Transformation system in the field of filamentous fungal hosts

• US 20040002136A1
• Filed: 03/21/2003
• Published: 01/01/2004
• Est. Priority Date: 10/06/1998
• Status: Active Grant

• Current Assignee: Danisco US, Inc. (International Flavors & Fragrances Incorporated)
• Original Assignee: Dyadic International (Usa), Inc.
• Inventors: Van Zeijl, Cornelia Maria Johanna, Sinitsyn, Arkady Panteleimonovich, Punt, Peter Jan, Emalfarb, Mark Aaron, Bousson, Jean Christophe, Pynnonen, Christine Marie, Parriche, Martine, Burlingame, Richard Paul, Olson, Philip Terry
• Granted Patent: US 7,399,627 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/80 for fungi
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12N 9/2462 Lysozyme (3.2.1.17)
• CPC Class Codes: C12N 9/2482 Endo-1,4-beta-xylanase (3.2.1.8)
• CPC Class Codes: C12N 9/58 derived from fungi
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01008 Endo-1,4-beta-xylanase (3.2.1.8)
• CPC Class Codes: C12Y 302/01091 Cellulose 1,4-beta-cellobiosidase (3.2.1.91)

Abstract

A novel transformation system in the field of filamentous fungal hosts for expressing and secreting heterologous proteins or polypeptides is described. The invention also covers a process for producing large amounts of polypeptide or protein in an economical manner. The system comprises a transformed or transfected fungal strain of the genus Chrysosporium, more particularly of Chrysosporium lucknowense and mutants or derivatives thereof. It also covers transformants containing Chrysosporium coding sequences, as well expression-regulating sequences of Chrysosporium genes. Also provided are novel fungal enzymes and their encoding sequences and expression-regulating sequences.

Claims

1. A mutant Chrysosporium strain comprising a nucleic acid sequence encoding a polypeptide of interest, said nucleic acid sequence being operably linked to an expression-regulating region and optionally a secretion signal sequence, said mutant strain expressing said polypeptide of interest at a higher level than the corresponding non-mutant strain under the same conditions.

2. A mutant Chrysosporium strain according to claim 1, said mutant being obtained by recombinant methods comprising stable introduction of at least one heterologous nucleic acid sequence selected from heterologous polypeptide-encoding nucleic acid sequences, heterologous signal sequences and heterologous expression-regulating sequences.

3. A mutant Chrysosporium strain according to claim 2, wherein said polypeptide of interest is a heterologous polypeptide of plant, animal (including human), insect, algal, bacterial, archaebacterial or fungal origin.

4. A mutant Chrysosporium strain according to claim 1, wherein said polypeptide of interest is a homologous polypeptide which is expressed at a higher level than in the corresponding non-mutant strain under the same conditions.

5. A mutant Chrysosporium strain according to claim 1, wherein said polypeptide of interest is selected from carbohydrate-degrading enzymes, proteases, lipases, esterases, other hydrolases, oxidoreductases and transferases.

6. A mutant Chrysosporium strain according to claim 1, wherein said polypeptide of interest is selected from fugal enzymes allowing (over)production of primary metabolites, including organic acids, and secondary metabolites, including antibiotics.

7. A mutant Chrysosporium strain according to claim 1, wherein said polypeptide of interest is inactivated at a pH below 6.

8. A mutant Chrysosporium strain according to claim 1, wherein said polypeptide of interest exhibits optimal activity and/or stability at a pH above 6, and/or has more than 70% of its activity and/or stability at a pH above 6.

9. A mutant Chrysosporium strain according to claim 1, comprising a heterologous signal sequence.

10. A mutant Chrysosporium strain according to claim 1, comprising a fungal signal sequence.

11. A mutant Chrysosporium strain according to claim 10, wherein the fungal signal sequence is a signal sequence of a cellulase, β

-galactosidase, xylanase, pectinase, esterase, protease, amylase, polygalacturonase or hydrophobin.

12. A mutant Chrysosporium strain according to claim 1, further comprising a selectable marker.

13. A mutant Chrysosporium strain according to claim 12, wherein the selectable marker confers resistance to a drug or relieves a nutritional defect.

14. A mutant Chrysosporium strain according to claim 1, comprising a heterologous expression-regulating region.

15. A mutant Chrysosporium strain according to claim 1, comprising a fungal expression-regulating region.

16. A mutant Chrysosporium strain according to claim 15, wherein the expression-regulating region comprises is an inducible promoter.

17. A mutant Chrysosporium strain according to claim 15, wherein the expression-regulating region comprises a high expression promoter.

18. A mutant Chrysosporium strain according to claim 1, said mutant being obtained by mutagenesis steps, the steps including at least one step chosen from the group consisting of UV irradiation and chemical mutagenesis.

19. A mutant Chrysosporium strain according to claim 18, wherein the mutagenesis steps comprise a first UV irradiation step, a N-methyl-N′

-nitro-N-nitrosoguanidine treatment step, and a second UV irradiation step.

20. A mutant Chrysosporium strain according to claim 1, said mutant being derived from Chrysosporium lucknowense.

21. A mutant Chrysosporium strain according to claim 20, wherein said mutant is or is derived from a Chrysosporium lucknowense mutant strain selected from the group consisting of C. lucknowense strain C1 (VKM F-3500 D), UV13-6 (VKM F-3632 D), NG7C-19 (VKM F-3633 D), and UV18-25 (VKM F-3631 D).

22. A mutant Chrysosporium strain according to claim 1, wherein, when a Trichodenna reesei strain and said Chrysosporium strain are cultured under equivalent optimal conditions, when the Trichoderma culture attains a viscosity of 200-600 cP, said Chrysosporium strain exhibits a biomass of less than half that of the Trichoderma.

23. A mutant Chrysosporium strain according to claim 1, said strain producing at least the amount of cellulase in moles per liter as produced by any of the Chrysosporium lucknowense mutant strains C1 (VKM F-3500 D), UV13-6 (VKM F-3632 D), NG7C-19 (VKM F-3633 D), and UV18-25 (VKM F-3631 D).

24. A mutant Chrysosporium strain according to claim 1, said strain producing less protease than produced by the Chrysosporium lucknowense strain C1 (VKM F-3500 D).

25. A mutant Chrysosporium strain according to claim 24, wherein the strain produces less than half the amount of protease produced by the C1 strain.

31. A method of producing a polypeptide of interest, comprising culturing a strain according to claim 1 under conditions permitting expression of the protein or polypeptide, and recovering the subsequently produced polypeptide of interest.

32. The method of claim 31, wherein the conditions further permit secretion of the protein or polypeptide of interest.

35. A method according to any one of claims 31-34, wherein the protein or polypeptide is expressed as a precursor protein, further comprising the step of cleavage of the precursor into the polypeptide or precursor of interest.

36. The method according to claim 35, wherein the cleavage step is cleavage with an enzyme selected from the group consisting of Kex-2 like proteases, basic amino acid paired proteases, or Kex-2.

26. A nucleic acid construct comprising a nucleic acid expression-regulatory region derived from Chrysosporium lucknowense, operably linked to a polypeptide-encoding nucleic acid sequence.

27. A nucleic acid construct acording to claim 26, wherein the expression-regulatory region is derived from a Chrysosporium lucknowense strain selected from the group consisting of C1 (VKM F-3500 D) and UV18-25 (VKM F-3631 D).

28. A nucleic acid construct according to claim 26, said expression-regulatory region comprising a promoter sequence associated with cellulase expression, xylanase expression, or gpdA expression.

29. A recombinant microorganism containing a nucleic acid construct according to claim 26, and capable of expressing the polypeptide encoded by the coding nucleic acid sequence.

30. The microorganism of claim 29, wherein the microorganism is a fungal strain.

33. A method of producing a polypeptide of interest, said method comprising culturing a strain according to claim 29 under conditions permitting expression of the protein or polypeptide and recovering the subsequently produced polypeptide of interest.

34. The method of claim 33, wherein the conditions further permit secretion of the protein or polypeptide of interest.

37. The method according to claim 33, wherein the cultivation occurs at pH in the range 6-9, and/or at a temperature between 25 and 43°

C.

38. A method for producing a mutant Chrysosporium strain according to claim I comprising stably introducing a nucleic acid sequence encoding a heterologous or homologous polypeptide into a Chrysosporium strain, said nucleic acid sequence being operably linked to an expression regulating region.

39. A method according to claim 38, wherein the nucleic acid is introduced by the protoplast transformation method.

40. A protein corresponding to a Chrysosporium glycosyl hydrolase family 7, exhibiting at least 75% amino acid identity as determined by the BLAST algorithm with the amino acid sequence of SEQ ID NO:

1 or a part thereof having at least 20 contiguous amino acids which are identical to the corresponding part of the amino acid sequence 1-246 or 394-526 of SEQ ID NO;

1.

49. A method for hydrolysing β

-glucosidic bonds, comprising contacting a β

-glucoside with an enzyme according to any one of claims 40, 45, 47, and 48.

51. A nucleic acid sequence encoding a protein according to claim 40.

62. A recombinant microbial strain, containing a nucleic acid sequence according to claim 51, and capable of expressing the polypeptide encoded by the coding nucleic acid sequence.

41. A protein corresponding to a Chrysosporium glycosyl hydrolase family 10, exhibiting at least 70% amino acid identity as determined by the BLAST algorithm with the amino acid sequence of SEQ ID NO:

2 or a part thereof having at least 20 contiguous amino acids which are identical to the corresponding part of the amino acid sequence 1-383 of SEQ ID NO;

2.

50. A method for hydrolysing β

-xylosidic bonds, comprising contacting a β

-xyloside with an enzyme according to claim 41.

52. A nucleic acid sequence encoding a protein according to claim 41.

63. A recombinant microbial strain, containing a nucleic acid sequence according to claim 52 and capable of expressing the polypeptide encoded by the coding nucleic acid sequence.

42. A protein corresponding to a Chrysosporium glycosyl hydrolase family 10, exhibiting at least 65% amino acid identity as determined by the BLAST algorithm with the amino acid sequence of SEQ ID NO:

2 or a part thereof having at least 20 contiguous amino acids which are identical to the corresponding part of the amino acid sequence 1-383 of SEQ ID NO;

2,

53. A nucleic acid sequence encoding a protein according to claim 42.

64. A recombinant microbial strain, containing a nucleic acid sequence according to claim 53, and capable of expressing the polypeptide encoded by the coding nucleic acid sequence.

43. A protein corresponding to a Chrysosporium glycosyl hydrolase family 10 and comprising a cellulose-binding domain selected from the group comprising (a) domains having at least 75% amino acid identity with the amino acid sequence 22-53, and (b) domains having at least 20 contiguous amino acids identical to a part of amino acid sequence 22-53 of SEQ ID NO:

2.

44. A fungal glycosyl hydrolase of family 10, comprising a cellulose-binding domain, not derived from Fusarium oxysporum.

45. A protein corresponding to a Chrysosporium glycosyl hydrolase family 12, exhibiting at least 65% amino acid identity as determined by the BLAST algorithm with the amino acid sequence of SEQ ID NO:

3 or a part thereof having at least 20 contiguous amino acids which are identical to the corresponding part of the amino acid sequence 1-247 of SEQ ID NO;

3.

54. A nucleic acid sequence encoding a protein according to claim 45.

65. A recombinant microbial strain, containing a nucleic acid sequence according to claim 54, and capable of expressing the polypeptide encoded by the coding nucleic acid sequence.

46. A protein corresponding to a Chrysosporium glyceraldehyde phosphate dehydrogenase, exhibiting at least 86% amino acid identity as determined by the BLAST algorithm with the partial amino acid sequence of SEQ ID NO:

4 or a part thereof having at least 20 contiguous amino acids which are identical to the corresponding part of the amino acid sequence 1-277 of SEQ ID NO;

4.

55. A nucleic acid sequence encoding a protein according to claim 46.

66. A recombinant microbial strain, containing a nucleic acid sequence according to claim 55, and capable of expressing the polypeptide encoded by the coding nucleic acid sequence.

47. A protein corresponding to a Chrysosporium glycosyl hydrolase family 45, exhibiting at least 83% amino acid identity as determined by the BLAST algorithm with the amino acid sequence of SEQ ID NO:

5 or a part thereof having at least 20 contiguous amino acids which are identical to the corresponding part of the amino acid sequence 1-225 of SEQ ID NO;

5.

56. A nucleic acid sequence encoding a protein according to claim 47.

67. A recombinant microbial strain, containing a nucleic acid sequence according to claim 56, and capable of expressing the polypeptide encoded by the coding nucleic acid sequence.

48. A protein corresponding to a Chrysosporium glycosyl hydrolase family 6, exhibiting at least 50% amino acid identity as determined by the BLAST algorithm with the amino acid sequence of SEQ ID NO:

6 or a part thereof having at least 20 contiguous amino acids which are identical to the corresponding part of the amino acid sequences 1-395 of SEQ ID NO;

6.

57. A nucleic acid sequence encoding a protein according to claim 48.

68. A recombinant microbial strain, containing a nucleic acid sequence according to claim 57, and capable of expressing the polypeptide encoded by the coding nucleic acid sequence.

58. A nucleic acid sequence comprising at least 70% of the nucleotides contained in the 5′

-noncoding region of the nucleic acid sequence of any one of SEQ ID NOS;

1-6.

59. A nucleic acid construct comprising a nucleic acid expression-regulatory region derived from Chrysosporium, contained in the 5′

-noncoding region of the nucleic acid sequence of any one of SEQ ID No'"'"'s 1-6, operationally linked to a nucleic acid sequence encoding a polypetide of interest.

60. A recombinant microbial strain, containing a nucleic acid construct according to claim 59, and capable of expressing the polypeptide encoded by the coding nucleic acid sequence.

61. A recombinant microbial strain according to claim 60, which is a fungal strain.

69. A process of producing a polypeptide using a construct according to claim 59.

70. A process of producing a polypeptide using a microbial strain according to any one of claims 60-68.

71. An oligonucleotide probe comprising at least 15 contiguous nucleotides of the nucleic acid sequence of any one of SEQ ID No'"'"'s 1-6, or the complement thereof.

72. An oligonucleotide probe according to claim 71, wherein the probe is 20-50 nucleotides in length.

73. An oligonucleotide probe according to either one of claims 71 or 72, wherein the probe is labelled with a detectable label.