FLUX TO ACETOLACTATE-DERIVED PRODUCTS IN LACTIC ACID BACTERIA

• US 20110136192A1
• Filed: 09/29/2010
• Published: 06/09/2011
• Est. Priority Date: 09/29/2009
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: Suh, Wonchul, Paul, Brian James
• Granted Patent: US 9,169,499 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: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12Y 101/01027 L-Lactate dehydrogenase (1.1.1.27)
• CPC Class Codes: C12Y 203/01054 Formate C-acetyltransferase (2.3.1.54), i.e. pyruvate formate-lyase or PFL
• CPC Class Codes: C12Y 401/01005 Acetolactate decarboxylase (4.1.1.5)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

An engineering method was developed to allow genetic modification and isolation of lactic acid bacteria cells that lack lactate dehydrogenase and acetolactate decarboxylase activities. In cells with these modifications and an isobutanol biosynthetic pathway, improved production of isobutanol was observed.

Claims

1. A recombinant lactic acid bacteria cell comprising at least one engineered genetic modification that reduces or eliminates enzyme activity of endogenously expressed acetolactate decarboxylase and at least one engineered genetic modification that eliminates enzyme activity of endogenously expressed lactate dehydrogenase.

2. The bacteria cell of claim 1 comprising at least one engineered genetic modification that eliminates enzyme activity of endogenously expressed acetolactate decarboxylase and at least one engineered genetic modification that eliminates enzyme activity of endogenously expressed lactate dehydrogenase.

3. The bacteria cell of claim 1 wherein each of the engineered genetic modifications is a deletion of at least a portion of a gene encoding acetolactate decarboxylase or lactate dehydrogenase

4. The bacteria cell of claim 3 wherein said gene encoding acetolactate decarboxylase is selected from the group consisting of aldB, aldC, and ald.

5. The bacteria cell of claim 4 wherein the gene encoding acetolactate decarboxylase encodes a protein having an amino acid sequence that has at least about 95% identity to a sequence selected from the group consisting of SEQ ID NOs:

24, 26, 28, 30, 32, 34, 36, and 38.

6. The bacteria cell of claim 3 wherein said gene encodes lactate dehydrogenase and is selected from the group consisting of ldhL, ldhD, ldhL1, and ldhL2.

7. The bacteria cell of claim 6 wherein the gene encoding lactate dehydrogenase encodes a protein having an amino acid sequence that has at least about 95% identity to a sequence selected from the group consisting of SEQ ID NOs:

2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22.

8. The bacteria 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.

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

10. The bacteria cell of claim 9 wherein the genetic modification is in a gene encoding pyruvate formate lyase, a gene encoding pyruvate formate lyase activating enzyme, or in both genes.

11. The bacteria cell of claim 10 wherein the said gene encoding pyruvate formate lyase is selected from the group consisting of pfl, pflB1 and pflB2 and said gene encoding formate C-acetyltransferase activating enzyme is selected from the group consisting of pflA, pflA1 and pflA2.

12. The bacteria cell of claim 1, 2, or 8 wherein the cell produces isobutanol.

13. The bacteria cell of claim 12 comprising an isobutanol biosynthetic pathway.

14. The bacteria cell of claim 13 wherein the isobutanol biosynthetic pathway comprises substrate to product conversions consisting of:

a) pyruvate to acetolactate;

b) acetolactate to 2,3-dihydroxyisovalerate;

c) 2,3-dihydroxyisovalerate to α

-ketoisovalerate;

d) α

-ketoisovalerate to isobutyraldehyde; and

e) isobutyraldehyde to isobutanol.

15. A method for producing a recombinant lactic acid bacteria cell comprising:

a) providing a lactic acid bacteria cell;

b) modifying by genetic engineering at least one endogenous gene encoding lactate dehydrogenase in the cell of (a) to eliminate enzyme activity of endogenously expressed lactate dehydrogenase;

c) expressing acetolactate decarboxylase activity from a plasmid in the cell of (b) to create a cell with non-chromosomally expressed acetolactate decarboxylase;

d) modifying by genetic engineering an endogenous gene encoding acetolactate decarboxylase in the cell of (c) to eliminate enzyme activity of endogenously expressed acetolactate decarboxylase; and

e) curing the plasmid expressing acetolactate decarboxylase activity from the cell of (d);

whereby a recombinant lactic acid bacteria cell lacking enzyme activity of endogenously expressed lactate dehydrogenase and acetolactate decarboxylase is produced.

17. The method of claim 15 wherein step (b) comprises a modification to a first gene encoding lactate dehydrogenase prior to (c), then a second gene encoding lactate dehydrogenase is modified by genetic engineering following step (c).

18. The method of claim 15, 16 or 17 further comprising modifying at least one endogenous gene to reduce pyruvate formate lyase activity.

16. A method for producing a recombinant lactic acid bacteria cell comprising:

a) providing a lactic acid bacteria cell;

b) modifying by genetic engineering an endogenous gene encoding acetolactate decarboxylase in the cell of (c) to eliminate enzyme activity of endogenously expressed acetolactate decarboxylase;

c) expressing lactate dehydrogenase activity from a plasmid in the cell of (b) to create a cell with non-chromosomally expressed lactate dehydrogenase;

d) modifying by genetic engineering at least one endogenous gene encoding lactate dehydrogenase in the cell of (a) to eliminate enzyme activity of endogenously expressed lactate dehydrogenase; and

e) curing the plasmid expressing lactate dehydrogenase activity from the cell of (d);

whereby a recombinant lactic acid bacteria cell lacking enzyme activity of endogenously expressed lactate dehydrogenase and acetolactate decarboxylase is produced.

19. A method for producing isobutanol comprising:

(a) providing a lactic acid bacteria cell comprising;

i) at least one genetic modification that eliminates enzyme activity of endogenously expressed acetolactate decarboxylase and at least one genetic modification that eliminates enzyme activity of endogenously expressed lactate dehydrogenase; and

ii) an isobutanol biosynthetic pathway; and

(b) culturing the cell of (a) under conditions wherein isobutanol is produced.

20. The method of claim 19 wherein the lactic acid bacteria cell of (a) further comprises at least one genetic modification that reduces pyruvate formate lyase activity.

21. An integration vector for lactic acid bacteria comprising:

a) a Tn-5 transposase coding region operably linked to a promoter that is active in lactic acid bacteria cells;

b) Tn5IE and TN5OE elements bounding a selection marker that is active in E. coli and lactic acid bacteria cells, and a DNA segment targeted for integration;

c) a second selection marker that is active in lactic acid bacteria cells;

d) an origin of replication for E. coli cells;

e) an origin of replication for lactic acid bacteria cells that is conditionally active;

wherein the Tn5IE and TN5OE elements direct random integration of the DNA segment of b) into the lactic acid bacteria cell genome.

22. A method for randomly integrating a DNA segment into a lactic acid bacteria cell genome comprising:

a) providing a vector comprising;

i) a Tn-5 transposase coding region operably linked to a promoter that is active in lactic acid bacteria cells;

ii) Tn5IE and TN5OE elements bounding a selection marker that is active in E. coli and lactic acid bacteria cells;

iii) a second selection marker active in lactic acid bacteria cells;

iv) an origin of replication for E. coli cells;

v) an origin of replication for lactic acid bacteria cells that is conditionally active;

b) placing a DNA segment for integration between the elements of step a (ii) creating an integration construction;

c) transforming the integration construction into a lactic acid bacteria cell whereby transformed cells are produced;

d) growing and selecting the transformed cells of step (c) in permissive conditions using the selection marker of step a (ii) to produce selected transformants; and

e) growing the selected transformants of step (d) in nonpermissive conditions;

wherein the vector is cured from the lactic acid bacteria cells and the DNA segment for integration is randomly integrated into the genome of said lactic acid bacteria cell.