Methods of producing 7-carbon chemicals via CoA-dependent carbon chain elongation associated with carbon storage

Patent metadata

Field	Value
Publication number	US-9790525-B2
Application number	US-201314106033-A
Country	US
Kind code	B2
Filing date	Dec 13, 2013
Priority date	Dec 14, 2012
Publication date	Oct 17, 2017
Grant date	Oct 17, 2017

Abstract

This document describes biochemical pathways for producing pimelic acid, 7-hydroxyheptanoic acid, 7-aminoheptanoic acid, heptamethylenediamine or 1,7-heptanediol by forming two terminal functional groups, comprised of carboxyl, amine or hydroxyl group, in a C7 aliphatic backbone substrate. These pathways, metabolic engineering and cultivation strategies described herein rely on the CoA-dependent elongation enzymes or analog enzymes associated with the carbon storage pathways from polyhydroxyalkanoate accumulating bacteria.

First claim

What is claimed is:

1. A method for biosynthesizing 7-aminoheptanoate in vitro or in a recombinant host, said method comprising:
providing acetyl-CoA and propanoyl-CoA;
enzymatically synthesizing heptanoyl-CoA from acetyl-CoA and propanoyl-CoA via two cycles of CoA-dependent carbon chain elongation;
enzymatically converting heptanoyl-CoA to heptanoate by contacting heptanoyl-CoA with a butanal dehydrogenase classified under EC 1.2.1.57 to form heptanal and contacting heptanal with an aldehyde dehydrogenase classified under EC 1.2.1.4 to form heptanoate, or contacting heptanoyl-CoA with a thioesterase classified under EC 3.1.2.- to form heptanoate;
enzymatically converting heptanoate to 7-hydroxyheptanoate by contacting heptanoate with a cytochrome P450 monooxygenase classified under EC 1.14.15.1 or EC 1.14.15.3 to form 7-hydroxyheptanoate;
enzymatically converting 7-hydroxyheptanoate to pimelate semialdehyde by contacting 7-hydroxyheptanoate with an alcohol dehydrogenase classified under EC 1.1.1.- or a cytochrome P450 monooxygenase classified under EC 1.14.15.1 or EC 1.14,15.3; and
enzymatically converting pimelate semialdehyde to 7-aminoheptanoate by contacting pimelate semialdehyde with a ω-transaminase classified under EC 2.6.1.-.

2. The method of claim 1 , wherein the cytochrome P450 monooxygenase classified under EC 1.14.15.1 or EC 1.14.15.3 has at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 14-16, the thioesterase classified under EC 3.1.2.- has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. and the ω-transaminase classified under EC 2.6.1.- has at least 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 8-13.

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

4. The method of claim 3 , wherein the principal carbon source fed to the fermentation derives from monosaccharides, disaccharides, levulinic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers'solubles, or municipal waste, natural gas, syngas, CO 2 /H 2 , methanol, ethanol, benzoate, non-volatile residue (NVR) from cyclohexane oxidation processes, caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams from terephthalic acid and/or polyethylene terephthalate manufacture.

5. The method of claim 3 , wherein the recombinant host is a prokaryote.

6. The method of claim 5 , wherein said prokaryote is from a genus selected from the genus Escherichia , the genus Clostridia , the genus Corynebacteria , the genus Cupriavidus , the genus Pseudomonas , the genus Delftia acidovorans , the genus Bacillus , the genus Lactobacillus , the genus Lactococcus , and the genus Rhodococcus.

7. The method of claim 3 , wherein the recombinant host is a eukaryote.

8. The method of claim 7 , wherein said eukaryote is selected from the genus Aspergillus , the genus Saccharomyces , the genus Pichia , the genus Yarrowia , the genus Issatchenkiau , the genus Debaryomyces , the genus Arxula , and the genus Kluyveromyces.

9. A recombinant host comprising one or more nucleic acids encoding each of the following enzymes: (i) a β-ketothiolase classified under EC 2.3.1.16; or a β-ketoacyl-[acp] synthase classified under EC 2.3.1.180 and an acetyl-CoA carboxylase classified under EC 6.4.1.2; (ii) a 3-hydroxyacyl-CoA dehydrogenase classified under EC 1.1.1.157, EC 1.1.1.36, or EC 1.1.1.35; or a 3-oxoacyl-CoA reductase classified under EC 1.1.1.100; (iii) an enoyl-CoA hydratase classified under EC 4.2.1.17 or EC 4.2.1.119; and (iv) a trans-2-enoyl-CoA reductase classified under EC 1.3.1.8, EC 1.3.1.38, or EC 1.3.1.44, wherein at least one enzyme is encoded by an exogenous nucleic acid, said host producing heptanoyl-CoA.

10. The recombinant host of claim 9 , said host further comprising one or more polypeptides selected from a thioesterase classified under EC 3.1.2.-, a butanal dehydrogenase classified under EC 1.2.1.57, and an aldehyde dehydrogenase classified under EC 1.2.1.4, said host further producing heptanal or heptanoate.

11. The recombinant host of claim 10 , said host further comprising one or more polypeptides selected from a cytochrome P450 monooxygenase classified under EC 1.14.15.1 or EC 1.14.15.3, a ω-transaminase classified under EC 2.6.1-, and an alcohol dehydrogenase classified under EC 1.1.1-, said host further producing 7-aminoheptanoate.

12. The method of claim 1 , wherein the two cycles of CoA-dependent carbon chain elongation comprise:
(i) contacting propanoyl-CoA and acetyl-CoA with a β-ketothielase classified under EC 2.3.1.16 to form 3-oxopentanoyl-CoA, or
contacting acetyl-CoA with an acetyl-CoA carboxylase classified under EC 6.4.1.2 to form malonyl-CoA and contacting malonyl-CoA with a β-ketoacyl-[acp]synthase classified under EC 2.3.1.180 to form 3-oxopentanoyl-CoA;
(ii) contacting 3-oxopentanoyl-CoA with an acetoacetyl-CoA reductase classified under EC 1.1.1.36 or a 3-oxoacyl-CoA reductase classified under EC 1.1.1.100 to form (R) 3-hydroxypentanoyl-CoA and contacting (R) 3-hydroxypentanoyl-CoA with an enoyl-CoA hydratase classified under EC 4.2.1.119 to form pent-2-enoyl-CoA;
(iii) contacting pent-2-enoyl-CoA with a trans-2-enoyl-CoA reductase classified under EC 1.3.1.38 or EC 1.3.1.8 to form pentanoyl-CoA and contacting pentanoyl-CoA with a β-ketothiolase classified under EC 2.3.1.16 to form 3-oxoheptanoyl-CoA;
(iv) contacting 3-oxoheptanoyl-CoA with an acetoacetyl-CoA reductase classified under EC 1.1.1.36 or a 3-oxoacyl-CoA reductase classified under EC 1.1.1.100 to form (R) 3-hydroxyheptanoyl-CoA and contacting (R) 3-hydroxvheptanoyl-CoA with an enoyl-CoA hydratase classified under EC 4.2.1.119 to form hept-2-enoyl-CoA; and
(v) contacting hept-2-enoyl-CoA with a trans-2-enoyl-CoA reductase classified under EC 1.3.1.38 or EC 1.3.1.8 to form heptanoyl-CoA.

13. The method of claim 1 , wherein the two cycles of CoA-dependent carbon chain elongation comprise:
(i) contacting propanoyl-CoA and acetyl-CoA with a β-ketothiolase classified under EC 2.3.1.16 to form 3-oxopentanoyl-CoA, or
contacting acetyl-CoA with an acetyl-CoA carboxylase classified under EC 6.4.1.2 to form malonyl-CoA and contacting malonyl-CoA with a β-ketoacyl-[acp]synthase classified under EC 2.3.1.180 to form 3-oxopentanoyl-CoA;
(ii) contacting 3-oxopentanoyl-CoA with a 3-hydroxyacyl-CoA dehydrogenase classified under EC 1.1.1.157 or EC 1.1.1.35 to form (S) 3-hydroxypentanoyl-CoA and contacting (S) 3-hydroxypentanoyl-CoA with an enoyl-CoA hydratase classified under EC 4.2.1.17 to form pent-2-enoyl-CoA;
(iii) contacting pent-2-enoyl-CoA with a trans-2-enoyl-CoA reductase classified under EC 1.3.1.44 to form pentanoyl-CoA and contacting pentanoyl-CoA with a β-ketothiolase classified under EC 2.3.1.16 to form 3-oxoheptanoyl-CoA;
(iv) contacting 3-oxoheptanoyl-CoA with a 3-hydroxyacyl-CoA dehydrogenase classified under EC 1.1.1.157 or EC 1.1.1.35 to form (S) 3-hydroxyheptanoyl-CoA and contacting (S) 3-hydroxyheptanoyl-CoA with an enoyl-CoA hydratase classified under EC 4.2.1.17 to form hept-2-enoyl-CoA; and
(v) contacting hept-2-enoyl-CoA with a trans-2-enoyl-CoA reductase classified under EC 1.3.1.44 to form heptanoyl-CoA.

14. The method of claim 1 , wherein said two cycles of CoA-dependent carbon chain elongation comprise:
(i) contacting propanoyl-CoA and acetyl-CoA with a β-ketothiolase classified under EC 2.3.1.16 to form 3-oxopentanoyl-CoA, or
contacting acetyl-CoA with an acetyl-CoA carboxylase classified under E

Assignees

• Invista North America S Ár L
• Invista North America Sarl

Inventors

• Conradie Alex Van Eck
• Botes Adriana Leonora

Classifications

• C12P7/44

  Polycarboxylic acids · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C12P7/42

  Hydroxy-carboxylic acids · CPC title

• C12N9/1029

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

• C12P7/18 Primary

  polyhydric · CPC title