Methods for producing polypeptides in enzyme-deficient mutants of Fusarium venentatum

Patent metadata

Field	Value
Publication number	US-9593342-B2
Application number	US-201514974879-A
Country	US
Kind code	B2
Filing date	Dec 18, 2015
Priority date	Sep 30, 2008
Publication date	Mar 14, 2017
Grant date	Mar 14, 2017

Abstract

The present invention relates to methods of producing a polypeptide, comprising: (a) cultivating a mutant of a parent Fusarium venenatum strain in a medium for the production of the polypeptide, wherein the mutant strain comprises a polynucleotide encoding the polypeptide and one or more (several) genes selected from the group consisting of pyrG, amyA, and alpA, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of orotidine-5′-monophosphate decarboxylase, alpha-amylase, and alkaline protease, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions; and (b) recovering the polypeptide from the cultivation medium. The present invention also relates to enzyme-deficient mutants of Fusarium venenatum strains and methods for producing such mutants.

First claim

What is claimed is:

1. A mutant of a parent Fusarium venenatum strain, comprising a polynucleotide encoding a polypeptide and pyrG and amyA genes, wherein one or both of the pyrG and amyA genes are modified rendering the mutant strain deficient in the production of orotine-5′-monophosphate decarboxylase, alpha-amylase, or orotine-5′-monophosphate decarboxylase and alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

2. The mutant strain of claim 1 , which produces at least 25% less of the orotine-5′-monophosphate decarboxylase, alpha-amylase, or orotine-5′-monophosphate decarboxylase and alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

3. The mutant strain of claim 1 , which is completely deficient in the orotine-5′-monophosphate decarboxylase, alpha-amylase, or orotine-5′-monophosphate decarboxylase and alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

4. The mutant strain of claim 1 , which further comprises an alpA gene, wherein the alpA gene is modified rendering the mutant strain deficient in the production of an alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

5. The mutant strain of claim 4 , which produces at least 25% less of the alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

6. The mutant strain of claim 4 , which is completely deficient in the alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

7. The mutant strain of claim 1 , which further comprises one or both of the genes tri5 and dps1, wherein the one or both of the genes are modified rendering the mutant strain deficient in the production of one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

8. The mutant strain of claim 7 , which produces at least 25% less of the one or both enzymes of trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

9. The mutant strain of claim 7 , which is completely deficient in the one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

10. The mutant strain of claim 1 , wherein the polypeptide is native or foreign to the Fusarium venenatum strain.

11. A method for obtaining the mutant of a parent Fusarium venenatum strain of claim 1 , comprising:
(a) modifying pyrG and amyA genes in the parent Fusarium venenatum strain; and
(b) identifying a mutant strain from step (a) wherein one or both of the pyrG and amyA genes are modified rendering the mutant strain deficient in the production of orotine-5′-monophosphate decarboxylase, alpha-amylase, or orotine-5′-monophosphate decarboxylase and alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

12. The method of claim 11 , wherein the mutant strain produces at least 25% less of the orotine-5′-monophosphate decarboxylase, alpha-amylase, or orotine-5′-monophosphate decarboxylase and alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

13. The method of claim 11 , wherein the mutant strain is completely deficient in the orotine-5′-monophosphate decarboxylase, alpha-amylase, or orotine-5′-monophosphate decarboxylase and alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

14. The method of claim 11 , further comprising modifying an alpA gene rendering the mutant strain deficient in the production of an alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

15. The method of claim 14 , wherein the mutant strain produces at least 25% less of the alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

16. The method of claim 14 , wherein the mutant strain is completely deficient in the alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

17. The method of claim 11 , further comprising modifying one or both of the genes tri5 and dps1, rendering the mutant strain deficient in the production of one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

18. The method of claim 17 , wherein the mutant strain produces at least 25% less of the one or both enzymes of trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

19. The method of claim 17 , wherein the mutant strain is completely deficient in the one or both enzymes trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

20. A method of producing a polypeptide, comprising:
(a) cultivating the mutant of a parent Fusarium venenatum strain of claim 1 in a medium for the production of the polypeptide, wherein the mutant strain comprises a polynucleotide encoding the polypeptide and pyrG and amyA genes, wherein one or both of the pyrG and amyA genes are modified rendering the mutant strain deficient in the production of orotine-5′-monophosphate decarboxylase, alpha-amylase, or orotine-5′-monophosphate decarboxylase and alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions; and
(b) recovering the polypeptide from the cultivation medium.

21. The method of claim 20 , wherein the mutant strain produces at least 25% less of the orotine-5′-monophosphate decarboxylase, alpha-amylase, or orotine-5′-monophosphate decarboxylase and alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

22. The method of claim 20 , wherein the mutant strain is completely deficient in the orotine-5′-monophosphate decarboxylase, alpha-amylase, or orotine-5′-monophosphate decarboxylase and alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

23. The method of claim 20 , wherein the mutant strain further comprises an alpA gene, wherein the alpA gene is modified rendering the mutant strain deficient in the production of an alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

24. The method of claim 23 , wherein the mutant strain produces at least 25% less of the alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

25. The method of claim 23 , wherein the mutant strain is completely deficient in the alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

26. The method of claim 20 , wherein the mutant strain further comprises one or both of the genes tri5 and dps

Assignees

• Novozymes Inc

Inventors

• Shasky Jeffrey
• Yoder Wendy

Classifications

• C12Y401/01023

  Orotidine-5'-phosphate decarboxylase (4.1.1.23) · CPC title

• C12Y302/01001

  Alpha-amylase (3.2.1.1) · CPC title

• C12N15/80 Primary

  for fungi · CPC title

• C12N9/58

  derived from fungi · CPC title

• C12N9/2414

  Alpha-amylase (3.2.1.1.) · CPC title