Methods, cells and reagents for production of isoprene, derivatives and intermediates thereof

Patent metadata

Field	Value
Publication number	US-10167487-B2
Application number	US-201615238234-A
Country	US
Kind code	B2
Filing date	Aug 16, 2016
Priority date	Aug 17, 2015
Publication date	Jan 1, 2019
Grant date	Jan 1, 2019

Abstract

This application describes methods, including non-naturally occurring methods, for biosynthesizing 3-hydroxy-3-methylglutaryl-coA and intermediates thereof, as well as non-naturally occurring hosts for producing 3-hydroxy-3-methylglutaryl-coA. This application also describes methods, including non-naturally occurring methods, for biosynthesizing isoprene and intermediates thereof, as well as non-naturally occurring hosts for producing isoprene.

First claim

What is claimed is:

1. A method for synthesizing 3-hydroxy-3-methylglutaryl-CoA comprising:
enzymatically converting 4-methyl-2-oxopentanoate to 3-methylbut-2-enoyl-CoA by:
(a) enzymatically converting 4-methyl-2-oxopentanoate to 3-methylbutanal; enzymatically converting 3-methylbutanal to 3-methylbutanoate; and enzymatically converting 3-methylbutanoate to 3-methylbutanoyl-CoA;
(b) enzymatically converting 4-methyl-2-oxopentanoate to 3-methylbutanoyl-CoA; or
(c) both (a) and (b);

enzymatically converting 3-methylbutanoyl-CoA to 3-methylbut-2-enoyl-CoA;
enzymatically converting 3-methylbut-2-enoyl-CoA to 3-methyl-glutaconyl CoA; and
enzymatically converting 3-methyl-glutaconyl-CoA to 3-hydroxy-3-methylglutaryl-CoA.

2. The method of claim 1 , further comprising synthesizing isoprene via a mevalonate pathway comprising one or more steps selected from the group consisting of:
enzymatically converting 3-hydroxy-3-methylglutaryl-CoA to (R)-mevalonate;
enzymatically converting (R)-mevalonate to (R)-5-phosphomevalonate;
enzymatically converting (R)-5-phosphomevalonate to (R)-5-diphosphomevalonate;
enzymatically converting (R)-5-diphosphomevalonate to isopentenyl diphosphate;
enzymatically converting isopentenyl diphosphate to dimethylallyl diphosphate; and
enzymatically converting dimethylallyl diphosphate to isoprene.

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

4. The method of claim 1 , wherein at least one of the enzymatic conversions is performed in a recombinant host.

5. The method of claim 4 , wherein the host is a prokaryotic host from the genus Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Bacillus , or Rhodococcus.

6. The method of claim 5 , wherein the host is Cupriavidus necator.

7. The method of claim 4 , wherein the host is a eukaryotic host from the genus Aspergillus, Saccharomvces, Pichia, Yarrowia, Issatchenkia, Debaryomvces, Arxula , or Kluvveromvces.

8. The method of claim 4 , wherein the host is capable of endogenously producing 3-hydroxy-3-methylglutaryl-CoA.

9. The method of claim 4 , wherein at least one of the enzymatic conversions comprises gas fermentation within the host.

10. The method of claim 9 , wherein the gas comprises at least one of natural gas, syngas, CO 2 /H 2 , methanol, ethanol, non-volatile residue, caustic wash from cyclohexane oxidation processes, or waste stream from a chemical or petrochemical industry.

11. The method of claim 10 , wherein the gas is CO 2 /H 2 .

12. A method for synthesizing 3-hydroxy-3-methylglutaryl-CoA comprising:
enzymatically converting 4-methyl-2-oxopentanoate to 3-methylbut-2-enoyl-CoA by:
(a) enzymatically converting 4-methyl-2-oxopentanoate to 3-methylbutanal using a polypeptide having the activity of an EC 4.1.1.74 or EC 4.1.1.43 enzyme;
enzymatically converting 3-methylbutanal to 3-methylbutanoate using a polypeptide having the activity of an EC 1.2.1.39 or EC 1.2.1.5 enzyme; and
enzymatically converting 3-methylbutanoate to 3-methylbutanoyl-CoA using a polypeptide having the activity of an EC 6.2.1.2 enzyme;
(b) enzymatically converting 4-methyl-2-oxopentanoate to 3-methylbutanoyl-CoA using a polypeptide having the activity of an EC 1.2.7.7 or EC 1.2.1 enzyme; or
(c) both (a) and (b);
enzymatically converting 3-methylbutanoyl-CoA to 3-methylbut-2-enoyl-CoA using a polypeptide having the activity of an EC 1.3.8.4 enzyme;
enzymatically converting 3-methylbut-2-enoyl-CoA to 3-methyl-glutaconyl CoA using a polypeptide having the activity of an EC 6.4.1.4 enzyme; and
enzymatically converting 3-methyl-glutaconyl-CoA to 3-hydroxy-3-methylglutaryl-CoA using a polypeptide having enzyme activity classified under EC 4.2.1.18.

13. The method of claim 12 , wherein the step of enzymatically converting 4-methyl-2-oxopentanoate to 3-methylbutanal comprises using a polypeptide having enzyme activity classified under EC 4.1.1.74.

14. The method of claim 13 , wherein the polypeptide having enzyme activity classified under EC 4.1.1.74 is the gene product of ipdC.

15. The method of claim 12 , wherein the step of enzymatically converting 3-methylbutanal to 3-methylbutanoate comprises using a polypeptide having enzyme activity classified under EC 1.2.1.39.

16. The method of claim 15 , wherein the polypeptide having enzyme activity classified under EC 1.2.1.39 is the gene product of padA.

17. The method of claim 12 , wherein the polypeptide having enzyme activity classified under EC 1.3.8.4 is the gene product of IiuA.

Assignees

• Invista North America Sarl

Inventors

• Conradie Alex Van Eck

Classifications

• C12Y108/01004

  Dihydrolipoyl dehydrogenase (1.8.1.4), i.e. lipoamide-dehydrogenase · CPC title

• C12Y402/03027

  Isoprene synthase (4.2.3.27) · CPC title

• C12Y207/01036

  Mevalonate kinase (2.7.1.36) · CPC title

• C12Y207/04002

  Phosphomevalonate kinase (2.7.4.2) · CPC title

• C12P5/007 Primary

  containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00) · CPC title