Transformation system in the field of filamentous fungal hosts

• US 6,573,086 B1
• Filed: 04/13/2000
• Issued: 06/03/2003
• Est. Priority Date: 10/06/1998
• Status: Expired due to Term

• Current Assignee: Danisco US, Inc. (International Flavors & Fragrances Incorporated)
• Original Assignee: Dyadic International (Usa), Inc.
• Inventors: Van Zeijl, Cornelia Marie Johanna, Pynnonen, Christine Marie, Burlingame, Richard Paul, Olson, Philip Terry, Emalfrab, Mark Aaron, Sinitsyn, Arkady Panteleimonovich, Punt, Peter Jan, Bousson, Jean Christophe, Parriche, Martine
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/548,938
• Time in Patent Office: 1,146 Days
• Field of Search: 435/254.11, 435/69.1, 435/209
• US Class Current: 435/254.11
• 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 expression-regulating region selected from the group consisting of a promoter sequence associated with cellulase expression, xylanase expression, or gpdA expression;

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 mutant strain expresses said polypeptide of interest at a higher level than the corresponding non-mutant strain under the same conditions.

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 fungal enzymes allowing production or overproduction of primary metabolites, organic acids, secondary metabolites, and 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, wherein said secretion signal sequence is 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, wherein said mutant Chrysosporium strain is a mutant Chrysosporium lucknowense strain.

21. A mutant Chrysosporium strain according to claim 20, wherein said mutant is or is a mutant of a 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 Trichoderma reesei and said Chrysosporium strain are cultured under conditions optimal for each strain, 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.