Yeast production host cells

• US 9,260,708 B2
• Filed: 09/29/2010
• Issued: 02/16/2016
• 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.
• Primary Examiner(s): PAK, YONG D
• Application Number:
  US12/893,089
• Publication Number:
  US 20110124060A1
• Time in Patent Office: 1,966 Days
• US Class Current: 1/1
• 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) a first genetic modification, which has the effect of reducing glucose repression, wherein the first genetic modification is a deletion in at least one endogenous gene encoding a hexokinase enzyme that has nuclear and cytoplasmic localization, wherein the deletion eliminates the activity of the hexokinase enzyme; and

(b) a second genetic modification, wherein the second genetic modification is a deletion in at least one endogenous gene encoding a pyruvate decarboxylase enzyme, wherein the deletion eliminates pyruvate decarboxylase activity; and

(c) a pyruvate utilizing biosynthetic pathway, wherein the pyruvate utilizing biosynthetic pathway is an isobutanol biosynthetic pathway comprising heterologous DNA molecules encoding polypeptides that catalyze the following substrate to product conversions;

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 the yeast production host cell produces isobutanol, and

wherein growth of the recombinant yeast production host cell is improved at least 2-fold as compared to a recombinant yeast production host cell without the first genetic modification.

2. 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.

3. The yeast production host cell of claim 1 comprising increased NADH-dependent enzyme activity.

4. The yeast production host cell of claim 3 wherein the increased NADH-dependent enzyme activity is an activity of the expressed pyruvate-utilizing biosynthetic pathway.

5. The yeast production host cell of claim 3 wherein the increased NADH-dependent enzyme activity is engineered by overexpressing an endogenous gene encoding a NADH-dependent enzyme.

6. The yeast production host cell of claim 3 wherein the increased NADH-dependent enzyme activity is engineered by expressing a heterologous gene encoding a NADH-dependent enzyme.

7. A method for the production of isobutanol comprising growing the yeast cell of claim 1 under conditions wherein the isobutanol is produced and optionally recovering the isobutanol.