Methods of producing 7-carbon chemicals via pyruvate and succinate semialdehyde aldol condensation

Patent metadata

Field	Value
Publication number	US-9738911-B2
Application number	US-201314138992-A
Country	US
Kind code	B2
Filing date	Dec 23, 2013
Priority date	Dec 31, 2012
Publication date	Aug 22, 2017
Grant date	Aug 22, 2017

Abstract

This document describes biochemical pathways for producing one or more of pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine and 1,7-heptanediol by forming one or two terminal functional groups, comprised of carboxyl, amine or hydroxyl groups, in a C7 aliphatic backbone substrate produced from succinate semialdehyde or pyruvate. These pathways, metabolic engineering and cultivation strategies described herein rely on the aldol condensation of succinate semialdehyde and pyruvate.

First claim

What is claimed is:

1. A method for biosynthesizing 7-aminoheptanoate in vitro or in a recombinant host cell, said method comprising enzymatically synthesizing a seven carbon chain aliphatic backbone from succinate semialdehyde and pyruvate via aldol condensation, wherein the aldol condensation comprises an aldol reaction catalyzed by a 4-hydroxy-2-oxopimelate aldolase classified under EC 4.1.2.52 and a dehydration catalyzed by a 2-hydroxyhepta-2,4-dienedioate hydratase, and enzymatically forming an amine terminal group in said backbone using a ω-transaminase classified under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82, thereby forming the product 7-aminoheptanoate, wherein the host cell is a bacterium, fungus, or yeast.

2. The method of claim 1 , wherein the seven carbon chain aliphatic backbone is pimeloyl-CoA or pimelate semialdehyde.

3. The method of claim 1 , wherein the product of the aldol reaction, 2,4-dihydroxyhept-2-enedioate or its tautomer 4-hydroxy-2-oxo-pimelate (2,4-dihydroxyhept-2-enedioate/4-hydroxy-2-oxo-pimelate), is converted to a) pimeloyl-CoA using a 2-hydroxyhepta-2,4-dienedioate hydratase and one or more of (i) an enoate reductase classified under EC 1.3.1.31, (ii) a 2-hydroxyglutarate dehydrogenase classified under EC 1.1.1.-, (iii) a glutaconate CoA-transferase classified under EC 2.8.3.12, (iv) a 2-hydroxyglutaryl-CoA dehydratase, or (v) an enoyl-CoA reductase or an enoyl-[acp] reductase classified under EC 1.3.1.-; or b) pimelate semialdehyde using a 2-hydroxyhepta-2,4-dienedioate hydratase and one or more of (vi) an enoate reductase classified under EC 1.3.1.31, (vii) a 2-hydroxyglutarate dehydrogenase classified under EC 1.1.1.-, (viii) a glutaconate CoA-transferase classified under EC 2.8.3.12, (ix) a 2-hydroxyglutaryl-CoA dehydratase, or (x) a carboxylate reductase classified under EC 1.2.99.6.

4. The method of claim 3 , wherein said enoate reductase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16 or SEQ ID NO:17.

5. The method of claim 1 , wherein said ω-transaminase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO. 8-13.

6. The method of claim 3 , wherein said carboxylate reductase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO. 2-7.

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

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

9. The method of claim 8 , 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.

10. The method of claim 8 , 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.

11. The method of claim 7 , wherein the host is a bacterium.

12. The method of claim 11 , wherein said bacterium is from a genus selected from Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus, and Rhodococcus.

13. The method of claim 7 , wherein the host is a fungus or yeast.

14. The method of claim 13 , wherein said fungus or yeast is from a genus selected from Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula, and Kluyveromyces.

15. The method of claim 7 , wherein the host's tolerance to high concentrations of a C7 building block is improved through continuous cultivation in a selective environment.

16. A method for biosynthesizing 7-aminoheptanoate in vitro or in a recombinant host cell, said method comprising enzymatically synthesizing a seven carbon chain aliphatic backbone from succinate semialdehyde and pyruvate via aldol condensation, wherein the aldol condensation comprises an aldol reaction catalyzed by a polypeptide having the activity of a 4-hydroxy-2-oxopimelate aldolase classified under EC 4.1.2.52 and a dehydration catalyzed by a polypeptide having the activity of a 2-hydroxyhepta-2,4-dienedioate hydratase, and enzymatically forming an amine terminal group in said backbone using a polypeptide having the activity of a ω-transaminase classified under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82, thereby forming the product 7-aminoheptanoate, wherein the host cell is a bacterium, fungus, or yeast.

17. The method of claim 16 , wherein the seven carbon chain aliphatic backbone is pimeloyl-CoA or pimelate semialdehyde.

18. The method of claim 16 , wherein the product of the aldol reaction, 2,4-dihydroxyhept-2-enedioate or its tautomer 4-hydroxy-2-oxo-pimelate (2,4-dihydroxyhept-2-enedioate/4-hydroxy-2-oxo-pimelate), is converted to a) pimeloyl-CoA using a polypeptide having the activity of 2-hydroxyhepta-2, 4-dienedioate hydratase and one or more of (i) a polypeptide having the activity of an enoate reductase classified under EC 1.3.1.31, (ii) a polypeptide having the activity of a 2-hydroxyglutarate dehydrogenase classified under EC 1.1.1.-, (iii) a polypeptide having the activity of a glutaconate CoA-transferase classified under EC 2.8.3.12, (iv) a polypeptide having the activity of a 2-hydroxyglutaryl-CoA dehydratase, or (v) a polypeptide having the activity of an enoyl-CoA reductase or an enoyl-[acp] reductase classified under EC 1.3.1.-; or b) pimelate semialdehyde using a polypeptide having the activity of a 2-hydroxyhepta-2,4-dienedioate hydratase and one or more of (vi) a polypeptide having the activity of an enoate reductase classified under EC 1.3.1.31, (vii) a polypeptide having the activity of a 2-hydroxyglutarate dehydrogenase classified under EC 1.1.1.-, (viii) a polypeptide having the activity of a glutaconate CoA-transferase classified under EC 2.8.3.12, (ix) a polypeptide having the activity of a 2-hydroxyglutaryl-CoA dehydratase, or (x) a polypeptide having the activity of a carboxylate reductase classified under EC 1.2.99.6.

19. The method of claim 18 , wherein said polypeptide having the activity of an enoate reductase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16 or SEQ ID NO:17, or 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 NO. 2-7.

20. The method of claim 12 , wherein said bacterium is Escherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans, Bacillus subtillis, Lactobacillus delbrueckii, Lactococcus lactis , or Rhodococcus equi.

21. The method of claim 14 , wherein said fungus or yeast is Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans , or Kluyveromyces lactis.

22. The method of claim 1 , wherein said ω-transaminase has at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO. 8-13.

Assignees

• Invista North America Sarl
• Invista North America Sarl

Inventors

• Conradie Alex Van Eck
• Botes Adriana Leonora
• Chen Changlin
• Pearlman Paul S

Classifications

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12P7/24

  containing a carbonyl group · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12P7/42

  Hydroxy-carboxylic acids · CPC title

• C12P13/001

  Amines; Imines · CPC title