Cloning and amplification of the .beta.-glucosidase gene of Trichoderma reesei

• US 6,022,725 A
• Filed: 06/05/1995
• Issued: 02/08/2000
• Est. Priority Date: 12/10/1990
• Status: Expired due to Term

• Current Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Barnett, Christopher C., Fowler, Timothy, Shoemaker, Sharon
• Primary Examiner(s): LeGuyader, John L.
• Application Number:
  US08/462,090
• Time in Patent Office: 1,709 Days
• Field of Search: 435/69.1, 435/203, 435/207, 435/6, 435/252.3, 435/254, 435/320.1, 435/172.3, 536/27, 536/23.2, 536/23.1, 536/23.74, 536/24.3
• US Class Current: 435/209
• CPC Class Codes: C11D 3/38645 containing cellulase
• CPC Class Codes: C12G 1/02 Preparation of must from grapes; Must treatment and fermentation
• CPC Class Codes: C12N 1/145 Fungal isolates
• CPC Class Codes: C12N 9/2408 acting on alpha -1,4-glucosidic bonds
• CPC Class Codes: C12N 9/2445 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12P 19/00 Preparation of compounds containing saccharide radicals ketoaldonic acids C12P7/58
• 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: C12R 2001/885 Trichoderma
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

A process for expressing extracellular β-glucosidase in a filamentous fungus by expressing a fungal DNA sequence encoding enhanced, deleted or altered β-glucosidase in a recombinant host microorganism is disclosed. Recombinant fungal cellulase compositions containing enhanced, deleted or altered expression of β-glucosidase is also disclosed.

Claims

1. A process for expressing extracellular β

-glucosidase from DNA encoding an extracellular β

-glucoidase which DNA is obtained from a microorganism selected from the group consisting of Trichoderma, Aspergillus, and Neurospora, and wherein said DNA sequence or a portion thereof is capable of amplification by PCR with SEQ ID NO;

3 and SEQ ID NO;

4, wherein the amplification conditions are denaturation at 95°

C. for 10 minutes, annealing at 50°

C. for 2 minutes and extension at 65°

C. for 10 minutes for 30 cycles and wherein the amplification product is a DNA sequence that comprises a DNA sequence of about 700 base pairs from about position 471 to about position 1171 of SEQ ID NO;

1, comprising;

expressing said extracellular β

-glucosidase from a DNA insert through recombinant techniques in a filamentous fungus, wherein the inserted DNA comprises all of the coding region of a fungal β

-glucosidase gene and sequence necessary for the β

-glucosidase gene'"'"'s transcription and translation, and wherein said filamentous fungus is selected from the group consisting of Trichoderma, Aspergillus, Neurospora, Humicola and Penicillium.

2. The process according to claim 1, further comprising the step of isolating transformants having at least one additional copy of a fungal gene encoding for extracellular β

-glucosidase.

3. The process according to claim 2, further comprising the steps of:

(a) culturing said transformants under conditions to permit growth of said transformants; and

(b) isolating a recombinant fungal cellulase composition produced from said transformant.

4. The process according to claims 3, further comprising the step of purifying the β

-glucosidase from said isolated recombinant fungal cellulase composition.

5. The process according to claim 1, wherein said filamentous fungus is Trichoderma reesei, Trichoderma viridae, Trichoderma koningii, Aspergillus niger, Aspergillus nidulans, Aspergillus wentii, Aspergillus oryzae, Aspergillus phoenicis, Neurospora crassa, Humicola grisea, Penicillium pinophilum, or Penicillium oxalicum.

6. The process according to claim 1 wherein said β

-glucosidase gene is a bgl1 gene derived from Trichoderma reesei.

7. The process according to claim 3, wherein said recombinant fungal cellulase composition is isolated by:

(a) centrifuging said culture medium containing said transformants having β

-glucosidase activity to form a supernatant and a pellet; and

(b) filtering said supernatant to obtain a recombinant fungal cellulase composition.

8. The process according to claim 7, wherein an antimicrobial agent is added to said recombinant fungal cellulase composition after filtration.

9. The process according to claim 6, wherein said deleted bgl1 gene comprises the amino acid sequence and nucleotide sequence from base nos. 311 to 2679 of SEQ ID NO:

1.

10. Transformants having at least one additional copy of a T. reesii fungal gene encoding for extracellular β

-glucosidase produced by the process according to claim 2.

11. A process for expressing extracellular β

-glucosidase, comprising a) inserting DNA encoding an extracellular β

-glucosidase gene obtained from a microorganism selected from the group consisting of Trichoderma, Aspergillus, and Neurospora, wherein said DNA sequence or a portion thereof is capable of amplification by PCR with SEQ ID NO;

3 and SEQ ID NO;

4, wherein the amplification conditions are denaturation at 95°

C. for 10 minutes, annealing at 50°

C. for 2 minutes and extension at 65°

C. for 10 minutes for 30 cycles and wherein the amplification product is a DNA sequence that comprises a DNA sequence of about 700 base pairs from about position 471 to about position 1171 of SEQ ID NO;

1, and wherein said host fungus is selected from the group consisting of Trichoderma, Aspergillus, Neurospora, Humicola and Penicillium;

b) isolating transformants having enhanced β

-glucosidase expression as compared to a non-transformed filamentous host;

c) culturing said transformants under conditions to permit growth of said transformants; and

d) isolating a fungal cellulase composition containing the β

-glucosidase from said transformants.

12. The process according to claim 11 wherein the extracellular β

-glucosidase gene is a bgl1 gene having the nucleotide sequence of SEQ ID NO;

1.

13. The process according to claim 11 wherein the extracellular β

-glucosidase gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO;

2.

14. The process according to claim 11 wherein said host fungus is a transformed Trichoderma.

15. A probe for use in detecting nucleic acid sequences encoding β

-glucosidase from a filamentous fungi comprising SEQ ID. NO;

1.

16. The probe of claim 15 wherein said probe is used to detect a nucleic acid sequence encoding a β

-glucosidase from a filamentous fungi selected from the group consisting of Trichoderma, Aspergillus, and Neurospora.

17. A polynucleotide sequence comprising the nucleic acid sequence of SEQ ID NO:

1.

18. An expression vector comprising the polynucleotide of claim 17.

19. A filamentous fungal host transformed with the polynucleotide of claim 17 wherein said host is selected from the group consisting of Trichoderma, Aspergillus, Neurospora, Humicola and Penicillium.