PRODUCTION OF ISOBUTANOL IN YEAST MITOCHONDRIA

• US 20100129886A1
• Filed: 11/12/2009
• Published: 05/27/2010
• Est. Priority Date: 11/13/2008
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: Huang, Lixuan Lisa, Ye, Rick W., Anthony, Larry Cameron
• Granted Patent: US 8,828,694 B2
• Time in Patent Office: Days
• US Class Current: 435/160
• CPC Class Codes: C12N 15/8273 for drought, cold, salt resistance
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Yeast cells with modified expression of certain enzyme activities in the mitochondria are described for isobutanol production. Modifications described provide an isobutanol biosynthesis pathway in the yeast mitochondria.

Claims

1. A recombinant yeast cell comprising a heterologous gene encoding a mitochondrial localized polypeptide having α

-keto acid decarboxylase activity.

2. The yeast cell of claim 1 wherein the cell produces isobutanol in the mitochondria.

3. The yeast cell of claim 2 comprising genes encoding mitochondrial localized polypeptides having ketol-acid reductoisomerase activity and dihydroxy-acid dehydratase activity which are overexpressed.

4. The yeast cell of claim 3 wherein either one or both of the mitochondrial localized polypeptides having ketol-acid reductoisomerase activity and dihydroxy-acid dehydratase activity are heterologous to the yeast cell.

5. The yeast cell of claim 3 comprising at least one gene encoding a mitochondrial localized polypeptide having acetolactate synthase activity which is overexpressed.

6. The yeast cell of claim 3 wherein the at least one mitochondrial localized polypeptide having acetolactate synthase activity is heterologous to the yeast cell.

7. The yeast cell of claim 3 wherein the polypeptide having dihydroxy-acid dehydratase activity is a [4Fe-4S] or a [2Fe-2S] dihydroxy-acid dehydratase.

8. The yeast cell of claim 3 wherein the mitochondria is substantially devoid of a functional polypeptide having an enzyme activity selected from the group consisting of:

threonine deaminase activity;

isopropylmalate synthase activity, branched chain amino acid transaminase activity and pyruvate dehydrogenase activity.

9. The yeast cell of claim 3 comprising a gene encoding a polypeptide localized in the mitochondria having ATP-NAD (NADH) kinase activity which is over-expressed.

10. The yeast cell of claim 1 selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia.

11. The yeast cell of claim 1 comprising reduced pyruvate decarboxylase activity.

12. The yeast cell of claim 1 wherein the α

-keto acid decarboxylase activity is defined by the enzyme classification number EC 4.1.1.72.

13. The yeast cell of claim 3 wherein the ketol-acid reductoisomerase activity is defined by the enzyme classification number EC EC 1.1.1.86 and the dihydroxy-acid dehydratase activity is defined by the enzyme classification number EC 4.2.1.9.

14. The yeast cell of claim 5 wherein the acetolactate synthase activity is defined by the enzyme classification number EC 2.2.1.6 9.

15. The yeast cell of claim 8 wherein:

a) the threonine deaminase activity is defined by the enzyme classification number EC 4.3.1.19;

b) the isopropylmalate synthase activity is defined by the enzyme classification number EC 2.3.3.13;

c) the branched chain amino acid transaminase activity is defined by the enzyme classification number EC 2.6.1.42; and

d) the pyruvate dehydrogenase activity is defined by the enzyme classification number EC 1.2.4.1.

16. The yeast cell of claim 8 wherein the polypeptide having the pyruvate dehydrogenase activity is a multienzyme complex comprising proteins selected from the group consisting of:

PDA1, PDA1, PDB1, LAT1, LPD1, and PDX1.

17. The yeast cell of claim 9 wherein the ATP-NAD (NADH) kinase activity is defined by the enzyme classification number EC 2.7.1.86.

26. A method for making isobutanol comprising growing the yeast host cell of any of claims 3, 5 or 8 under conditions whereby isobutanol is produced.

18. A method of making a recombinant yeast cell for the production of isobutanol in the mitochondria comprising the steps of:

a) providing a yeast comprising mitochondrial biosynthetic pathways for the production of isoleucine from threonine and production of isopropyl malate, valine, and acetyl-CoA from pyruvate wherein the pathway for the production of isopropyl malate, valine, and acetyl-CoA from pyruvate comprises genes encoding polypeptides having the following activities;

i) ketol-acid reductoisomerase activity

ii) dihydroxy-acid dehydratase activity

iii) acetolactate synthase;

iv) isopropylmalate synthase activity,

v) branched chain amino acid transaminase activity;

vi) pyruvate dehydrogenase activity; and

vii) ATP-NAD (NADH) kinase activity;

and wherein the pathway for the production of isoleucine from threonine comprises at least one gene encoding a polypeptide having threonine deaminase activity;

b) introducing into the the yeast of (a) at least one gene encoding a mitochondria localized polypeptide having α

-keto acid decarboxylase activity;

c) increasing the expression of the polypeptides having ketol-acid reductoisomerase and dihydroxy-acid dehydratase activities;

wherein isobutanol is produced in the mitochondria of the yeast cell.

19. The method of claim 18 wherein the expression of the polypeptide having acetolactate synthase is increased.

20. The method of claim 18 wherein the expression of the polypeptide having an ATP-NAD (NADH) kinase activity is increased.

21. The method of claim 18 wherein at least one gene encoding a polypeptide having at least one of the following activities is down-regulated:

a) an isopropylmalate synthase activity,

b) a branched chain amino acid transaminase activity;

c) a pyruvate dehydrogenase activity; and

d) threonine deaminase activity.

22. The method of claim 18 wherein the polypeptide having a ketol-acid reductoisomerase activity comprises a mitochondrial targeting signal sequence.

23. The method of claim 18 wherein the polypeptide having a dihydroxy-acid dehydratase activity comprises a mitochondrial targeting signal sequence.

24. The method of claim 18 wherein the polypeptide having a α

-keto acid decarboxylase activity comprises a mitochondrial targeting signal sequence.

25. The method of claim 19 wherein the polypeptide having a ATP-NAD (NADH) kinase activity comprises a mitochondrial targeting signal sequence.