Host cells and methods for producing isopentenol from mevalonate

Patent metadata

Field	Value
Publication number	US-10273506-B2
Application number	US-201715682325-A
Country	US
Kind code	B2
Filing date	Aug 21, 2017
Priority date	Feb 20, 2015
Publication date	Apr 30, 2019
Grant date	Apr 30, 2019

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 isopentenol and/or 3-methyl-3-butenol, comprising (a) an increased expression of phosphomevalonate decarboxylase (PMD), wherein the PMD has an amino acid sequence having at least 90% identity with SEQ ID NO:1, and (i) amino acid residue at position 74 is histidine, (ii) amino acid residue at position 145 is phenylalanine, or (iii) amino acid residue at position 74 is histidine and amino acid residue at position 145 is phenylalanine, (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 dihydroneopterin triphosphate diphosphate (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; wherein the host cell is a bacterial or fungal cell.

2. The genetically modified host cell of claim 1 , wherein the decreased expression is a disruption of the promoter or knock out of the gene encoding NudB, PMK, and/or PMD.

3. The genetically modified host cell of claim 1 , wherein the third compound is isoprene.

4. The genetically modified host cell of claim 1 , further comprising an increased expression of one or more of acetyl-CoA acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), and/or mevalonate kinase (MK).

5. The genetically modified host cell of claim 1 , wherein the PMD is encoded on a nucleotide sequence which is in nucleic acids which is transformed into the genetically modified host cell, or host cell prior to genetic modification.

6. The genetically modified host cell of claim 3 , wherein one or more of the PMD, phosphatase, AtoB, HMGS, HMGR, and MK, 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, or host cell prior to genetic modification.

7. A method for producing isopentenol and/or 3-methyl-3-butenol and/or the third compound, comprising: (a) providing a genetically modified host cell of claim 1 , 2 , or 3 , (b) culturing the genetically modified host cell under a condition wherein phosphomevalonate decarboxylase (PMD) and/or phosphatase are expressed, and (c) optionally recovering the isopentenol and/or 3-methyl-3-butenol and/or the third compound.

8. The method of claim 7 , wherein the (b) culturing step further comprises expressing acetyl-CoA acetyltransferase (AtoB), hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), and/or mevalonate kinase (MK).

9. The method of claim 7 , wherein the (b) culturing step is under an anaerobic or microaerobic condition.

10. The method of claim 8 , wherein the (b) culturing step is under an anaerobic or microaerobic condition.

11. 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.

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

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

14. The genetically modified host cell of claim 13 , wherein the yeast cell is a species of the Saccharomyces genus.

15. The genetically modified host cell of claim 1 , 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; or (b) E at position 73, S at position 94, N at position 96, A at position 105, S at position 106, S at position 107, A at position 108, S at position 141, R at position 144, S at position 192, and D at position 283 corresponding to SEQ ID NO:2.

16. 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.

17. The genetically modified host cell of claim 16 , wherein the PMD has an amino acid sequence having at least 99% identity with SEQ ID NO:1.

Assignees

• Univ California

Inventors

• Lee Taek Soon
• Kang Aram

Classifications

• C07F9/091 Primary

  with hydroxyalkyl compounds with further substituents on alkyl · CPC title

• C12P5/007

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

• C07F9/098

  Esters of polyphosphoric acids or anhydrides · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12P7/04 Primary

  acyclic · CPC title