YEAST PRODUCTION HOST CELLS

• US 20110124060A1
• Filed: 09/29/2010
• Published: 05/26/2011
• Est. Priority Date: 09/29/2009
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: ANTHONY, LARRY CAMERON, Nelson, Mark J.
• Granted Patent: US 9,260,708 B2
• Time in Patent Office: Days
• US Class Current: 435/115
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/815 for yeasts other than Saccharomyces
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/1022 transferring aldehyde or ketonic groups (2.2)
• 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

Crabtree positive yeast cells that have endogenous expressed pyruvate decarboxylase genes inactivated and an engineered biosynthetic pathway utilizing pyruvate were found to have improved growth and product yield when glucose repression was reduced. These cells were able to grow in media containing a high glucose concentration.

Claims

1. A recombinant yeast production host cell comprising a genetic modification which has the effect of reducing glucose repression wherein the yeast production host cell is pdc- and wherein the unmodified yeast host cell is crabtree-positive.

2. The yeast production host cell of claim 1 comprising at least one inactivated endogenous gene encoding a pyruvate decarboxylase enzyme.

3. The yeast production host cell of claim 1 wherein the genetic modification which has the effect of reducing glucose repression is a modification of a gene encoding a protein selected from the group consisting of nuclear and cytoplasmic localized hexokinase, transcription activator Hap1, transcription repressor Mig1, transcription repressor Mig2, and SCF ubiquitin-ligase complex component GRR1.

4. The yeast production host cell of claim 3 wherein the genetic modification is a disruption in at least one endogenous gene encoding a hexokinase enzyme that has nuclear and cytoplasmic localization.

5. The yeast production host cell of claim 1 wherein the yeast is selected from the group consisting of Saccharomyces, Zygosaccharomyces, Schizosaccharomyces, Dekkera, Torulopsis, Brettanomyces, and Candida.

6. The yeast production host cell of claim 1 wherein the cell expresses a pyruvate-utilizing biosynthetic pathway wherein said cell is capable of producing a product of the pathway.

7. The yeast production host cell of claim 6 wherein the pyruvate-utilizing biosynthetic pathway comprises at least one heterologous gene.

8. The yeast production host cell of claim 6 comprising increased NADH-dependent enzyme activity.

9. The yeast production host cell of claim 7 wherein the increased NADH-dependent enzyme activity is an activity of the expressed pyruvate-utilizing biosynthetic pathway.

10. The yeast production host cell of claim 7 wherein the increased NADH-dependent enzyme activity is engineered by overexpressing an endogenous gene encoding a NADH-dependent enzyme.

11. The yeast production host cell of claim 7 wherein the increased NADH-dependent enzyme activity is engineered by expressing a heterologous gene encoding a NADH-dependent enzyme.

12. The yeast production host cell of claim 6 wherein the product of the pyruvate-utilizing biosynthetic pathway is selected from the group consisting of 2,3-butanediol, isobutanol, 2-butanol, 1-butanol, 2-butanone, valine, leucine, lactic acid, malate, isoamyl alcohol and isoprenoids.

13. The yeast production host cell of claim 6 wherein said pyruvate-utilizing biosynthetic pathway is an isobutanol biosynthetic pathway comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of:

i) pyruvate to acetolactate;

ii) acetolactate to 2,3-dihydroxyisovalerate;

iii) 2,3-dihydroxyisovalerate to α

-ketoisovalerate;

iv) α

-ketoisovalerate to isobutyraldehyde; and

v) isobutyraldehyde to isobutanol;

wherein yeast host cell produces isobutanol.

14. The yeast production host cell of claim 6 wherein said pyruvate-utilizing biosynthetic pathway is a 2-butanone biosynthetic pathway comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of:

i) pyruvate to acetolactate;

ii) acetolactate to acetoin;

iii) acetoin to 2,3-butanediol; and

iv) 2,3-butanediol to 2-butanone;

wherein said yeast host cell produces 2-butanone.

15. The yeast production host cell of claim 6 wherein said pyruvate-utilizing biosynthetic pathway is a 2-butanol biosynthetic pathway comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of:

i) pyruvate to acetolactate;

ii) acetolactate to acetoin;

iii) acetoin to 2,3-butanediol;

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

v) 2-butanone to 2-butanol;

wherein said yeast host cell produces 2-butanol.

16. The yeast production host cell of claim 6 wherein said pyruvate-utilizing biosynthetic pathway is a 1-butanol biosynthetic pathway comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of:

i) acetyl-CoA to acetoacetyl-CoA;

ii) acetoacetyl-CoA to 3-hydroxybutyryl-CoA;

iii) 3-hydroxybutyryl-CoA to crotonyl-CoA;

iv) crotonyl-CoA to butyryl-CoA

v) butyryl-CoA to butyraldehyde; and

v) butyraldehyde to1-butanol;

wherein said yeast host cell produces 1-butanol.

17. A method for the production of a product selected from the group consisting of 2,3-butanediol, isobutanol, 2-butanol, 1-butanol, 2-butanone, valine, leucine, lactic acid, malic acid, isoamyl alcohol, and isoprenoids comprising growing the yeast cell of any one of claims 1-9 under conditions wherein the product is produced and optionally recovering the product.

18. The method of claim 17 wherein said yeast host cell comprises an isobutanol biosynthetic pathway comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of:

i) pyruvate to acetolactate;

ii) acetolactate to 2,3-dihydroxyisovalerate;

iii) 2,3-dihydroxyisovalerate to α

-ketoisovalerate;

iv) α

-ketoisovalerate to isobutyraldehyde; and

v) isobutyraldehyde to isobutanol;

and wherein said product is isobutanol.

19. The method of claim 17 wherein said yeast host cell comprises a 2-butanone biosynthetic pathway comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of:

i) pyruvate to acetolactate;

ii) acetolactate to acetoin;

iii) acetoin to 2,3-butanediol; and

iv) 2,3-butanediol to 2-butanone;

and wherein said product is 2-butanone.

20. The method of claim 17 wherein said yeast host cell comprises a 2-butanol biosynthetic pathway comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of:

i) pyruvate to acetolactate;

ii) acetolactate to acetoin;

iii) acetoin to 2,3-butanediol;

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

v) 2-butanone to 2-butanol;

and wherein said product is 2-butanol.

21. The method of claim 17 wherein said yeast host cell comprises a 1-butanol biosynthetic pathway comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of:

i) acetyl-CoA to acetoacetyl-CoA;

ii) acetoacetyl-CoA to 3-hydroxybutyryl-CoA;

iii) 3-hydroxybutyryl-CoA to crotonyl-CoA;

iv) crotonyl-CoA to butyryl-CoA

v) butyryl-CoA to butyraldehyde; and

v) butyraldehyde to 1-butanol;

and wherein said product is 1-butanol.