Recombinant microorganisms for enhanced production of mevalonate, isoprene, and isoprenoids

Patent metadata

Field	Value
Publication number	US-10138498-B2
Application number	US-201514806343-A
Country	US
Kind code	B2
Filing date	Jul 22, 2015
Priority date	Apr 29, 2011
Publication date	Nov 27, 2018
Grant date	Nov 27, 2018

Abstract

The invention features compositions and methods for the increased production of mevalonate, isoprene, isoprenoid precursor molecules, and/or isoprenoids in microorganisms by engineering a microorganism for increased carbon flux towards mevalonate production in the following enzymatic pathways: (a) citrate synthase, (b) phosphotransacetylase, (c) acetate kinase, (d) lactate dehydrogenase, (e) malic enzyme, and (f) pyruvate dehydrogenase such that one of more of the enzyme activity is modulated. In addition, production of mevalonate, isoprene, isoprenoid precursor molecules, and/or isoprenoids can be further enhanced by the heterologous expression of the mvaE and mvaS genes (such as, but not limited to, 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 bacterial or yeast cells capable of increased production of isoprenoids, wherein the cells are engineered by
(i) modulation of citrate synthase activity such that the activity of citrate synthase is Decreased;
(ii) modulation of the activities of one or more of the following enzymes such that:
(a) phosphotransacetylase activity is attenuated;
(b) acetate kinase activity is attenuated; and/or
(c) lactate dehydrogenase activity is attenuated; and

(iii) modulation of the activity of two or more of the following enzymes such that (d) malate dehydrogenase activity is increased, (e) pyruvate dehydrogenase activity is increased, and/or (f) phosphoenolpyruvate carboxylase activity is attenuated, resulting in increased carbon flux towards mevalonate production;
wherein the cells further comprise (A) one or more nucleic acids encoding one or more mevalonate (MVA) pathway polypeptides; and (B) one or more nucleic acids encoding a polyprenyl pyrophosphate synthase; and
wherein the cells produce increased amounts of isoprenoids compared to isoprenoid-producing cells that do not comprise (i-iii).

2. The cells of claim 1 , wherein the one or more nucleic acids encoding MVA pathway polypeptides are from the upper MVA pathway, wherein the upper MVA pathway nucleic acids are selected from the group consisting of acetyl-CoA acetyltransferase (AA-CoA thiolase), 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) nucleic acids.

3. The cells of claim 1 , wherein the one or more nucleic acids encoding MVA pathway polypeptides are an mvaE gene and an mvaS gene.

4. The cells of claim 3 , wherein the mvaE gene and the mvaS gene are selected from the group consisting of: (a) an mvaE gene and an mvaS gene from L. grayi ; (b) an mvaE gene and an mvaS gene from E. faecium ; (c) an mvaE gene and an mvaS gene from E. gallinarum ; (d) an mvaE gene and an mvaS gene from E. casseliflavus ; and (e) an mvaE gene and an mvaS gene from E. faecalis.

5. The cells of claim 1 , wherein the one or more nucleic acids encoding MVA pathway polypeptides are from the lower MVA pathway, wherein the lower MVA pathway nucleic acids are selected from the group consisting of mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and diphosphomevalonte decarboxylase (MVD) nucleic acids.

6. The cells of claim 5 , wherein the MVK 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 cells further comprise one or more heterologous nucleic acids encoding one or more 1-deoxy-d-xylulose 5-phosphate (DXP) pathway polypeptides.

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

9. The cells of claim 1 , wherein the cells are gram-positive bacterial cells, Streptomyces cells, gram-negative bacterial cells, Escherichia cells, Pantoea cells, or yeast cells.

10. The cells of claim 1 , wherein the activity of citrate synthase is modulated by decreasing the activity of an endogenous citrate synthase gene.

11. The cells of claim 1 , wherein the activity of malate dehydrogenase is modulated by increasing the activity of a malate dehydrogenase gene.

12. The cells of claim 1 , wherein the activity of pyruvate dehydrogenase is modulated by increasing the activity of one or more genes of the pyruvate dehydrogenase complex consisting of (a) pyruvate dehydrogenase (E 1 ), (b) dihydrolipoyl transacetylase, and (c) dihydrolipoyl dehydrogenase.

13. The cells of claim 1 , wherein the activity of phosphoenolpyruvate carboxylase is modulated by attenuating the activity of an endogenous phosphoenolpyruvate carboxylase gene.

14. The cells of claim 1 , wherein the modulation of the activity of (iii) two or more enzymes comprises (e) increasing the activity of pyruvate dehydrogenase.

15. The cells of claim 9 , wherein the yeast cells are Saccharomyces cerevisiae cells.

16. The cells of claim 9 , wherein the gram-positive bacterial cells or gram-negative bacterial cells are Bacillus subtilis cells, Escherichia coli cells or Pantoea citrea cells.

17. The cells of claim 1 , wherein the isoprenoid is one or more isoprenoid selected from the group consisting of monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, triterpenoids, tetraterpenoids, and higher polyterpenoids.

18. The cells of claim 17 , wherein the monoterpenoid is geranyl pyrophosphate, eucalyptol, limonene, or pinene.

19. The cells of claim 17 , wherein the sesquiterpenoid is farnesyl pyrophosphate, artemisinin, or bisabolol.

20. The cells of claim 17 , wherein the diterpenoid is geranylgeranyl pyrophosphate, retinol, retinal, phytol, taxol, forskolin, or aphidicolin.

21. The cells of claim 17 , wherein the triterpenoid is squalene or lanosterol.

22. The cells of claim 17 , wherein the isoprenoid is selected from the group consisting of abietadiene, amorphadiene, carene, α-farnesene, β-farnesene, farnesol, geraniol, geranylgeraniol, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, β-pinene, sabinene, γ-terpinene, terpindene and valencene.

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

24. The method of claim 23 , further comprising (c) recovering the isoprenoids.

25. The method of claim 23 , wherein the isoprenoid is one or more isoprenoid selected from the group consisting of hemiterpenoids, monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, triterpenoids, tetraterpenoids, and higher polyterpenoids.

26. The method of claim 25 , wherein the hemiterpenoid is prenol (3-methyl-2-buten-1-ol), isoprenol (3-methyl-3-buten-1-ol), 2-methyl-3-buten-2-ol, or isovaleric acid.

27. The method of claim 25 , wherein the sesquiterpenoid is farnesyl pyrophosphate, artemisinin, or bisabolol.

28. The method of claim 25 , wherein the diterpenoid is geranylgeranyl pyrophosphate, retinol, retinal, phytol, taxol, forskolin, or aphidicolin.

29. The method of claim 25 , wherein the triterpenoid is squalene or lanosterol.

30. The method of claim 25 , wherein the isoprenoid is selected from the group consisting of abietadiene, amorphadiene, carene, α-farnesene, β-farnesene, farnesol, geraniol, geranylgeraniol, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, -pinene, sabinene, γ-terpinene, terpindene and valencene.

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

• Y02E50/343

  Cross-Sectional Technologies · mapped topic

• C12Y101/01034

  Hydroxymethylglutaryl-CoA reductase (NADPH) (1.1.1.34) · CPC title

• C12Y203/01016

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

• C12P5/002

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

• C12N9/1217

  Phosphotransferases with a carboxyl group as acceptor (2.7.2) · CPC title