Flux to acetolactate-derived products in lactic acid bacteria

• US 9,169,499 B2
• Filed: 09/29/2010
• Issued: 10/27/2015
• Est. Priority Date: 09/29/2009
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax Advanced Biofuels LLC (International Flavors & Fragrances Incorporated)
• Inventors: Paul, Brian James, Suh, Wonchul
• Primary Examiner(s): PAK, YONG D
• Application Number:
  US12/893,065
• Publication Number:
  US 20110136192A1
• Time in Patent Office: 1,854 Days
• US Class Current: 1/1
• 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 Lactobacillus cell comprising at least one engineered genetic modification that eliminates enzyme activity of endogenously expressed acetolactate decarboxylase, wherein the at least one engineered genetic modification is a deletion of at least a portion of an endogenous gene encoding acetolactate decarboxylase, and at least one engineered genetic modification that eliminates enzyme activity of endogenously expressed lactate dehydrogenase, wherein the at least one engineered genetic modification is a deletion of at least a portion of an endogenous gene encoding lactate dehydrogenase.

2. The recombinant Lactobacillus cell of claim 1 wherein said gene encoding acetolactate decarboxylase is selected from the group consisting of aldB, aldC, and ald.

3. The recombinant Lactobacillus cell of claim 2 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.

4. The recombinant Lactobacillus cell of claim 1 wherein said gene encodes lactate dehydrogenase and is selected from the group consisting of IdhL, IdhD, IdhL1, and IdhL2.

5. The recombinant Lactobacillus cell of claim 4 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.

6. The recombinant Lactobacillus cell of claim 1 further comprising at least one genetic modification that eliminates pyruvate formate lyase activity, wherein the at least one genetic modification is a deletion of at least a portion of an endogenous gene encoding pyruvate formate lyase.

7. The recombinant Lactobacillus cell of claim 1 further comprising at least one genetic modification that eliminates pyruvate formate lyase activating enzyme activity, wherein the at least one genetic modification is a deletion of at least a portion of an endogenous gene encoding pyruvate formate lyase activating enzyme.

8. The recombinant Lactobacillus cell of claim 6 or 7 wherein the said gene encoding pyruvate formate lyase is selected from the group consisting of pfl, pflB1 and pflB2and said gene encoding formate C-acetyltransferase activating enzyme is selected from the group consisting of pflA, pflA 1 and pflA2.

9. The recombinant Lactobacillus cell of claim 1 comprising an isobutanol biosynthetic pathway, wherein the isobutanol biosynthetic pathway comprises heterologous polynucleotides encoding polypeptides that catalyze the following substrate to product conversions:

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,

wherein

i) the substrate to product conversion of step (a) is performed by an acetolactate synthase enzyme;

ii) the substrate to product conversion of step (b) is performed by a ketol-acid reductoisomerase enzyme;

iii) the substrate to product conversion of step (c) is performed by a dihydroxy-acid dehydratase enzyme;

iv) the substrate to product conversion of step (d) is performed by a branched-chain α

-keto acid decarboxylase; and

v) the substrate to product conversion of step (e) is performed by an alcohol dehydrogenase enzyme.

10. A method for producing isobutanol comprising:

(a) providing the Lactobacillus cell of claim 9; and

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

11. The method of claim 10 wherein the Lactobacillus cell of (a) further comprises at least one genetic modification that eliminates pyruvate formate lyase activity, wherein the at least one genetic modification is a deletion of at least a portion of an endogenous gene encoding pyruvate formate lyase.