Host cells and methods for producing isopentenol from mevalonate

Patent metadata

Field	Value
Publication number	US-10814724-B2
Application number	US-201916388556-A
Country	US
Kind code	B2
Filing date	Apr 18, 2019
Priority date	Feb 20, 2015
Publication date	Oct 27, 2020
Grant date	Oct 27, 2020

Abstract

The present invention provides for a genetically modified host cell capable of producing isopentenol and/or 3-methyl-3-butenol, comprising (a) an increased expression of phosphomevalonate decarboxylase (PMD) (b) an increased expression of a phosphatase capable of converting isopentenol into 3-methyl-3-butenol, (c) optionally the genetically modified host cell does not express, or has a decreased expression of one or more of NudB, phosphomevalonate kinase (PMK), and/or PMD, and (d) optionally one or more further enzymes capable of converting isopentenol and/or 3-methyl-3-butenol into a third compound, such as isoprene.

First claim

What is claimed is:

1. A genetically modified host cell capable of producing 3-methyl-3-butenol, comprising:
(a) expression of acetyl-CoA acetyltransferase (AtoB),
(b) expression of hydroxymethylglutaryl-CoA synthase (HMGS),
(c) expression of hydroxymethylglutaryl-CoA reductase (HMGR),
(d) expression of phosphomevalonate decarboxylase (PMD), wherein the PMD has an amino acid sequence having at least 90% identity with SEQ ID NO:1, and
(e) the genetically modified host cell does not express phosphomevalonate kinase (PMK);
wherein the host cell is a bacterial or fungal cell.

2. The genetically modified host cell of claim 1 , comprising wherein the PMD comprises (i) a histidine at position 74, (ii) a phenylalanine at position 145, or (iii) a histidine at position 74 and a phenylalanine at position 145, corresponding to the numbering of SEQ ID NO:1.

3. The genetically modified host cell of claim 2 , wherein the PMD comprises the following amino acid residues: (a) E at position 71, S at position 108, N at position 110, A at position 119, S at position 120, S at position 121, A at position 122, S at position 155, R at position 158, S at position 208, and D at position 302 corresponding to SEQ ID NO:1.

4. The genetically modified host cell of claim 1 , wherein the PMD has an amino acid sequence having at least 95% identity with SEQ ID NO:1.

5. The genetically modified host cell of claim 4 , wherein the PMD has an amino acid sequence having at least 99% identity with SEQ ID NO:1.

6. The genetically modified host cell of claim 1 , comprising one or more further enzymes capable of converting 3-methyl-3-butenol into a third compound.

7. The genetically modified host cell of claim 6 , wherein the third compound is isoprene.

8. The genetically modified host cell of claim 1 , wherein one or more of the PMD, AtoB, HMGS, and HMGR, are encoded on one or more nucleotide sequences which are in one or more nucleic acids which are transformed into the genetically modified host cell prior to genetic modification.

9. The genetically modified host cell of claim 1 , wherein the genetically modified host cell is a species of the genus Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Paracoccus , or Clostridia.

10. The genetically modified host cell of claim 9 , wherein the genetically modified host cell is a species of the genus Escherichia.

11. The genetically modified host cell of claim 1 , wherein the genetically modified host cell is a yeast cell.

12. The genetically modified host cell of claim 11 , wherein the yeast cell is a species of the Saccharomyces genus.

13. A method for producing 3-methyl-3-butenol, comprising:
(a) providing a genetically modified host cell comprising:
(i) expression of acetyl-CoA acetyltransferase (AtoB),
(ii) expression of hydroxymethylglutaryl-CoA synthase (HMGS),
(iii) expression of hydroxymethylglutaryl-CoA reductase (HMGR),
(iv) expression of phosphomevalonate decarboxylase (PMD), wherein the PMD has an amino acid sequence having at least 90% identity with SEQ ID NO:1, and
(v) the genetically modified host cell does not express phosphomevalonate kinase (PMK), wherein the host cell is a bacterial or fungal cell; and

(b) culturing the genetically modified host cell under a condition wherein phosphomevalonate decarboxylase (PMD), acetyl-CoA acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), and hydroxymethylglutaryl-CoA reductase (HMGR) are expressed.

14. The method of claim 13 , wherein the PMD comprises (i) a histidine at position 74, (ii) a phenylalanine at position 145, or (iii) a histidine at position 74 and a phenylalanine at position 145, corresponding to the numbering of SEQ ID NO:1.

15. The method of claim 13 , comprising (c) recovering the 3-methyl-3-butenol.

16. The method of claim 13 , wherein the genetically modified host cell comprises one or more further enzymes capable of converting 3-methyl-3-butenol into a third compound.

17. The method of claim 16 , comprising (c) recovering the third compound.

18. The method of claim 16 , wherein the third compound is isoprene.

19. The method of claim 18 , comprising (c) recovering the isoprene.

20. The method of claim 13 , wherein the (b) culturing step is under an anaerobic or microaerobic condition.

Assignees

• Univ California

Inventors

• Lee Taek Soon
• Kang Aram

Classifications

• C07F9/091 Primary

  with hydroxyalkyl compounds with further substituents on alkyl · CPC title

• C12N9/1025

  Acyltransferases (2.3) · CPC title

• C07F9/098

  Esters of polyphosphoric acids or anhydrides · CPC title

• C08F136/08

  Isoprene · CPC title

• C12Y207/01036

  Mevalonate kinase (2.7.1.36) · CPC title