Expression of hexose kinase in recombinant host cells

• US 8,637,289 B2
• Filed: 12/29/2010
• Issued: 01/28/2014
• Est. Priority Date: 12/29/2009
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: Anthony, Larry Cameron, Kruckeberg, Arthur Leo
• Primary Examiner(s): Carlson, Karen Cochrane
• Application Number:
  US12/980,607
• Publication Number:
  US 20120015416A1
• Time in Patent Office: 1,126 Days
• Field of Search: 435/194, 435/254.1
• US Class Current: 435/194
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The invention relates to a recombinant host cell having (a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity; (b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and optionally (c) a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. Additionally, the invention relates to methods of making and using such recombinant host cells including, for example, methods of increasing glucose consumption, methods of improving redox balance, and/or methods of increasing the production of a product of a pyruvate-utilizing pathway.

Claims

1. A recombinant yeast host cell comprising:

(a) a modification in an endogenous polynucleotide encoding a polypeptide having dual-role hexokinase activity in the host cell wherein the activity of the polypeptide of (a) is reduced or substantially eliminated; and

(b) a heterologous polynucleotide encoding a polypeptide having hexose kinase activity; and

(c) a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity wherein pyruvate decarboxylase activity is reduced or substantially eliminated.

2. The recombinant host cell of claim 1 wherein the glucose consumption rate is increased as compared to that of the host cell comprising (a) but not (b).

3. The recombinant host cell of claim 1 wherein the modification of (a) is a deletion.

4. The recombinant host of claim 1 wherein the polypeptide of (a) is HXK2, and wherein the recombinant host cell is S. cerevisiae.

5. The recombinant host cell of claim 4 wherein the heterologous polynucleotide of (b) encodes a polypeptide of SEQ ID NO:

4, 6, 8, 121, or 123.

6. The recombinant host cell of claim 4 wherein the heterologous polynucleotide of (b) encodes a polypeptide that has at least about 85% identity to SEQ ID NO:

4, 6, 8, 121, or 123.

7. The recombinant host cell of claim 4 wherein the heterologous polynucleotide of (b) en codes a polypeptide that has at least about 85% identity to SEQ ID NO:

130.

8. The recombinant host cell of claim 4 wherein the heterologous polynucleotide of (b) comprises a conditional promoter and encodes a polypeptide having at least 85% identity to SEQ ID NO:

4 or SEQ ID NO;

2.

9. The recombinant host cell of claim 8 wherein the conditional promoter comprises a sequence derived from the OLE1 promoter region.

10. The recombinant host cell of claim 4 comprising an isobutanol biosynthetic pathway, the isobutanol biosynthetic pathway comprising a heterologous polynucleotide encoding a polypeptide that catalyzes a substrate to product conversion of:

(i) pyruvate to acetolactate;

(ii) acetolactate to 2,3-dihydroxyisovalerate;

(iii) 2,3-dihydroxyisovalerate to 2-ketoisovalerate;

(iv) 2-ketoisovalerate to isobutyraldehyde; and

(v) isobutyraldehyde to isobutanol; and

wherein the host cell produces isobutanol.

11. The recombinant host cell of claim 1,

wherein the polypeptide of (a) is RAG5, and wherein the recombinant host cell is K. lactis, or

wherein the polypeptide of (a) is HPGLK1, and wherein the recombinant host cell is H. polymorpha, or

wherein the polypeptide of (a) is HXK2, and wherein the recombinant host cell is S. pombe.

12. The recombinant host cell of claim 1 wherein the polynucleotide of (b) comprises a promoter such that the polypeptide of (b) is conditionally expressed.

13. The recombinant host cell of claim 1 wherein the polynucleotide of (b) comprises a promoter such that the polypeptide of (b) is constitutively expressed.

14. The recombinant host cell of claim 1 wherein the heterologous polynucleotide of (b) comprises the polypeptide of (a) having a deletion of a protein interaction domain that prevents function as a transcriptional regulator.

15. The recombinant host cell of claim 1 wherein the heterologous polynucleotide of (b) encodes a polypeptide that has at least about 85% identity to SEQ ID NO:

2, 115, 117, 119, 4, 6, 8, 121, or 123.

16. The recombinant host cell of claim 1 wherein the polypeptide encoded by the heterologous polynucleotide of (b) is constitutively expressed.

17. The recombinant host cell of claim 1 wherein the heterologous polynucleotide of (b) comprises i) a promoter region derived from S. cerevisiae ADH1 promoter region or ii) a promoter region having at least about 85% identity to SEQ ID NO:

131.

18. The recombinant host cell of claim 1 further comprising a pyruvate-utilizing biosynthetic pathway which forms a product 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.

19. The recombinant host cell of claim 18 wherein the product is isobutanol.

20. The recombinant host cell of claim 18 wherein the pyruvate-utilizing biosynthetic pathway is an isobutanol biosynthetic pathway comprising a heterologous polynucleotide encoding a polypeptide for each of the following substrate to product conversions:

(i) pyruvate to acetolactate;

(ii) acetolactate to 2,3-dihydroxyisovalerate;

(iii) 2,3-dihydroxyisovalerate to 2-ketoisovalerate;

(iv) 2-ketoisovalerate to isobutyraldehyde; and

(v) isobutyraldehyde to isobutanol; and

wherein the host cell produces sobutanol.

21. The recombinant host cell of claim 19 wherein the pyruvate-utilizing biosynthetic pathway is a 2-butanone biosynthetic pathway comprising a heterologous polynucleotide encoding a polypeptide for each the following substrate to product conversions:

(i) pyruvate to acetolactate;

(ii) acetolactate to acetoin;

(iii) acetoin to 2,3-butanediol; and

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

wherein the host cell produces 2-butanone.

22. The recombinant host cell of claim 18 wherein the pyruvate-utilizing biosynthetic pathway is a 2-butanol biosynthetic pathway comprising a heterologous polynucleotide encoding a polypeptide for each the following substrate to product conversions:

(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 the host cell produces 2-butanol.

23. The recombinant host cell of claim 18 wherein the pyruvate-utilizing biosynthetic pathway is a 1-butanol biosynthetic pathway comprising a heterologous polynucleotide encoding a polypeptide for each the following substrate to product conversions:

(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

(vi) butyraldehyde to 1-butanol; and

wherein the host cell produces isobutanol.

24. A method for the production of isobutanol comprising:

(a) providing a recombinant host cell of claim 10; and

(b) growing the recombinant host cell of claim 10 under conditions wherein isobutanol is produced.

25. A method of increasing glucose consumption of a recombinant host cell comprising:

(i) providing the recombinant host cell of claim 1; and

(ii) growing the recombinant host cell under conditions wherein the heterologous polynucleotide of (b) is expressed in functional form;

wherein the glucose consumption of the recombinant host cell is greater than the glucose consumption of a host cell comprising (a) but not (b).

26. A method of increasing the formation of a product of a pyruvate-utilizing biosynthetic pathway comprising:

(i) providing the recombinant host cell of claim 18, or combinations thereof; and

(ii) growing the recombinant host cell under conditions wherein the product of the pyruvate-utilizing pathway is formed;

wherein the amount of product formed by the recombinant host cell is greater than the amount of product formed by a host cell comprising (a) but not (b).

27. The method of claim 26 wherein the product is butanol.

28. The method of claim 27, wherein said butanol is isobutanol.

29. A method for improving the redox balance of a recombinant host cell comprising:

(i) providing the recombinant host cell of claim 1; and

(ii) growing the recombinant host cell of (i) under conditions wherein the heterologous polynucleotide of (b) is expressed in functional form wherein the redox balance of such a recombinant host cell is improved compared to the redox balance of a recombinant host cell comprising (i)(a) but not (i)(b).

30. A method for the production of isobutanol comprising:

(a) providing a recombinant host cell of claim 20; and

(b) growing the recombinant host cell of claim 20 under conditions wherein isobutanol is produced.