Expression of granular starch hydrolyzing enzyme in Trichoderma

• US 20050208623A1
• Filed: 11/18/2004
• Published: 09/22/2005
• Est. Priority Date: 11/21/2003
• Status: Active Grant

• Current Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Lantz, Suzanne E., Pepsin, Michael J., Dunn-Coleman, Nigel, Bower, Benjamin S., Baldwin, Toby M.
• Granted Patent: US 7,262,041 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C12N 1/14 Fungi culture of mushrooms A01G18/00; as new plants A01H15/00; Culture media therefor
• CPC Class Codes: C12N 9/242 Fungal source
• CPC Class Codes: C12N 9/2428 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• CPC Class Codes: C12P 19/14 produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase , e.g. by cellulase, hemicellulase
• CPC Class Codes: C12P 19/20 produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
• CPC Class Codes: C12P 19/24 produced by the action of an isomerase, e.g. fructose
• 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 present invention relates to filamentous fungal host cells and particularly Trichoderma host cells useful for the production of heterologous granular starch hydrolyzing enzymes having glucoamylase activity.

Claims

1. A method for producing a granular starch hydrolyzing enzyme having glucoamylase activity (GSHE) in a filamentous fungal host cell comprising

a) transforming a filamentous fungal host cell with a DNA construct comprising a promoter having transcriptional activity in the filamentous fungal host cell operably linked to a heterologous polynucleotide encoding a GSHE,

b) cultivating the transformed filamentous fungal host cell in a suitable culture medium to allow expression of said GSHE, and

c) producing the GSHE.

2. The method according to claim 1 further comprising recovering the produced GSHE.

3. The method according to claim 1, wherein the heterologous polynucleotide encoding the GSHE is derived from a strain of Humicola grisea or a strain of Aspergillus awamori.

4. The method according to claim 3, wherein the GSHE has at least 90% sequence identity with the sequence of SEQ ID NO:

3.

5. The method according to claim 4, wherein the GSHE has at least 95% sequence identity with the sequence of SEQ ID NO:

3.

6. The method according to claim 5, wherein the GSHE has the amino acid sequence of SEQ ID NO:

3.

7. The method according to claim 3, wherein the GSHE has at least 90% sequence identity with the sequence of SEQ ID NO:

6.

8. The method according to claim 7, wherein the GSHE has at least 95% sequence identity with the sequence of SEQ ID NO:

6.

9. The method according to claim 8, wherein the GSHE has the amino acid sequence of SEQ ID NO:

6.

10. The method according to claim 1, wherein the filamentous fungal host cell is a Trichoderma cell.

11. The method according to claim 10, wherein the Trichoderma cell is a T. reesei cell.

12. The method according to claim 1, wherein the filamentous fungal host cell is an Aspergillus cell.

13. The method according to claim 12, wherein the Aspergillus cell is an A. awamori, A. niger or A. oryzae cell.

14. The method according to claim 1, wherein one or more genes encoding cellulytic enzymes has been deleted in the filamentous fungal host cell.

15. The method according to claim 1, wherein a level of glycosylation of the expressed GSHE from the transformed filamentous fungal host cell is less than the level of glycosylation of the GSHE expressed in a native fungal host.

16. The method according to claim 15, wherein the native fungal host is a strain of Humicola grisea.

17. The method according to claim 1, wherein the amount of GSHE expressed is greater than 1.0 g/L of culture media.

18. The method according to claim 1, wherein the amount of GSHE expressed is greater than 10 g/L of culture media.

19. The method according to claim 1, wherein at a pH level of about 3.0 to 4.0 enzyme activity is greater for the produced GSHE than enzyme activity of the corresponding GSHE expressed and produced in a native fungal host.

20. The method according to claim 1, wherein the transformed fungal host cell is cultured under continuous fermentation conditions.

21. The method according to claim 1, wherein the transformed fungal host is cultured under batch fermentation conditions.

22. A recombinant Trichoderma cell comprising a heterologous polynucleotide encoding a granular starch hydrolyzing enzyme having glucoamylase activity (GSHE).

23. The recombinant Trichoderma cell of claim 22, wherein the Trichoderma cell is a T. reesei cell.

24. The recombinant Trichoderma cell of claim 22, wherein one or more genes encoding cellulytic enzymes has been deleted from the Trichoderma cell.

25. The recombinant Trichoderma cell of claim 22, wherein said heterologous polynucleotide is derived from a strain of Humicola grisea.

26. The recombinant Trichoderma cell of claim 25, wherein the strain is a Humicola grisea var. thermoidea.

27. The recombinant Trichoderma cell of claim 22, wherein the GSHE encoded by the heterologous polynucleotide has an amino acid sequence with at least 95% identity to SEQ ID NO:

3.

28. The recombinant Trichoderma cell of claim 22, wherein said heterologous polynucleotide is derived from a strain of Aspergillus awamori.

29. The recombinant Trichoderma cell of claim 28, wherein the strain is an Aspergillus awamori var. kawachi.

30. The recombinant Trichoderma cell of claim 22, wherein the GSHE encoded by the heterologous polynucleotide has an amino acid sequence with at least 95% identity to SEQ ID NO:

6.

31. A fermentation medium comprising a granular starch hydrolyzing enzyme having glucoamylase activity (GSHE) produced from a culture of Trichoderma reesei, wherein the Trichoderma reesei comprises a heterologous polynucleotide encoding a GSHE having at least 90% amino acid sequence identity with SEQ ID NO:

3 or a GSHE having at least 90% amino acid sequence identity with SEQ ID NO;

6.

32. The fermentation medium of claim 31, wherein the T. reesei comprises a heterologous polynucleotide encoding a GSHE having at least 95% amino acid sequence identity with SEQ ID NO:

3.

33. The fermentation medium of claim 32, wherein the T. reesei comprises a heterologous polynucleotide encoding a GSHE having the amino acid sequence of SEQ ID NO:

3.

34. The fermentation medium of claim 31, wherein the T. reesei comprises a heterologous polynucleotide encoding a GSHE having at least 95% amino acid sequence identity with SEQ ID NO:

6.

35. The fermentation medium of claim 34, wherein the T. reesei comprises a heterologous polynucleotide encoding a GSHE having the amino acid sequence of SEQ ID NO:

6.

36. A method for producing a granular starch hydrolyzing enzyme having glucoamylase activity (GSHE) in a Trichoderma host cell comprising

a) transforming a Trichoderma host cell with a DNA construct comprising a promoter having transcriptional activity in the host cell operably linked to a heterologous polynucleotide encoding a GSHE having at least 95% sequence identity to SEQ ID NO:

3 or SEQ ID NO;

6,

b) cultivating the transformed Trichoderma host cell to allow expression of said GSHE, and

C) producing the GSHE.

37. The method according to claim 36, wherein the heterologous polynucleotide encodes a GSHE having the sequence of SEQ ID NO:

3.

38. The method according to claim 36, wherein the heterologous polynucleotide encodes a GSHE having the sequence of SEQ ID NO:

6.

39. The method according to claim 36 further comprising recovering the produced GSHE.

40. The method according to claim 36, wherein the Trichoderma host cell is a T. reesei cell.