STRAIN FOR BUTANOL PRODUCTION WITH INCREASED MEMBRANE UNSATURATED TRANS FATTY ACIDS

• US 20100136641A1
• Filed: 11/25/2009
• Published: 06/03/2010
• Est. Priority Date: 12/03/2008
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: FLINT, DENNIS, VAN DYK, TINA K.
• Granted Patent: US 8,652,823 B2
• Time in Patent Office: Days
• US Class Current: 435/160
• CPC Class Codes: C12N 1/38 Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound C12N1/34 takes precedence
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Bacteria that are not natural butanol producers were found to have increased tolerance to butanol when the membrane content of unsaturated trans fatty acids was increased. Feeding cells with unsaturated trans fatty acids increased their concentration in the membrane, which may also be accomplished by expressing a fatty acid cistrans isomerase.

Claims

1. A butanol tolerant bacterial cell comprising an engineered butanol biosynthetic pathway and having an increased concentration of membrane unsaturated trans fatty acids as compared with a wildtype cell.

2. The butanol tolerant bacterial cell of claim 1 wherein the concentration of at least one unsaturated trans fatty acid selected from the group consisting of elaidic acid, vaccenic acid, and C16:

1 trans fatty acid is increased as compared with a wildtype cell.

3. The butanol tolerant bacterial cell of claim 1 wherein the cell is a member of a genus selected from the group consisting of Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Leuconostoc, and Brevibacterium.

4. The butanol tolerant bacterial cell of claim 1 wherein the cell is a member of the genus Lactobacillus, said cell produces isobutanol and the growth yield of the cell is at least about 1.6 to about 3.5-fold higher in 2.5% isobutanol than when the cell does not have an increased concentration of membrane unsaturated trans fatty acids.

5. The butanol tolerant bacterial cell of claim 1 wherein the cell is a member of the genus Lactobacillus, said cell produces 1-butanol and the growth yield of the cell is at least about 1.6 to about 3.0-fold higher in 2.25% 1-butanol than when the cell does not have an increased concentration of membrane unsaturated trans fatty acids.

6. The butanol tolerant bacterial cell of claim 1 wherein the cell is a member of the genus Lactobacillus, said cell produces 2-butanol, and the growth yield of the cell is at least about 2.2 to about 4-fold higher in 4.0% 2-butanol than when the cell does not have an increased concentration of membrane unsaturated trans fatty acids

7. The butanol tolerant bacterial cell of claim 1 wherein the concentration of at least one membrane unsaturated trans fatty acid is about 44 fold higher than a wildtype cell.

8. The butanol tolerant bacterial cell of claim 1 comprising at least one gene encoding fatty acid cistrans isomerase.

9. The butanol tolerant bacterial cell of claim 1 wherein the butanol biosynthetic pathway is selected from the group consisting of:

a) 1-butanol biosynthetic pathway

b) a 2-butanol biosynthetic pathway; and

c) an isobutanol biosynthetic pathway.

10. A method for the production of a butanol producing butanol tolerant bacterial cell comprising:

a) providing a bacterial cell comprising an engineered butanol biosynthetic pathway; and

b) feeding the bacterial cell of step (a) at least one trans fatty acid under conditions wherein the concentration of trans unsaturated fatty acids in the membrane of the cell are increased.

11. The method of claim 10 wherein the at least one fatty acid is selected from the group consisting of elaidic acid, vaccenic acid and C16:

1 trans fatty acid.

12. A method for the production of a butanol producing butanol tolerant bacterial cell comprising:

a) providing a bacterial cell comprising an engineered butanol biosynthetic pathway and at least one gene encoding a fatty acid cistrans isomerase; and

b) expressing the at least one gene encoding a fatty acid cistrans isomerase whereby the concentration of unsaturated trans fatty acids in the membrane of the cell are increased.

13. A method for the production of isobutanol comprising:

a) providing a bacterial cell comprising an engineered isobutanol biosynthetic pathway;

b) feeding the bacterial cell of step (a) at least one trans fatty acid under conditions wherein the concentration of unsaturated trans fatty acids in the membrane of the cell are increased; and

c) growing the bacterial cell of step (b) under conditions wherein isobutanol is produced.

14. The method of claim 13 wherein the isobutanol biosynthetic pathway comprises:

a) at least one gene encoding acetolactate synthase;

b) at least one gene encoding acetohydroxy acid isomeroreductase;

c) at least one gene encoding acetohydroxy acid dehydratase;

d) at least one gene encoding a branched-chain keto acid decarboxylase; and

e) at least one gene encoding branched-chain alcohol dehydrogenase.

15. A method for the production of isobutanol comprising:

a) providing a bacterial cell comprising an engineered isobutanol biosynthetic pathway and at least one gene encoding encoding cistrans isomerase;

b) expressing the at least one gene encoding fatty acid cistrans isomerase whereby the concentration of unsaturated trans fatty acids in the membrane of the cell are increased; and

c) growing the bacterial cell of step (b) under conditions wherein isobutanol is produced.

16. The method of claim 15 wherein the isobutanol biosynthetic pathway comprises:

a) at least one gene encoding acetolactate synthase;

b) at least one gene encoding acetohydroxy acid isomeroreductase;

c) at least one gene encoding acetohydroxy acid dehydratase;

d) at least one gene encoding a branched-chain keto acid decarboxylase; and

e) at least one gene encoding branched-chain alcohol dehydrogenase.