ENHANCED PYRUVATE TO 2,3-BUTANEDIOL CONVERSION IN LACTIC ACID BACTERIA

• US 20100112655A1
• Filed: 09/29/2009
• Published: 05/06/2010
• Est. Priority Date: 09/29/2008
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: PAUL, BRIAN JAMES
• Granted Patent: US 8,455,224 B2
• Time in Patent Office: Days
• US Class Current: 435/160
• CPC Class Codes: C12N 15/746 for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/18 polyhydric
• CPC Class Codes: C12P 7/26 Ketones
• CPC Class Codes: C12Y 101/02004 D-lactate dehydrogenase (cytochrome) (1.1.2.4)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

A high flux of metabolites from pyruvate to 2,3-butanediol in Lactobacillus plantarum was achieved through genetic engineering. Substantial elimination of lactate dehydrogenase activity in the presence of heterologously expressed butanediol dehydrogenase activity led to 2,3 butanediol production that was at least 49% of the total of major pyruvate-derived products.

Claims

1. A recombinant lactic acid bacterial cell comprising at least one gene encoding a heterologous polypeptide having butanediol dehydrogenase activity wherein the bacterial cell is substantially free of lactate dehydrogenase activity and wherein the cell produces 2,3-butanediol.

2. The bacterial cell of claim 1 comprising a disruption in at least one endogenous gene encoding a polypeptide having lactate dehydrogenase activity.

3. The bacterial cell of claim 1 wherein the cell is a member of a genus selected from the group consisting of Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus.

4. The bacterial cell of claim 1 further comprising at least one genetic modification that reduces pyruvate formate lyase activity.

5. The bacterial cell of claim 4 wherein the genetic modification affects a gene encoding pyruvate formate lyase, a gene encoding pyruvate formate lyase activating enzyme, or both.

6. The bacterial cell of claim 5 wherein the gene encoding pyruvate formate lyase is selected from the group consisting of pfl, pflB1 and pfl B2 and the gene encoding formate C-acetyltransferase activating enzyme is selected from the group consisting of pflA, pflA1, and pflA2.

7. The bacterial cell of claim 1 wherein the cell produces a product selected from the group consisting of lactate, acetoin, ethanol, succinate, and formate.

8. The bacterial cell of claim 7 wherein 2,3-butanediol comprises at least about 49 Mol % of all products produced from pyruvate.

9. The bacterial cell of claim 2 wherein the polypeptide having lactate dehydrogenase activity is encoded by a gene selected from the group consisting of ldhL, ldhD, ldhL1, and ldhL2.

10. The bacterial cell of claim 9 wherein the lactic acid host cell is Lactobacillus plantarum Lactococcus lactis, Leuconostoc mesenteroides, Streptococcus thermophilus, Pediococcus pentosaceus, or Lactobacillus acidophilus.

11. The bacterial cell of claim 1 wherein the cell produces 2-butanone.

12. The bacterial cell of claim 1 wherein the cell produces 2-butanol.

13. The bacterial cell of claim 12 comprising a 2-butanol biosynthetic pathway.

14. The bacterial cell of claim 11 comprising a 2-butanone biosynthetic pathway.

15. A method for the production of 2-butanol comprising:

a) providing the recombinant lactic acid bacterial cell of claim 1 comprising a 2-butanol biosynthetic pathway; and

b) growing the bacterial cell of step (a) under conditions whereby 2-butanol is produced.

16. A method for the production of 2-butanone comprising:

a) providing the recombinant lactic acid bacterial cell of claim 1 comprising a 2-butanone biosynthetic pathway; and

b) growing the bacterial cell of step (a) under conditions whereby 2-butanone is produced.