EGVII endoglucanase and nucleic acids encoding the same

• US 20030113734A1
• Filed: 12/18/2001
• Published: 06/19/2003
• Est. Priority Date: 12/18/2001
• Status: Active Grant

• Current Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Ward, Michael, Goedegebuur, Frits, Dunn-Coleman, Nigel, Yao, Jian
• Granted Patent: US 7,045,331 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C11D 3/38 Products with no well-defined composition , e.g. natural products
• CPC Class Codes: C11D 3/38636 containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
• CPC Class Codes: C11D 3/38645 containing cellulase
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/11 Antisense
• 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 9/2445 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• 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 2201/00 Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12P 7/14 Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/133 Renewable energy sources, e.g. sunlight

Abstract

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

Claims

1. An isolated polynucleotide derived from a fungal source, which polynucleotide comprises a nucleotide sequence encoding an enzyme having endoglucanase activity.

2. An isolated polynucleotide selected from the group consisting of:

(a) a nucleic acid sequence which encodes or is complementary to a sequence which encodes an EGVII polypeptide having at least 85% sequence identity to the amino acid sequence presented in FIG. 2 (SEQ ID NO;

2);

(b) a nucleic acid sequence which encodes or is complementary to a sequence which encodes an EGVII polypeptide having at least 90% sequence identity to the amino acid sequence presented in FIG. 2 (SEQ ID NO;

2);

(c) a nucleic acid sequence which encodes or is complementary to a sequence which encodes an EGVII polypeptide having at least 95% sequence identity to the amino acid sequence presented in FIG. 2;

(d) a nucleic acid sequence which encodes or is complementary to a sequence which encodes an EGVII polypeptide having the amino acid sequence presented in FIG. 2;

(e) a nucleic acid sequence which encodes or is complementary to a sequence which encodes an EGVII polypeptide having at least 95% sequence identity to the amino acid sequence presented as SEQ ID NO;

2;

(f) a nucleic acid sequence which encodes or is complementary to a sequence which encodes an EGVII polypeptide having the amino acid sequence presented as SEQ ID NO;

2;

(g) a nucleic acid sequence presented as SEQ ID NO;

4, or the complement thereof; and

(h) a nucleic acid sequence that hybridizes, under high stringency conditions to the sequence presented as SEQ ID NO;

4, or the complement or a fragment thereof, wherein said isolated polynucleotide encodes a polypeptide having the biological activity of an endoglucanase.

3. The isolated polynucleotide of claim 2, wherein % identity is calculated using the CLUSTAL-W program in MacVector version 6.5, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

4. The isolated polynucleotide of claim 2, wherein hybridization is conducted at 42°

C. in 50% formamide, 6×

SSC, 5×

Denhardt'"'"'s solution, 0.5% SDS and 100 μ

g/ml denatured carrier DNA followed by washing two times in 2×

SSPE and 0.5% SDS at room temperature and two additional times in 0.1 SSPE and 0.5% SDS at 42°

C.

5. The isolated polynucleotide of claim 2, wherein said polynucleotide is an RNA molecule.

10. A vector comprising an isolated polynucleotide of claim 2, operably linked to control sequences recognized by a host cell transformed with the vector.

12. A host cell transformed with the vector of claim 10.

13. The host cell of claim 12, which is a prokaryotic cell.

14. The host cell of claim 12, which is a eukaryotic cell.

15. A recombinant host cell comprising a polynucleotide of claim 2.

16. The recombinant host cell of claim 15, which is a prokaryotic cell.

17. The recombinant host cell of claim 15, which is a eukaryotic cell.

19. A method of producing an enzyme having endoglucanase activity, comprising:

(a) stably transforming a host cell with an expression vector comprising a polynucleotide as defined in claim 2;

(b) cultivating said transformed host cell under condition suitable for said host cell to produce said endoglucanase; and

(c) recovering said endoglucanase.

20. The method of claim 19 wherein the host cell is a filamentous fungi or yeast cell.

21. A purified enzyme having endoglucanase activity prepared by the method of claim 19.

6. The isolated polynucleotide encoding an enzyme having endoglucanase activity, wherein the enzyme is derived from a Trichoderma source.

7. The isolated polynucleotide of claim 6, wherein the enzyme is derived from Trichoderma reesei.

8. An expression construct including a polynucleotide sequence (i) having at least 85% sequence identity to the amino acid sequence presented in FIG. 2 (SEQ ID NO:

2), or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence disclosed in FIG. 2 under conditions of intermediate to high stringency, or (iii) being complementary to a nucleotide sequence having at least 85% sequence identity to the amino acid sequence presented in FIG. 2 (SEQ ID NO;

2).

9. A vector including the expression construct of claim 8.

11. A host cell transformed with the vector of claim 9.

18. A substantially purified EGVII polypeptide with the biological activity of an endoglucanase, comprising a sequence selected from the group consisting of:

(a) an amino acid sequence having at least 85% sequence identity to the amino acid sequence presented in FIG. 2 (SEQ ID NO;

2);

(b) an amino acid sequence having at least 90% sequence identity to the amino acid sequence presented in FIG. 2 (SEQ ID NO;

2);

(c) an amino acid sequence having at least 95% sequence identity to the amino acid sequence presented in FIG. 2;

(d) an amino acid sequence presented in FIG. 2;

(e) an amino acid sequence having at least 95% sequence identity to the amino acid sequence presented as SEQ ID NO;

2;

(f) an amino acid sequence presented as SEQ ID NO;

2;

(g) a substantially purified biologically active fragment of the amino acid sequence presented as SEQ ID NO;

2.

22. A recombinant host cell comprising a deletion or insertion or other alteration in the egl7 gene which inactivates the gene and prevents EGVII polypeptide production.

23. An antisense oligonucleotide complementary to a messenger RNA that encodes an EGVII polypeptide having the sequence presented as SEQ ID NO:

2, wherein upon exposure to a endoglucanase-producing host cell, said oligonucleotide decreases or inhibits the production of endoglucanase by said host cell.

24. The antisense oligonucleotide of claim 23, wherein the host cell is a filamentous fungi.

25. A detergent composition, said composition comprising a polypeptide selected from the group consisting of:

(a) an amino acid sequence having at least 85% sequence identity to the amino acid sequence presented in FIG. 2 (SEQ ID NO;

2);

(b) an amino acid sequence having at least 90% sequence identity to the amino acid sequence presented in FIG. 2 (SEQ ID NO;

2);

(c) an amino acid sequence having at least 95% sequence identity to the amino acid sequence presented in FIG. 2;

(d) an amino acid sequence presented in FIG. 2;

(e) an amino acid sequence having at least 95% sequence identity to the amino acid sequence presented as SEQ ID NO;

2;

(f) an amino acid sequence presented as SEQ ID NO;

2;

(g) a substantially purified biologically active fragment of the amino acid sequence presented as SEQ ID NO;

2.

26. A method of expressing a heterologous polypeptide having β

-glucosidase activity in an Aspergillus species, comprising;

(a) Providing a host Aspergillus with an expression vector comprising a polynucleotide encoding a signal sequence linked to a polynucleotide encoding a heterologous -glucosidase, thereby encoding a chimeric polypeptide;

(b) Cultivating said host Aspergillus under conditions suitable for said Aspergillus to produce said chimeric polypeptide, wherein said chimeric polypeptide is produced.

27. A method of producing ethanol, said method comprising the steps of:

a) contacting a biomass composition with an enzymatic composition comprising β

-glucosidase 4 to yield a sugar solution;

b) adding to the sugar solution a fermentative microorganism; and

c) culturing the fermentative microorganism under conditions sufficient to produce ethanol,

wherein the biomass composition may be optionally pretreated.

28. The method of claim 27 wherein step (a) further comprises the addition of at least one endoglucanase.

29. The method of claim 27 wherein step (a) further comprises the addition of at least one cellbiohydrolase.

30. The method of claim 28 wherein step (a) further comprises the addition of at least one cellbiohydrolase.

31. The method of claim 27 wherein the pretreatment is with a dilute acid.

32. A method of producing ethanol, said method comprising the steps of:

a) contacting a biomass composition with an enzymatic composition comprising a β

-glucosidase 4 and a fermentative microorganism; and

b) culturing the fermentative microorganism under conditions sufficient to produce ethanol,

wherein the biomass composition may be optionally pretreated.

33. The method of claim 32 wherein step (a) further comprises the addition of at least one endoglucanase.

34. The method of claim 32 wherein step (a) further comprises the addition of at least one cellbiohydrolase.

35. The method of claim 33 wherein step (a) further comprises the addition of at least one cellbiohydrolase.

36. The method of claim 32 wherein the pretreatment is with a dilute acid.