Enhanced pyruvate to 2,3-butanediol conversion in lactic acid bacteria

• US 8,455,224 B2
• Filed: 09/29/2009
• Issued: 06/04/2013
• 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
• Primary Examiner(s): FRONDA, CHRISTIAN L
• Application Number:
  US12/569,136
• Publication Number:
  US 20100112655A1
• Time in Patent Office: 1,344 Days
• Field of Search: None
• 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 and at least one genetic modification that reduces pyruvate formate lyase activity, wherein the bacterial cell is substantially free of lactate dehydrogenase activity, and wherein the cell comprises a 2-butanol or 2-butanone biosynthetic pathway.

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 wherein the genetic modification affects a gene encoding pyruvare formate lyase, a gene encoding pyruvate formate lyase activating enzyme, or both.

5. The bacterial cell of claim 4 wherein the gene encoding pyruvate formate lyase is selected from the group consisting of pfl, pflB1 and pfl B2 and the gene encoding pyruvate formate lyase activating enzyme is selected from the group consisting of pflA, pflA1, and pflA2.

6. The bacterial cell of claim 2 wherein the polypeptide having lactate dehydrogenase activity is encoded by a gene selected from the group consisting of IdhL, IdhD, IdhL1, and IdhL2.

7. The bacterial cell of claim 6 herein the lactic acid host cell is Lactobacillus plantarum, Lactococcus lactis, Leuconostoc mesenteroides, Streptococcus thermophilus, Pediococcus pentosaceus, or Lactobacillus acidophilus.

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

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

10. The bacterial cell of claim 9 comprising a 2-butanol biosynthetic pathway, wherein the biosynthetic pathway comprises the following substrate to product conversions:

a) pyruvate to acetolactate;

b) acetolactate to acetoin;

c) acetoin to 2,3-butanediol;

d) 2,3-butanediol to 2-butanone; and

e) 2-butanone to 2-butanol.

11. The bacterial cell of claim 8 comprising a 2-butanone biosynthetic pathway, wherein the biosynthetic pathway comprises the following substrate to product conversions:

a) pyruvate to acetolactate;

b) acetolactate to acetoin;

c) acetoin to 2,3-butanediol; and

d) 2,3-butanediol to 2-butanone.

12. 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.

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

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.

14. The lactic acid bacterial cell of claim 1 comprising a 2-butanol biosynthetic pathway, wherein the heterologous polypeptide having butanediol dehydrogenase activity comprises an amino acid sequence with at least about 95% sequence identity to SEQ ID NO:

13, SEQ ID NO;

64, or SEQ ID NO;

66.

15. The lactic acid bacterial cell of claim 14 wherein the heterologous polypeptide having butanediol dc:

hydrogenase activity comprises the amino acid sequence of SEQ ID NO;

13.

16. The lactic acid bacterial cell of claim 10 wherein the substrate to product conversion e) is catalyzed by a heterologous butanol dehydrogenase comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO:

10.