Expression of granular starch hydrolyzing enzymes in Trichoderma and process for producing glucose from granular starch substrates

• US 20050136525A1
• Filed: 11/18/2004
• Published: 06/23/2005
• 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: Chotani, Gopal K., Lantero, Oreste J. Jr., Lantz, Suzanne E., Shetty, Jayarama K., Pepsin, Michael J., Dunn-Coleman, Nigel, Bower, Benjamin S., Strohm, Bruce A., Baldwin, Toby M.
• Granted Patent: US 7,303,899 B2
• Time in Patent Office: Days
• US Class Current: 435/204
• 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 (GSHE). Further the invention relates to a method for producing a glucose syrup comprising contacting a granular starch slurry obtained from a granular starch substrate simultaneously with an alpha amylase and a GSHE at a temperature equal to or below the gelatinization temperature of the granular starch to obtain a composition of a glucose syrup.

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 or SEQ ID NO;

6.

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

6. 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.

7. The method according to claim 1, wherein enzyme activity at a pH level of 3.0 to 4.0 is greater for the produced GSHE in the transformed filamentous fungal host cell than the corresponding produced GSHE from a native fungal host.

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

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

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

11. The recombinant Trichoderma cell of claim 10, wherein said heterologous polynucleotide is derived from a strain of Humicola grisea or from a strain of Aspergillus awamori.

12. 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 with SEQ ID NO;

6.

13. A process for producing a glucose syrup from a granular starch slurry, said process comprising contacting a granular starch substrate simultaneously with the fermentation medium of claim 12 and an alpha amylase derived from a Bacillus strain at a temperature below the gelatinization temperature of the granular starch and allowing the alpha amylase and GSHE to act for a period of time between 2 and 100 hours to hydrolyze the granular starch to obtain a glucose syrup.

14. An enzyme composition having granular starch hydrolyzing activity comprising an alpha amylase and a glucoamyalse having granular starch hydrolyzing activity (GSHE), wherein the ratio of the alpha amylase to GSHE is between 15:

1 and 1;

15.

15. The enzyme composition of claim 14, wherein the alpha amylase to GSHE ratio is 10:

1 to 1;

10.

16. A one-step process for producing a glucose syrup from a granular starch slurry, said process comprising

contacting a granular starch slurry obtained from a granular starch substrate simultaneously with an alpha amylase and a glucoamylase having granular starch hydrolyzing activity (GSHE) at a temperature equal to or below the gelatinization temperature of the granular starch and

allowing the alpha amylase and the GSHE to act for a period of time sufficient to hydrolyze the granular starch to obtain a composition comprising a glucose syrup.

17. The process according to claim 16, wherein the granular starch substrate is obtained from corn, wheat, barley or rice.

18. The process according to claim 17, wherein the granular starch substrate is corn starch obtained from whole grain.

19. The process according to claim 16, wherein the granular starch substrate is dry milled or wet milled.

20. The process according to claim 16, wherein the granular starch slurry has a dry solids content of 15-55%.

21. The process according to claim 20, wherein the granular starch slurry has a dry solids content of 15-45%.

22. The process according to claim 16, wherein the temperature is less than 65°

C. and greater than 50°

C.

23. The process according to claim 22, wherein the temperature is less than 65°

C. and greater than 55°

C.

24. The process according to claim 16, wherein the period of time sufficient to hydrolyze the granular starch is 2 to 100 hours.

25. The process according to claim 16, wherein the contacting step is conducted at a pH of 5.0 to 6.0.

26. The process according to claim 16, wherein the alpha amylase is derived from a Bacillus.

27. The process according to claim 16, wherein the amount of alpha amylase per metric ton of starch is in the range of 0.05 to 5.0 kg.

28. The process according to claim 27, wherein the amount of alpha amylase per metric ton of starch is in the range of 0.05 to 2.0 kg.

29. The process according to claim 16, wherein the GSHE is obtained from the heterologous expression of a polynucleotide encoding a GSHE having at least 90% sequence identity to the sequence of SEQ ID NO:

3 or SEQ ID NO;

6.

30. The process according to claim 16, wherein the amount of GSHE is in the range of 0.01 to 10.0 GSHE U/g starch dry solids.

31. The process according to claim 30, wherein the amount of GSHE is in the range of 0.1 to 5.0 GSHE Units/g starch dry solids.

32. The process according to claim 31, wherein the amount of GSHE is in the range of 0.25 to 2.5 GSHE Units/g starch dry solids.

33. The process according to claim 16, wherein at least 90% of the granular starch is hydrolyzed within 24 hours.

34. The process according to claim 16 further comprising recovering the glucose syrup.

35. The process according to claim 34, wherein the glucose syrup is recovered by filtration separation.

36. The process according to claim 16 further comprising converting the glucose syrup to a desired end product.

37. The process according to claim 36, wherein the desired end product is a fructose syrup.

38. The process according to claim 36, wherein the desired end product is ethanol.

39. The process according to claim 36, wherein the desired end product is a crystallized glucose.

40. The process according to claim 35, wherein residual starch is obtained as a by product of the glucose syrup separation.

41. The process according to claim 40 further comprising recycling the residual starch by contacting the residual starch with the GSHE and alpha amylase at a temperature below the gelatinization temperature of the granular starch.

42. The residual starch obtained according to the process of claim 40.

43. A process for the production of a high fructose starch-base syrup comprising enzymatically converting the glucose syrup obtained according to the process of claim 16 to a fructose syrup.

44. The process according to claim 43, wherein the converting step includes use of a glucose isomerase.

45. A process for production of a residual starch composition comprising, contacting a granular starch substrate according to claim 16 to produce a composition comprising a glucose syrup and separating the glucose syrup from the composition to obtain a mixture including residual starch.

46. A method of using the residual starch obtained according to claim 45 in a starch saccharification processes.

47. A process for the production of ethanol comprising subjecting the glucose syrup produced according to claim 16 to fermentation with an ethanolgenic microorganism.

48. The process according to claim 47, wherein the fermentation is carried out separately and sequentially to the contacting step.

49. The process according to claim 47, wherein the fermentation is carried out simultaneously with the contacting step.

50. The process according to claim 47 further comprising recovering the ethanol from the fermentation.