PROTEIN PRODUCTION IN FILAMENTOUS FUNGAL CELLS IN THE ABSENCE OF INDUCING SUBSTRATES

• US 20190309276A1
• Filed: 10/03/2017
• Published: 10/10/2019
• Est. Priority Date: 10/04/2016
• Status: Active Grant

• Current Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated), VTT Technical Research Centre Of Finland Ltd.
• Original Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated), VTT Technical Research Centre Of Finland Ltd.
• Inventors: Ward, Michael, Luo, Yun, Bendezu, Felipe Oseas, Valkonen, Mari, Saloheimo, Markku, Aro, Nina, Pakula, Tiina
• Granted Patent: US 10,876,103 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 14/37 from fungi
• CPC Class Codes: C12N 1/14 Fungi culture of mushrooms A01G18/00; as new plants A01H15/00; Culture media therefor
• 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: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Abstract

Certain embodiments of the disclosure are directed to variant filamentous fungal cells, compositions thereof and methods thereof for increased production of one or more proteins of interest. More particularly, in certain embodiments, the disclosure is directed to variant filamentous fungal (host) cells derived from parental filamentous fungal cells, wherein the variant host cells comprise a genetic modification which enables the expression/production of a protein of interest (POI) in the absence of inducing substrate. In certain embodiments, a variant fungal host cell of the disclosure comprises a genetic modification which increases the expression of a variant activator of cellulase expression 3 (ace3) gene encoding an Ace3 protein referred to herein as Ace3-L.

Claims

1. A variant filamentous fungal cell derived from a parental filamentous fungal cell, the variant cell comprising an introduced polynucleotide construct encoding an Ace3 protein comprising about 90% sequence identity to an Ace3 protein of SEQ ID NO:

6, wherein the variant cell produces an increased amount of a protein of interest (POI) in the absence of an inducing substrate relative to the parental cell, wherein the variant and parental cells are cultivated under similar conditions.

2. The variant cell of claim 1, wherein the variant cell further produces an increased amount of a protein of interest (POI) in the presence of an inducing substrate relative to the parental cell, wherein the variant and parental cells are cultivated under similar conditions.

3. The variant cell of claim 1, wherein the POI is an endogenous POI or a heterologous POI.

4. The variant cell of claim 1, wherein the variant cell comprises an introduced polynucleotide construct encoding a heterologous POI.

5. The variant cell of claim 3, wherein the endogenous POI is a lignocellulose degrading enzyme.

6. The variant cell of claim 5, wherein the lignocellulose degrading enzyme is selected from the group consisting of cellulase enzymes, hemi-cellulase enzymes, or a combination thereof.

7. The variant cell of claim 5, wherein the lignocellulose degrading enzyme is selected from the group consisting of cbh1, cbh2, egl1, egl2, egl3, egl4, egl5, egl6, bgl1, bgl2, xyn1, xyn2, xyn3, bxl1, abf1, abf2, abf3, axe1, axe2, axe3, man1, agl1, agl2, agl3, glr1, swo1, cip1 and cip2.

8. The variant cell of claim 3, wherein the heterologous POI is selected from the group consisting of an α

-amylase, an alkaline α

-amylase, a β

-amylase, a cellulase, a dextranase, an α

-glucosidase, an α

-galactosidase, a glucoamylase, a hemicellulase, a pentosanase, a xylanase, an invertase, a lactase, a naringanase, a pectinase, a pullulanase, an acid protease, an alkali protease, a bromelain, a neutral protease, a papain, a pepsin, a peptidase, a rennet, a rennin, a chymosin, a subtilisin, a thermolysin, an aspartic proteinase, a trypsin, a lipase, an esterase, a phospholipase, a phosphatase, a phytase, an amidase, an iminoacylase, a glutaminase, a lysozyme, a penicillin acylase;

an isomerase, an oxidoreductases, a catalase, a chloroperoxidase, a glucose oxidase, a hydroxysteroid dehydrogenase, a peroxidase, a lyase, an aspartic β

-decarboxylase, a fumarase, a histadase, a transferase, a ligase, an aminopeptidase, a carboxypeptidase, a chitinase, a cutinase, a deoxyribonuclease, an α

-galactosidase, a β

-galactosidase, a β

-glucosidase, a laccase, a mannosidase, a mutanase, a polyphenol oxidase, a ribonuclease and a transglutaminase.

9. The variant cell of claim 1, wherein the polynucleotide construct is integrated into the fungal cell genome.

10. The variant cell of claim 9, wherein the polynucleotide construct is integrated into a telomere site of the fungal cell genome.

11. The variant cell of claim 9, wherein the polynucleotide construct is integrated into a glucoamylase (gla1) gene locus of the fungal cell genome.

12. The variant cell of claim 1, wherein the polynucleotide construct comprises a nucleotide sequence comprising about 90% sequence identity to SEQ ID NO:

4, SEQ ID NO;

11 or SEQ ID NO;

13.

13. The variant cell of claim 1, wherein the encoded Ace3 protein comprises an amino acid sequence comprising 95% sequence identity to SEQ ID NO:

6, SEQ ID NO;

12 or SEQ ID NO;

14.

15. A lignocellulose degrading enzyme produced by the variant cell of claim 1.

16. A heterologous POI produced by the variant cell of claim 1.

14. A polynucleotide ORF encoding an Ace3 protein comprising about 90% sequence identity to SEQ ID NO:

6, SEQ ID NO;

12 or SEQ ID NO;

14.

17. A method for producing an endogenous protein of interest in a Trichoderma sp. fungal cell in the absence of an inducing substrate, the method comprising:

(i) introducing into the fungal cell a polynucleotide construct comprising in the 5′

to 3′

direction;

(a) a first nucleic acid sequence comprising a promoter and (b) a second nucleic acid sequence operably linked to the first nucleic acid sequence, wherein the second nucleic acid sequence encodes an Ace3 protein comprising about 90% sequence identity to SEQ ID NO;

6 and comprises “

Lys-Ala-Ser-Asp”

as the last four C-terminal amino acids, and

(ii) fermenting the cells of step (i) under conditions suitable for fungal cell growth and protein production, wherein such suitable growth conditions do not include an inducing substrate.

18. The method for claim 17, wherein the polynucleotide construct further comprises a third nucleic acid sequence 3′

and operably linked to the second nucleic acid, wherein the third nucleic acid sequence comprises a native ace3 terminator sequence.

19. The method of claim 17, wherein the polynucleotide construct is integrated into the fungal cell genome.

20. The method of claim 17, wherein the polynucleotide construct is integrated into a telomere site of the fungal cell genome.

21. The method of claim 17, wherein the polynucleotide construct is integrated into a glucoamylase (gla1) gene locus of the fungal cell genome.

22. The method cell of claim 17, wherein the polynucleotide construct comprises a nucleotide sequence comprising about 90% sequence identity to SEQ ID NO:

4, SEQ ID NO;

11 or SEQ ID NO;

13.

23. The method of claim 17, wherein the encoded Ace3 protein comprises an amino acid sequence comprising 95% sequence identity to SEQ ID NO:

6, SEQ ID NO;

12 or SEQ ID NO;

14.

24. The method of claim 17, wherein the promoter is selected from the group consisting of a rev3 promoter (SEQ ID NO:

15), a bxl promoter (SEQ ID NO;

     16), a tkl1 promoter (SEQ ID NO;

     17), a PID104295 promoter (SEQ ID NO;

     18), a dld1 promoter (SEQ ID NO;

     19), a xyn4 promoter (SEQ ID NO;

     20), a PID72526 promoter (SEQ ID NO;

     21), an axe1 promoter (SEQ ID NO;

     22), a hxk1 promoter (SEQ ID NO;

     23), a dic1 promoter (SEQ ID NO;

     24), an opt promoter (SEQ ID NO;

     25), a gut1 promoter (SEQ ID NO;

     26) and a pki1 promoter (SEQ ID NO;

     27).

25. A method for producing an endogenous protein of interest in a Trichoderma sp. fungal cell in the absence of an inducing substrate, the method comprising:

(i) introducing into the fungal cell a polynucleotide construct comprising in the 5′

to 3′

direction;

(a) a first nucleic acid sequence comprising a promoter and (b) a second nucleic acid sequence operably linked to the first nucleic acid sequence, wherein the second nucleic acid sequence encodes an Ace3 protein comprises about 90% sequence identity to SEQ ID NO;

12, and

(ii) fermenting the cells of step (i) under conditions suitable for fungal cell growth and protein production, wherein such suitable growth conditions do not include an inducing substrate.

26. The method of claim 25, wherein the polynucleotide construct comprises a third nucleic acid sequence 3′

and operably linked to the second nucleic acid, wherein the third nucleic acid sequence comprises a native ace3 terminator sequence.

27. The method of claim 25, wherein the polynucleotide construct is integrated into the fungal cell genome.

28. The method of claim 25, wherein the polynucleotide construct is integrated into a telomere site of the fungal cell genome.

29. The method of claim 25, wherein the polynucleotide construct is integrated into a glucoamylase (gla1) gene locus of the fungal cell genome.

30. The method cell of claim 25, wherein the polynucleotide construct comprises a nucleotide sequence comprising about 90% sequence identity to SEQ ID NO:

4, SEQ ID NO;

11 or SEQ ID NO;

13.

31. The method of claim 25, wherein the encoded Ace3 protein comprises an amino acid sequence comprising 95% sequence identity to SEQ ID NO:

6, SEQ ID NO;

12 or SEQ ID NO;

14.

32. The method of claim 25, wherein the promoter is selected from the group consisting of a rev3 promoter (SEQ ID NO:

15), a bxl promoter (SEQ ID NO;

     16), a tkl1 promoter (SEQ ID NO;

17, a PID104295 promoter (SEQ ID NO;

     18), a dld1 promoter (SEQ ID NO;

     19), a xyn4 promoter (SEQ ID NO;

     20), a PID72526 promoter (SEQ ID NO;

     21), an axe1 promoter (SEQ ID NO;

     22), a hxk1 promoter (SEQ ID NO;

     23), a dic1 promoter (SEQ ID NO;

     24), an opt promoter (SEQ ID NO;

     25), a gut1 promoter (SEQ ID NO;

     26) and a pki1 promoter (SEQ ID NO;

     27).

33. A method for producing a heterologous protein of interest in a Trichoderma sp. fungal cell in the absence of an inducing substrate, the method comprising:

(i) introducing into the fungal cell a polynucleotide construct comprising in the 5′

to 3′

direction;

(a) a first nucleic acid sequence comprising a constitutive promoter and (b) a second nucleic acid sequence operably linked to the first nucleic acid sequence, wherein the second nucleic acid sequence encodes an Ace3 protein comprising about 90% sequence identity to SEQ ID NO;

6 and comprises “

Lys-Ala-Ser-Asp”

as the last four C-terminal amino acids, and

(ii) fermenting the cells of step (i) under conditions suitable for fungal cell growth and protein production, wherein such suitable growth conditions do not include an inducing substrate.

34. The method of claim 33, wherein the fungal cell comprises an introduced polynucleotide construct encoding a heterologous POI, wherein the construct is introduced into in the fungal cell prior to step (i), during step (i) or after step (i).

35. The method for claim 33, wherein the polynucleotide construct comprises a third nucleic acid sequence 3′

and operably linked to the second nucleic acid, wherein the third nucleic acid sequence comprises a native ace3 terminator sequence.

36. The method of claim 33, wherein the polynucleotide construct is integrated into the fungal cell genome.

37. The method of claim 33, wherein the polynucleotide construct is integrated into a telomere site of the fungal cell genome.

38. The method of claim 33, wherein the polynucleotide construct is integrated into a glucoamylase (gla1) gene locus of the fungal cell genome.

39. The method cell of claim 33, wherein the polynucleotide construct comprises a nucleotide sequence comprising about 90% sequence identity to SEQ ID NO:

4, SEQ ID NO;

11 or SEQ ID NO;

13.

40. The method of claim 33, wherein the encoded Ace3 protein comprises an amino acid sequence comprising 95% sequence identity to SEQ ID NO:

6, SEQ ID NO;

12 or SEQ ID NO;

14.

41. The method of claim 33, wherein the heterologous POI is selected from the group consisting of an α

-amylase, an alkaline α

-amylase, a β

-amylase, a cellulase, a dextranase, an α

-glucosidase, an α

-galactosidase, a glucoamylase, a hemicellulase, a pentosanase, a xylanase, an invertase, a lactase, a naringanase, a pectinase, a pullulanase, an acid protease, an alkali protease, a bromelain, a neutral protease, a papain, a pepsin, a peptidase, a rennet, a rennin, a chymosin, a subtilisin, a thermolysin, an aspartic proteinase, a trypsin, a lipase, an esterase, a phospholipase, a phosphatase, a phytase, an amidase, an iminoacylase, a glutaminase, a lysozyme, a penicillin acylase;

an isomerase, an oxidoreductases, a catalase, a chloroperoxidase, a glucose oxidase, a hydroxysteroid dehydrogenase, a peroxidase, a lyase, an aspartic β

-decarboxylase, a fumarase, a histadase, a transferase, a ligase, an aminopeptidase, a carboxypeptidase, a chitinase, a cutinase, a deoxyribonuclease, an α

-galactosidase, a β

-galactosidase, a β

-glucosidase, a laccase, a mannosidase, a mutanase, a polyphenol oxidase, a ribonuclease and a transglutaminase.

42. The method of claim 33, wherein the promoter is selected from the group consisting of a rev3 promoter (SEQ ID NO:

15), a bxl promoter (SEQ ID NO;

     16), a tkl1 promoter (SEQ ID NO;

17, a PID104295 promoter (SEQ ID NO;

     18), a dld1 promoter (SEQ ID NO;

     19), a xyn4 promoter (SEQ ID NO;

     20), a PID72526 promoter (SEQ ID NO;

     21), an axe1 promoter (SEQ ID NO;

     22), a hxk1 promoter (SEQ ID NO;

     23), a dic1 promoter (SEQ ID NO;

     24), an opt promoter (SEQ ID NO;

     25), a gut1 promoter (SEQ ID NO;

     26) and a pki1 promoter (SEQ ID NO;

     27).

43. A method for producing a heterologous protein of interest in a Trichoderma sp. fungal cell in the absence of an inducing substrate, the method comprising:

(i) introducing into the fungal cell a polynucleotide construct comprising in the 5′

to 3′

direction;

(a) a first nucleic acid sequence comprising a constitutive promoter and (b) a second nucleic acid sequence operably linked to the first nucleic acid sequence, wherein the second nucleic acid sequence encodes an Ace3 protein comprises about 90% sequence identity to SEQ ID NO;

12, and

(ii) fermenting the cells of step (i) under conditions suitable for fungal cell growth and protein production, wherein such suitable growth conditions do not include an inducing substrate.

44. The method of claim 43, wherein the fungal cell comprises an introduced polynucleotide construct encoding a heterologous POI, wherein the construct is introduced into in the fungal cell prior to step (i), during step (i) or after step (i).

45. The method for claim 43, wherein the polynucleotide construct comprises a third nucleic acid sequence 3′

and operably linked to the second nucleic acid, wherein the third nucleic acid sequence comprises a native ace3 terminator sequence.

46. The method of claim 43, wherein the polynucleotide construct is integrated into the fungal cell genome.

47. The method of claim 43, wherein the polynucleotide construct is integrated into a telomere site of the fungal cell genome.

48. The method of claim 43, wherein the polynucleotide construct is integrated into a glucoamylase (gla1) gene locus of the fungal cell genome.

49. The method cell of claim 43, wherein the polynucleotide construct comprises a nucleotide sequence comprising about 90% sequence identity to SEQ ID NO:

4, SEQ ID NO;

11 or SEQ ID NO;

13.

50. The method of claim 43, wherein the encoded Ace3 protein comprises an amino acid sequence comprising 95% sequence identity to SEQ ID NO:

6, SEQ ID NO;

12 or SEQ ID NO;

14.

51. The method of claim 43, wherein the polynucleotide construct encoding the heterologous POI is expressed under the control of a cellulose-inducible gene promoter.

52. The method of claim 43, wherein the heterologous POI is selected from the group consisting of an α

-amylase, an alkaline α

-amylase, a β

-amylase, a cellulase, a dextranase, an α

-glucosidase, an α

-galactosidase, a glucoamylase, a hemicellulase, a pentosanase, a xylanase, an invertase, a lactase, a naringanase, a pectinase, a pullulanase, an acid protease, an alkali protease, a bromelain, a neutral protease, a papain, a pepsin, a peptidase, a rennet, a rennin, a chymosin, a subtilisin, a thermolysin, an aspartic proteinase, a trypsin, a lipase, an esterase, a phospholipase, a phosphatase, a phytase, an amidase, an iminoacylase, a glutaminase, a lysozyme, a penicillin acylase;

an isomerase, an oxidoreductases, a catalase, a chloroperoxidase, a glucose oxidase, a hydroxysteroid dehydrogenase, a peroxidase, a lyase, an aspartic β

-decarboxylase, a fumarase, a histadase, a transferase, a ligase, an aminopeptidase, a carboxypeptidase, a chitinase, a cutinase, a deoxyribonuclease, an α

-galactosidase, a β

-galactosidase, a β

-glucosidase, a laccase, a mannosidase, a mutanase, a polyphenol oxidase, a ribonuclease and a transglutaminase.

53. The method of claim 43, wherein the promoter is selected from the group consisting of a rev3 promoter (SEQ ID NO:

15), a bxl promoter (SEQ ID NO;

     16), a tkl1 promoter (SEQ ID NO;

17, a PID104295 promoter (SEQ ID NO;

     18), a dld1 promoter (SEQ ID NO;

     19), a xyn4 promoter (SEQ ID NO;

     20), a PID72526 promoter (SEQ ID NO;

     21), an axe I promoter (SEQ ID NO;

     22), a hxk1 promoter (SEQ ID NO;

     23), a dic1 promoter (SEQ ID NO;

     24), an opt promoter (SEQ ID NO;

     25), a gut1 promoter (SEQ ID NO;

     26) and a pki1 promoter (SEQ ID NO;

     27).

54. A method for genetically modifying a Trichoderma reesei strain for increased production of an endogenous protein in the absence of an inducing substrate, the method comprising:

(i) screening and identifying a T. reesei strain comprising a genomic copy of an ace3 gene encoding an Ace3-S protein of SEQ ID NO;

3, an Ace3-SC protein of SEQ ID NO;

8 or an Ace3-LC protein of SEQ ID NO;

10, wherein the T. reesei strain identified does not comprise a genomic copy of an ace3 gene encoding an Ace3-L protein of SEQ ID NO;

6, an Ace3-LN protein of SEQ ID NO;

14 or an Ace3-EL protein of SEQ ID NO;

12,

(ii) introducing into the T. reesei strain identified in step (i) a polynucleotide construct comprising in the 5′

to 3′

direction;

(a) a first nucleic acid sequence comprising a promoter and (b) a second nucleic acid sequence operably linked to the first nucleic acid sequence, wherein the second nucleic acid sequence encodes an Ace3 protein comprising about 90% sequence identity to SEQ ID NO;

6 and comprises “

Lys-Ala-Ser-Asp”

as the last four C-terminal amino acids or the second nucleic acid sequence encodes an Ace-3 protein comprising about 90% sequence identity to SEQ ID NO;

12, and

(iii) fermenting the cells of step (ii) under conditions suitable for fungal cell growth and protein production, wherein such suitable growth conditions do not include an inducing substrate.

55. A variant filamentous fungal cell derived from a parental filamentous fungal cell, the variant cell comprising a native ace3 gene promoter replaced by an alternative promoter, wherein the variant cell produces an increased amount of a protein of interest (POI) in the absence of an inducing substrate relative to the parental cell, wherein the variant and parental cells are cultivated under similar conditions.

56. The variant of claim 55, wherein the alternative promoter is a Trichoderma reesei promoter.

57. The variant of claim 56, wherein the alternative promoter is a promoter selected from the group consisting of a rev3 promoter (SEQ ID NO:

15), a bxl promoter (SEQ ID NO;

     16), a tkl1 promoter (SEQ ID NO;

17, a PID104295 promoter (SEQ ID NO;

     18), a dld1 promoter (SEQ ID NO;

     19), a xyn4 promoter (SEQ ID NO;

     20), a PID72526 promoter (SEQ ID NO;

     21), an axe1 promoter (SEQ ID NO;

     22), a hxk1 promoter (SEQ ID NO;

     23), a dic1 promoter (SEQ ID NO;

     24), an opt promoter (SEQ ID NO;

     25), a gut1 promoter (SEQ ID NO;

     26) and a pki1 promoter (SEQ ID NO;

     27).