Hosts and fermentation processes for cellulase production

• US 8,323,931 B2
• Filed: 11/03/2009
• Issued: 12/04/2012
• Est. Priority Date: 11/03/2008
• Status: Active Grant

• Current Assignee: Iogen Energy Corporation (Iogen Corporation)
• Original Assignee: Iogen Energy Corporation (Iogen Corporation)
• Inventors: Gudynaite-Savitch, Loreta, Hindle, Christopher D., White, Theresa C.
• Primary Examiner(s): RAGHU, GANAPATHIRAM
• Application Number:
  US12/611,486
• Publication Number:
  US 20100129880A1
• Time in Patent Office: 1,127 Days
• Field of Search: 435/99, 435/209, 435/69.1, 435/254.1, 435/254.3, 435/254.6, 435/254.7, 435/274, 435/277, 435/262, 536/23.2, 530/350
• US Class Current: 435/99
• CPC Class Codes: C12N 1/38 Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound C12N1/34 takes precedence
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)

Abstract

A fermentation process for the production of cellulase mixtures is provided. The process comprises providing a genetically modified host filamentous fungus that overexpresses a Xyr1 transcription factor and/or that is partially or completely deficient in expressing one or more hemicellulase enzyme. The host filamentous fungus is cultured in a medium comprising a carbon source. The carbon source contains from about 60 wt % to about 100 wt % hemicellulose-derived carbohydrate and less than 5% of a cellulase-inducing carbohydrate or contains from about 25 wt % to about 100% wt % hemicellulose-derived sugar alcohol in combination with from about 0 wt % to about 75 wt % glucose, glycerol or a combination thereof.

Claims

1. A fed-batch or continuous fermentation process for the production of a cellulase mixture, said process comprising:

a) providing a modified host filamentous fungus that overexpresses a Xyr1 transcription factor, said Xyr1 transcription factor being a protein (i) having an amino acid sequence exhibiting from about 90% to about 100% identity to SEQ ID NO;

27 (ii) containing a class III zinc binuclear cluster with a conserved amino acid motif (CX₂CX₆CX_5-12CX₂CX_6-8C) at the N-terminal part of the protein, and (iii) exhibiting DNA binding activity specific to a consensus sequence GGC(T/A)₃-like motif within cellulase and/or hemicellulase promoter sequences; and

b) culturing the modified host filamentous fungus of step a) in a medium comprising a carbon source, wherein the carbon source contains from about 60 wt % to about 100 wt % of a hemicellulose-derived carbohydrate selected from the group consisting of xylo-oligosaccharides, arabinoxylo-oligosaccharides, D-xylose, xylobiose, L-arabinose, and combinations thereof, and about 0 wt % of a cellulase-inducing carbohydrate selected from the group consisting of cellulose, lactose, cellobiose, sophorose, gentiobiose and a combination thereof, to produce a cellulase mixture;

wherein the fermentation process produces at least 10 g/L of the cellulase mixture, cellulase components represent at least 40 wt % of the total protein in the cellulase mixture, and the cellulase mixture exhibits at least 1.7-fold higher cellulase activity than a cellulase mixture produced by a parental filamentous fungus that does not overexpress the Xyr1 transcription factor when cultured in the same medium.

2. The fermentation process of claim 1, wherein the Xyr1 protein comprises the amino acid sequence of SEQ ID NO:

27.

3. The fermentation process of claim 1, wherein the modified host filamentous fungus is partially or completely deficient in expressing one or more hemicellulase enzyme.

4. The fermentation process of claim 3, wherein the one or more hemicellulase enzyme is selected from the group consisting of a xylanase, a beta-xylosidase, an alpha-arabinofuranosidase, a beta-mannanase, an alpha-glucuronidase, a acetyl xylan esterase and a combination thereof.

5. The fermentation process of claim 4, wherein the one or more hemicellulase enzyme is selected from the group consisting of a xylanase, a beta-xylosidase and a combination thereof.

15. The fermentation process of claim 1, wherein the process is characterized by having at least about a 2-fold increase in specific productivity (q_p) relative to an equivalent process utilizing a parental filamentous fungus that does not overexpress Xyr1.

17. The fermentation process of claim 1, wherein the host filamentous fungus is a species of Trichoderma, Hypocrea, Aspergillus, Humicola, Fusarium, Penicillium, Neurospora, Phanerochaete, Agaricus, Chaetomium, or Magnaporthe.

18. The fermentation process of claim 17, wherein the host filamentous fungus is Trichoderma reesei or Hypocrea jecorina.

21. The fermentation process of claim 1, wherein the medium comprises one or more additional carbon sources.

22. The fermentation process of claim 21, wherein the one or more additional carbon source is glycerol, one or more sugar alcohols or an organic acid.

23. The fermentation process of claim 22, wherein the one or more additional carbon source comprises xylitol.

27. The fermentation process of claim 1, wherein the step of culturing is conducted at a temperature of from about 20°

C. to about 35°

C. and a pH of from about 3.0 to about 6.5.

29. The fermentation process of claim 1, wherein the process is fed-batch.

31. The fermentation process of claim 1, wherein the process is continuous.

33. The fermentation process of claim 1, wherein the process is conducted aerobically.

35. The fermentation process of claim 1, wherein the carbon source contains 0 wt % cellulase-inducing carbohydrate.

37. A process for the hydrolyzing a cellulose substrate comprising contacting said substrate with a cellulase mixture produced by the fermentation process of claim 1.

38. The process of claim 37, wherein the cellulose substrate is a pretreated lignocellulosic feedstock.

44. The fermentation process of claim 1, wherein said Xyr1 transcription factor has an amino acid sequence exhibiting from about 95% to about 100% identity to SEQ ID NO:

27.

6. A fermentation process for the production of a cellulase mixture, said process comprising:

a) genetically modifying a host filamentous fungus to overexpress a Xyr1 transcription factor, said Xyl transcription factor being a protein (i) having an amino acid sequence exhibiting from about 90% to about 100% identity to SEQ ID NO;

27 (ii) containing a class III zinc binuclear cluster with a conserved amino acid motif (CX₂CX₆CX_5-12CX₂CX_6-8C) at the N-terminal part of the protein, and (iii) exhibiting DNA binding activity specific to a consensus sequence GGC(T/A)₃-like motif within cellulase and/or hemicellulase promoter sequences; and

b) culturing the host filamentous fungus of step a) in a medium comprising a carbon source, wherein the carbon source contains from about 60 wt % to about 100 wt % of a hemicellulose-derived carbohydrate selected from the group consisting of xylo-oligosaccharides, arabinoxylo-oligosaccharides, D-xylose, xylobiose, L-arabinose, and combinations thereof, and about 0 wt % of a cellulase-inducing carbohydrate selected from the group consisting of cellulose, lactose, cellobiose, sophorose, gentiobiose and a combination thereof, to produce a cellulase mixture;

wherein the fermentation process produces at least 10 g/L of the cellulase mixture, and wherein cellulase components represent at least 40 wt % of the total protein in the cellulase mixture and the cellulase mixture exhibits at least 1.7-fold higher cellulase activity than a cellulase mixture produced by a parental filamentous fungus that does not overexpress the Xyr1 transcription factor when cultured in the same medium.

7. The fermentation process of claim 6, wherein the step of genetically modifying comprises

a) transforming the host filamentous fungus with a Xyr1 genetic construct in which a nucleic acid sequence encoding the Xyr1 transcription factor is operatively linked to a promoter nucleic acid sequence;

and

b) selecting those transformants from step a) containing the Xyr1 genetic construct.

8. The fermentation process of claim 6, wherein the Xyr1 protein comprises the amino acid sequence of SEQ ID NO:

27.

9. The fermentation process of claim 7, wherein the promoter nucleic acid sequence is native or heterologous to the host filamentous fungus.

10. The fermentation process of claim 7, wherein the promoter nucleic acid sequence is derived from a gene whose expression is induced during growth of the host filamentous fungus on a carbon source comprising hemicellulose derived carbohydrates.

11. The fermentation process of claim 10, wherein the promoter nucleic acid sequence is derived from a gene selected from the group consisting of T. reesei bxl1, T. reesei xln1 and T. reesei xln2.

12. The fermentation process of claim 7, wherein the promoter nucleic acid sequence is from a gene whose expression is constitutive during growth of the host filamentous fungus.

13. The fermentation process of claim 6, wherein the step of genetically modifying further comprises modifying one or more gene in said host filamentous fungus encoding a hemicellulase enzyme selected from the group consisting of xylanases, beta-xylosidases, alpha-arabinofuranosidases, beta-mannanases, alpha-glucuronidases, acetylxylan esterase and a combination thereof, so that said host filamentous fungus is partially or completely deficient in expressing the one or more hemicellulase enzyme.

14. The fermentation process of claim 13, wherein said one or more hemicellulase enzyme is selected from the group consisting of a xylanase and a beta-xylosidase.

16. The fermentation process of claim 6, wherein the process is characterized by having at least about a 2-fold increase in specific productivity (q_p) relative to an equivalent process utilizing a parental filamentous fungus that does not overexpress Xyr1.

19. The fermentation process of claim 6, wherein the host filamentous fungus is a species of Trichoderma, Hypocrea, Aspergillus, Humicola, Fusarium, Penicillium, Neurospora, Phanerochaete, Agaricus, Chaetomium, or Magnaporthe.

20. The fermentation process of claim 19, wherein the host filamentous fungus is Trichoderma reesei or Hypocrea jecorina.

24. The fermentation process of claim 6, wherein the medium comprises one or more additional carbon sources.

25. The fermentation process of claim 24, wherein the one or more additional carbon source is glycerol, one or more sugar alcohols or an organic acid.

26. The fermentation process of claim 25, wherein the one or more additional carbon source comprises xylitol.

28. The fermentation process of claim 6, wherein the step of culturing is conducted at a temperature of from about 20°

C. to about 35°

C. and a pH of from about 3.0 to about 6.5.

30. The fermentation process of claim 6, wherein the process is fed-batch.

32. The fermentation process of claim 6, wherein the process is continuous.

34. The fermentation process of claim 6, wherein the process is conducted aerobically.

36. The fermentation process of claim 6, wherein the carbon source contains 0 wt % cellulase-inducing carbohydrate.

39. A process for the hydrolyzing a cellulose substrate comprising contacting said substrate with a cellulase mixture produced by the fermentation process of claim 6.

40. The process of claim 39, wherein the cellulose substrate is a pretreated lignocellulosic feedstock.

45. The fermentation process of claim 6, wherein said Xyr1 transcription factor has an amino acid sequence exhibiting from about 95% to about 100% identity to SEQ ID NO:

27.

41. A fermentation process for the production of a cellulase mixture, said process comprising:

a. providing a modified host filamentous fungus that overexpresses a Xyr1 transcription factor, said Xyr1 transcription factor being a protein (i) having an amino acid sequence exhibiting from about 90% to about 100% identity to SEQ ID NO;

27, (ii) containing a class III zinc binuclear cluster with a conserved amino acid motif (CX₂CX₆CX_5-12CX₂CX_6-8C) at the N-terminal part of the protein, and (iii) exhibiting DNA binding activity specific to a consensus sequence GGC(T/A)₃-like motif within cellulase and/or hemicellulase promoter sequences; and

b. culturing the modified host filamentous fungus of step a) in a medium comprising a carbon source, wherein the carbon source contains from about 25 wt % to about 100 wt % of a hemicellulose-derived sugar alcohol, about 0 wt % of a cellulase-inducing carbohydrate and from about 0 wt % to about 75 wt % glucose, glycerol or a combination thereof, to produce a cellulase mixture;

wherein the fermentation process produces at least 10 g/L of the cellulase mixture, cellulase components represent at least 40 wt % of the total protein in the cellulase mixture, and the cellulase mixture exhibits at least 1.7-fold higher cellulase activity than a cellulase mixture produced by a parental filamentous fungus that does not overexpress the Xyr1 transcription factor when cultured in the same medium.

42. The fermentation process of claim 41, wherein the hemicellulose-derived sugar alcohol is xylitol.

43. The fermentation process of claim 42, wherein the carbon source contains from about 0 wt % to about 25 wt % glycerol and from about 0 wt % to about 50 wt % glucose.

46. The fermentation process of claim 41, wherein said Xyr1 transcription factor has an amino acid sequence exhibiting from about 95% to about 100% identity to SEQ ID NO:

27.