Production of mevalonate, isoprene, and isoprenoids using genes encoding polypeptides having thiolase, HMG-CoA synthase and HMG-CoA reductase enzymatic activities

Patent metadata

Field	Value
Publication number	US-9752162-B2
Application number	US-201414514962-A
Country	US
Kind code	B2
Filing date	Oct 15, 2014
Priority date	Apr 29, 2011
Publication date	Sep 5, 2017
Grant date	Sep 5, 2017

Abstract

The invention features compositions and methods for the increased production of mevalonate, isoprene, isoprenoid precursor molecules, and/or isoprenoids in microorganisms via the heterologous expression of the mvaE and mvaS genes from the organisms Listeria grayi DSM 20601, Enterococcus faecium, Enterococcus gallinarum EG2, and Enterococcus casseliflavus.

First claim

What is claimed is:

1. Recombinant cells with increased production of isoprene, the cells comprising one or more heterologous nucleic acids comprising a mevalonate E (mvaE) gene and a mevalonate S (mvaS) gene selected from the organisms Listeria grayi ( L. grayi ), Enterococcus faecium ( E. faecium ), Enterococcus gallinarum ( E. gallinarum ), and Enterococcus casseliflavus ( E. casseliflavus ), wherein the mvaE gene and the mvaS gene encode polypeptides having thiolase, HMG-CoA synthase, and HMG-CoA reductase catalytic activities, and wherein the cells further comprise:
i. one or more nucleic acids encoding polypeptides of the lower MVA pathway; and
ii. a heterologous nucleic acid encoding an isoprene synthase polypeptide,

wherein the cells produce increased amounts of isoprene compared to isoprene-producing cells that do not comprise the mvaE gene and the mvaS gene.

2. The cells of claim 1 , wherein the mvaE gene and mvaS gene are from different organisms.

3. The cells of claim 1 , wherein:
a) the mvaE gene is from L. grayi and the mvaS gene is from E. faecium;
b) the mvaE gene is from L. grayi and the mvaS gene is from E. gallinarum;
c) the mvaE gene is from L. grayi and the mvaS gene is from E. casseliflavus;
d) the mvaE gene is from E. faecium and the mvaS gene is from L. grayi;
e) the mvaE gene is from E. faecium and the mvaS gene is from E. gallinarum;
f) the mvaE gene is from E. faecium and the mvaS gene is from E. casseliflavus;
g) the mvaE gene is from E. gallinarum and the mvaS gene is from L. grayi;
h) the mvaE gene is from E. gallinarum and the mvaS gene is from E. faecium;
i) the mvaE gene is from E. gallinarum and the mvaS gene is from E. casseliflavus;
j) the mvaE gene is from E. casseliflavus and the mvaS gene is from L. grayi;
k) the mvaE gene is from E. casseliflavus and the mvaS gene is from E. faecium ; or
l) the mvaE gene is from E. casseliflavus and the mvaS gene is from E. gallinarum.

4. The cells of claim 1 , wherein the cells produce increased amounts of isoprene compared to isoprene-producing cells that (A) contain a mvaE gene from Enterococcus faecalis and (B) do not contain the mvaE gene and the mvaS gene from L. grayi, E. faecium, E. gallinarum , or E. casseliflavus.

5. The cells of claim 1 , wherein the nucleic acids encoding polypeptides of the lower MVA pathway comprise enzymes selected from: (a) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate; (b) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate; and (c) an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate.

6. The cells of claim 5 , wherein the enzyme that phosphorylates mevalonate to mevalonate 5-phosphate is selected from the group consisting of M. mazei mevalonate kinase, M. burtonii mevalonate kinase polypeptide, Lactobacillus mevalonate kinase polypeptide, Lactobacillus sakei mevalonate kinase polypeptide, yeast mevalonate kinase polypeptide, Saccharomyces cerevisiae mevalonate kinase polypeptide, Streptococcus mevalonate kinase polypeptide, Streptococcus pneumoniae mevalonate kinase polypeptide, Streptomyces mevalonate kinase polypeptide, and Streptomyces CL190 mevalonate kinase polypeptide.

7. The cells of claim 1 , wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide or a variant thereof.

8. The cells of claim 7 , wherein the isoprene synthase polypeptide is a polypeptide from Pueraria or Populus or a hybrid, Populus alba x Populus tremula , or a variant thereof.

9. The cells of claim 7 , wherein the plant isoprene synthase polypeptide is a Populus alba isoprene synthase polypeptide.

10. The cells of claim 1 , further comprising one or more nucleic acids encoding an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide.

11. The cells of claim 1 , wherein any one or more nucleic acids is placed under an inducible promoter or a constitutive promoter.

12. The cells of claim 1 , wherein any one or more nucleic acids is cloned into a multicopy plasmid.

13. The cells of claim 1 , wherein any one or more nucleic acids is integrated into a chromosome of the cells.

14. The cells of claim 1 , wherein the cells are gram-positive bacterial cells, gram-negative bacterial cells, Escherichia cells, Pantoea cells, fungal cells, filamentous fungal cells, Trichoderma cells, Aspergillus cells, or yeast cells.

15. The cells of claim 14 , wherein the cells are selected from the group consisting of E. coli, P. citrea, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., and P. alcaligenes cells.

16. The cells of claim 15 , wherein the cells are E. coli cells.

17. The cells of claim 14 , wherein the cells are yeast cells.

18. The cells of claim 17 , wherein the yeast cells are selected from the group consisting of Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., and Candida sp. cells.

19. The cells of claim 18 , wherein the yeast cells are Saccharomyces cerevisiae cells.

20. A method of producing isoprene, comprising: (a) culturing the host cells of claim 1 under suitable culture conditions for the production of isoprene; and (b) producing the isoprene.

21. The method of claim 20 , further comprising (c) recovering the isoprene.

Assignees

• Danisco Us Inc
• Goodyear Tire & Rubber

Inventors

• Beck Zachary Q
• Miller Michael C
• Peres Caroline M
• Primak Yuliya A
• Pucci Jeff P
• Wells Derek H

Classifications

• C12N9/1025

  Acyltransferases (2.3) · CPC title

• C12N15/81

  for yeasts · CPC title

• C12P5/002

  cyclic (compounds containing at least three condensed carbocyclic rings C12P15/00) · CPC title

• C12P5/007 Primary

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

• C12Y203/01016

  Acetyl-CoA C-acyltransferase (2.3.1.16) · CPC title