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

Patent metadata

Field	Value
Publication number	US-2017145462-A1
Application number	US-201715418280-A
Country	US
Kind code	A1
Filing date	Jan 27, 2017
Priority date	Sep 30, 2008
Publication date	May 25, 2017
Grant date	—

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

1 - 29 . (canceled).

30 . A mutant of a parent Fusarium venenatum strain, comprising a polynucleotide encoding a polypeptide and pyrG and alpA genes, wherein both of the pyrG and alpA genes are modified rendering the mutant strain deficient in the production of orotine-5′-monophosphate decarboxylase and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

31 . The mutant strain of claim 30 , which produces at least 25% less of the orotine-5′-monophosphate decarboxylase and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

32 . The mutant strain of claim 30 , which is completely deficient in the production of the orotine-5′-monophosphate decarboxylase and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

33 . The mutant strain of claim 30 , which further comprises an amyA gene, wherein the amyA gene is modified rendering the mutant strain deficient in the production of an alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

34 . The mutant strain of claim 33 , which produces at least 25% less of the alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

35 . The mutant strain of claim 33 , which is completely deficient in the production of the alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

36 . The mutant strain of claim 30 , 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 of trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

37 . The mutant strain of claim 36 , which produces at least 25% less of the one or both of the trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

38 . The mutant strain of claim 36 , which is completely deficient in the production of the one or both of the trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

39 . The mutant strain of claim 30 , wherein the polypeptide is native or foreign to the Fusarium venenatum strain.

40 . A method for obtaining the mutant of a parent Fusarium venenatum strain of claim 30 , comprising:
(a) modifying pyrG and alpA genes in the parent Fusarium venenatum strain; and (b) identifying a mutant strain from step (a) wherein both of the pyrG and alpA genes are modified rendering the mutant strain deficient in the production of orotine-5′-monophosphate decarboxylase and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

41 . The method of claim 40 , wherein the mutant strain produces at least 25% less of the orotine-5′-monophosphate decarboxylase and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

42 . The method of claim 40 , wherein the mutant is completely deficient in the production of the orotine-5′-monophosphate decarboxylase and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

43 . The method of claim 40 , wherein the mutant further comprises an amyA gene, and wherein the amyA gene is modified rendering the mutant strain deficient in the production of an alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

44 . The method of claim 43 , wherein the mutant strain produces at least 25% less of the alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

45 . The method of claim 43 , wherein the mutant strain is completely deficient in the production of the alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

46 . The method of claim 40 , wherein the mutant strain further comprises one or both of the genes tri5 and dps1, and wherein the one or both of the genes are modified rendering the mutant strain deficient in the production of one or both of trichodiene synthase and cyclohexadepsipeptide synthetase, respectively, compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

47 . The method of claim 46 , wherein the mutant strain produces at least 25% less of the one or both of the trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

48 . The method of claim 46 , wherein the mutant strain is completely deficient in the production of the one or both of the trichodiene synthase and cyclohexadepsipeptide synthetase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

49 . A method of producing a polypeptide, comprising:
(a) cultivating the mutant of a parent Fusarium venenatum strain of claim 30 in a medium for the production of the polypeptide, wherein the mutant strain comprises a polynucleotide encoding the polypeptide and pyrG and alpA genes, and wherein both of the pyrG and alpA genes are modified rendering the mutant strain deficient in the production of orotine-5′-monophosphate decarboxylase and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions; and (b) recovering the polypeptide from the cultivation medium.

50 . The method of claim 49 , wherein the mutant strain produces at least 25% less of the orotine-5′-monophosphate decarboxylase and alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

51 . The method of claim 49 , wherein the mutant strain is completely deficient in the production of the orotine-5′-monophosphate decarboxylase and alkaline protease compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

52 . The method of claim 49 , wherein the mutant strain further comprises an amyA gene, and wherein the amyA gene is modified rendering the mutant strain deficient in the production of an alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

53 . The method of claim 52 , wherein the mutant strain produces at least 25% less of the alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

54 . The method of claim 52 , wherein the mutant strain is completely deficient in the production of the alpha-amylase compared to the parent Fusarium venenatum strain when cultivated under identical conditions.

55 . The method of claim 49 , wherein the mutant strain further comprises one or both of the genes tri5 and dps1, and wherein the one or both of the genes are modified rendering the mutant strain deficient in the production of one or both of trichodiene synthase and cyclohexadepsipeptide synthetase, respectivel

Assignees

• Novozymes Inc

Inventors

• Shasky Jeffrey
• Yoder Wendy

Classifications

• C12Y401/01023

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

• C12N9/242 Primary

  Fungal source · CPC title

• C12N9/58

  derived from fungi · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12Y302/01001

  Alpha-amylase (3.2.1.1) · CPC title