Methods, reagents and cells for biosynthesizing compounds

Patent metadata

Field	Value
Publication number	US-9957535-B2
Application number	US-201514741342-A
Country	US
Kind code	B2
Filing date	Jun 16, 2015
Priority date	Jun 16, 2014
Publication date	May 1, 2018
Grant date	May 1, 2018

Abstract

This document describes biochemical pathways for producing glutaric acid, 5-aminopentanoic acid, 5-hydroxypentanoic acid, cadaverine or 1,5-pentanediol by forming one or two terminal functional groups, comprised of carboxyl, amine or hydroxyl group, in a C5 backbone substrate such as 2-oxoglutarate.

First claim

What is claimed is:

1. A method of biosynthesizing a C5 building block selected from the group consisting of glutaric acid, 5-hydroxypentanoic acid, 5-aminopentanoic acid, cadaverine, and 1,5-pentanediol, in a recombinant host, the method comprising:
(a) (i) enzymatically converting 2-oxo-adipate to 2-amino-adipate using at least one polypeptide having the activity of an alpha-aminotransaminase classified under EC 2.6.1.7 or EC 2.6.1.39, (ii) enzymatically converting 2-oxo-adipate to 5-oxopentanoate using at least one polypeptide having the activity of a 2-oxoacid decarboxylase classified under EC 4.1.1.71, EC 4.1.1.72, EC 4.1.1.43 or EC 4.1.1.74, or (iii) enzymatically converting 2-oxo-adipate to glutaryl-CoA using at least one polypeptide having the activity of a 2-oxoglutarate dehydrogenase complex classified under EC 1.2.4.2, EC 1.8.1.4 and EC 2.3.1.61;
(b) enzymatically converting the 2-amino-adipate, 5-oxopentanoate, or glutaryl-CoA to the C5 building block, wherein:
at least one polypeptide having the activity of a thioesterase classified under EC 3.1.2.-, a glutaconate CoA-transferase classified under EC 2.8.3.12, a succinate-CoA ligase classified under EC 6.2.1.5, an aldehyde dehydrogenase classified under EC 1.2.1.-, an acyl-[acp] thioesterase classified under EC 3.1.2.-, a 5-oxopentanoate dehydrogenase classified under EC 1.2.1.-, a 7-oxoheptanoate dehydrogenase classified under EC 1.2.1.-, or a 6-oxohexanoate dehydrogenase classified under EC 1.2.1.63 enzymatically forms a terminal carboxyl group of the C5 building block;
at least one polypeptide having the activity of an alpha-amino decarboxylase classified under EC 4.1.1.-, a ω-transaminase classified under EC 2.6.1.-, or a deacetylase classified under EC 3.5.1.17 or EC 3.5.1.62 enzymatically forms a terminal amine group of the C5 building block; and/or
at least one polypeptide having the activity of a 6-hydroxyhexanoate dehydrogenase classified under EC 1.1.1.258, a 5-hydroxypentanoate dehydrogenase classified under EC 1.1.1-, a 4-hydroxybutyrate dehydratase classified under EC 1.1.1-, or an alcohol dehydrogenase classified under EC 1.1.1- enzymatically forms a terminal hydroxyl group of the C5 building block.

2. The method of claim 1 , wherein the polypeptide having the activity of a 2-oxoacid decarboxylase has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1.

3. The method of claim 1 , wherein the polypeptide having the activity of a 2-oxoglutarate dehydrogenase complex has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16.

4. The method of claim 1 , wherein the polypeptide having the activity of a thioesterase activity has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17 or 18.

5. The method of claim 1 , wherein the polypeptide having the activity of a ω-transaminase activity has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 8-13.

6. The method of claim 1 , wherein the polypeptide having the activity of an alpha-amino decarboxylase has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 19-22.

7. The method of claim 1 , wherein a polypeptide having the activity of a carboxylate reductase classified under EC 1.2.99.6 enzymatically forms a terminal aldehyde group as an intermediate in forming the C5 building block.

8. The method of claim 7 , wherein the polypeptide having the activity of a carboxylate reductase has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-7.

9. The method of claim 7 , wherein the polypeptide having the activity of a carboxylate reductase is used in combination with a polypeptide having the activity of a phosphopantetheine transferase, wherein the polypeptide having the activity of a phosphopantetheine transferase has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14 or 15.

10. The method of claim 1 , wherein the 2-oxo-adipate is obtained by enzymatically converting 2-oxo-glutarate to 2-oxo-adipate using at least one polypeptide having an activity selected from the group consisting of a 2-isopropylmalate synthase or homocitrate synthase classified under EC 2.3.3.14 or EC 2.3.3.13, a homoaconitate hydratase or 3-isopropylmalate dehydratase classified under EC 4.2.1.114, EC 4.2.1.36 or EC 4.2.1.33, and a homoisocitrate dehydrogenase or 3-isopropylmalate dehydrogenase classified under EC 1.1.1.85, EC 1.1.1.87 or EC 1.1.1.286.

11. The method of claim 1 , wherein glutaryl-CoA is enzymatically converted to glutaric acid using at least one polypeptide having an activity selected from the group consisting of (i) a thioesterase classified under EC 3.1.2.-, (ii) a succinate-CoA-ligase classified under EC 6.2.1.5, (iii) a CoA-transferase classified under EC 2.8.3.-, (iv) an acylating dehydrogenase classified under EC 1.2.1.10 or EC 1.2.1.76 and an aldehyde dehydrogenase classified under EC 1.2.1.-, and (v) an aldehyde dehydrogenase classified under EC 1.2.1.3.

12. The method of claim 1 , wherein 2-amino-adipate is enzymatically converted to 5-aminopentanoic acid using a polypeptide having the activity of an alpha-amino decarboxylase classified under EC 4.1.1.-.

13. The method of claim 1 , wherein 5-oxopentanoic acid is enzymatically converted to 5-hydroxypentanoic acid using at least one polypeptide having an activity selected from the group consisting of a 5-hydroxypentanoate dehydrogenase classified under EC 1.1.1.-, a 6-hydroxyhexanoate dehydrogenase classified under EC 1.1.1.258, and an alcohol dehydrogenase classified under EC 1.1.1.-.

14. The method of claim 1 , wherein 5-oxopentanoic acid is enzymatically converted to cadaverine using at least one polypeptide having an activity selected from the group consisting of a carboxylate reductase classified under EC 1.2.99.6 and a ω-transaminase classified under EC 2.6.1.-.

15. The method of claim 1 , wherein the C5 building block is glutaric acid and the method further comprises enzymatically converting glutaric acid to 5-aminopentanoic acid using at least one polypeptide having an activity selected from the group consisting of a carboxylate reductase classified under EC 1.2.99.6 and a ω-transaminase classified under EC 2.6.1.-.

16. The method of claim 1 , wherein the C5 building block is glutaric acid and the method further comprises enzymatically converting glutaric acid to 5-hydroxypentainoic acid using at least one polypeptide having an activity selected from the group consisting of a carboxylate reductase classified under EC 1.2.99.6 and an alcohol dehydrogenase classified under EC 1.1.1.-.

17. The method of claim 1 , wherein the C5 building block is 5-aminopentanoic acid and the method further comprises enzymatically converting 5-aminopentanoic acid to cadaverine using at least one polypeptide having an activity selected from the group consisting of a carboxylate reductase classified under EC 1.2.99.6 and a ω-transaminase classified under EC 2.6.1.-.

18. The method of claim 1 , wherein the C5 building block is 5-hydroxypentanoic acid and the method further comprises enzymatically converting 5-hydroxypentanoic acid to cadaverine using at least one polypeptide having an activity selected from the group consisting of a carboxylate reductase classified under EC 1.2.99.6, a ω-transaminase classified under EC 2.6.1.-, and an alcohol dehydrogenase classified under EC 1.1.1.-.

19. The method of claim 1 , wherein the C5 building block is 5-hydroxypentanoic acid and the method further comprises enzymatically converting the 5

Assignees

• Invista North America Sarl
• Invista North America Sarl

Inventors

• Conradie Alex Van Eck
• Botes Adriana Leonora

Classifications

• C12Y206/01018

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

• C12N9/0008

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

• C12N9/1096

  transferring nitrogenous groups (2.6) · CPC title

• C12P7/44

  Polycarboxylic acids · CPC title

• C12P7/62

  Carboxylic acid esters · CPC title