decarboxylating 2-aminopimelate into a six carbon chain aliphatic backbone and enzymatically forming one or two terminal functional groups, comprised of carboxyl, amine or hydroxyl group, in the backbone.

First claim

What is claimed is:

1. A method of biosynthesizing 2-aminopimelate in a recombinant host, the method comprising enzymatically converting 2,6-diaminopimelate to 2-aminopimelate using at least one polypeptide having an activity selected from 2-hydroxyacyl-CoA dehydratase activity, mutase activity, ammonia lyase activity, and enoate reductase activity, wherein said polypeptide having enoate reductase activity has at least 85% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 16-22, wherein said polypeptide having 2-hydroxyacyl-CoA dehydratase activity has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 28, wherein said polypeptide having mutase activity has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26, and wherein said polypeptide having ammonia lyase activity has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, the method optionally further comprising using at least one polypeptide having an activity selected from diaminopimelate dehydrogenase activity, 2-hydroxycarboxylate dehydrogenase activity, CoA-transferase activity, and carboxylate reductase activity to enzymatically convert 2,6-diaminopimelate to 2-aminopimelate, wherein said polypeptide having diaminopimelate dehydrogenase activity is classified under EC 1.4.1.16, wherein said polypeptide having 2-hydroxycarboxylate dehydrogenase activity is classified under EC 1.1.1.337, wherein said polypeptide having CoA-transferase activity is classified under EC 2.8.3, and wherein said polypeptide having carboxylate reductase activity has at least 85% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 3-7.

2. The method of claim 1 , wherein (S) 2-aminopimelate is biosynthesized.

3. The method of claim 1 , said method comprising:
using said polypeptide having 2-hydroxyacyl-CoA dehydratase activity and said polypeptide having enoate reductase activity to enzymatically convert 2,6-diaminopimelate to 2-aminopimelate; or
using said polypeptide having mutase activity, said polypeptide having ammonia lyase activity, and said polypeptide having enoate reductase activity to enzymatically convert 2,6-diaminopimelate to 2-aminopimelate.

4. The method of claim 1 , wherein (R) 2-aminopimelate is biosynthesized.

5. The method of claim 1 , said method further comprising using at least one polypeptide having an activity selected from CoA ligase activity, CoA-transferase activity, carboxylate reductase activity, and aldehyde dehydrogenase activity to enzymatically convert 2,6-diaminopimelate to 2-aminopimelate, wherein said polypeptide having CoA ligase activity is classified under EC 6.2.1.5, wherein said polypeptide having CoA-transferase activity is classified under EC 2.8.3, wherein said polypeptide having carboxylate reductase activity has at least 85% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 3-7, and wherein said aldehyde dehydrogenase is classified under EC 1.2.1.-.

6. The method of claim 1 , wherein the host is subjected to a cultivation strategy under aerobic or micro-aerobic cultivation conditions.

7. The method of claim 1 , wherein the host is cultured under conditions of nitrogen, phosphate or oxygen limitation.

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

9. The method of claim 1 , further comprising a principal carbon source fed to the fermentation derived from a biological feedstock.

10. The method of claim 9 , wherein the biological feedstock is or derives from monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid and formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste.

11. The method of claim 1 , further comprising a principal carbon source fed to the fermentation derived from a non-biological feedstock.

12. The method of claim 11 , wherein the non-biological feedstock is, or derives from, natural gas, syngas, CO 2 /H 2 , methanol, ethanol, benzoate, non-volatile residue (NVR) or a caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.

13. The method of claim 1 , wherein the 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 Kluyveromyces.

14. The method of claim 1 , wherein the host exhibits tolerance to high concentrations of a C6 building block, and wherein the tolerance to high concentrations of a C6 building block is improved through continuous cultivation in a selective environment.

15. The method of claim 1 , wherein the host comprises one or more of the following: the intracellular concentration of oxaloacetate for biosynthesis of a C6 building block is increased in the host by overexpressing recombinant genes forming oxaloacetate; wherein an imbalance in NADPH is generated that can be balanced via the formation of a C6 building block; wherein an exogenous lysine biosynthesis pathway synthesizing lysine from 2-oxoglutarate via 2-oxoadipate is introduced in a host using the meso 2,6 diaminopimelate pathway for lysine synthesis; wherein an exogenous lysine biosynthesis pathway synthesizing lysine from oxaloacetate to meso 2,6 diaminopimelate is introduced in a host using the 2-oxoadipate pathway for lysine synthesis; wherein endogenous degradation pathways of central metabolites and central precursors leading to and including C6 building blocks are attenuated in the host; or wherein the efflux of a C6 building block across the cell membrane to the extracellular media is enhanced or amplified by genetically engineering structural modifications to the cell membrane or increasing any associated transporter activity for a C6 building block.

16. A recombinant host cell comprising at least one exogenous nucleic acid encoding at least one polypeptide having an activity selected from 2-hydroxyacyl-CoA dehydratase activity, mutase activity, ammonia lyase activity, and enoate reductase activity, wherein said polypeptide having enoate reductase activity has at least 85% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 16-22, said polypeptide having 2-hydroxyacyl-CoA dehydratase activity has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 28, said polypeptide having mutase activity has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26, said polypeptide having ammonia lyase activity has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, said host producing 2-aminopimelate from 2,6-diaminopimelate,
the host optionally further comprising one or more exogenous polypeptides having an activity selected from aldehyde dehydrogenase activity, alcohol dehydrogenase activity, CoA-transferase activity, carboxylate reductase activity, α-aminotransferase activity, thioesterase activity, hydrolase activity, ω-transaminase activity, N-acetyltransferase activity, and deacylase activity, the host producing a product selected from adipic acid, adipate semialdehyde, 6-aminohexanoic acid, 6-hydroxyhexanoic acid, caprolactam, hexamethylenediamine, and 1,6-hexanediol, wherein said polypeptide having aldehyde dehydrogenase activ

Assignees

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

Inventors

• Conradie Alex Van Eck
• Botes Adriana Leonora

Classifications

• C12N9/0008

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

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12Y402/01

  Hydro-lyases (4.2.1) · CPC title

• C12N9/88

  Lyases (4.) · CPC title

• C12P13/04 Primary

  Alpha- or beta- amino acids {(other amino acids C12P13/005)} · CPC title