Vectors in use in filamentous fungi

• US 5,198,345 A
• Filed: 12/20/1985
• Issued: 03/30/1993
• Est. Priority Date: 04/15/1985
• Status: Expired due to Term

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: Gist-Brocades N.V. (DSM-Firmenich AG)
• Inventors: Gwynne, David I., Buxton, Francis P., Pickett, Mark H., Scazzocchio, Claudio, Davies, Roger W.
• Primary Examiner(s): Schwartz, Richard A.
• Application Number:
  US06/811,404
• Time in Patent Office: 2,657 Days
• Field of Search: 435/68, 435/91, 435/171, 435/172.3, 435/252.3, 435/254, 435/320, 435/913, 435/917, 435/911, 435/320.1, 435/254, 435/69.1, 536/27, 935/6, 935/22, 935/24, 935/34, 935/36, 935/48, 935/60, 935/61, 935/68, 935/10, 935/69.1, 935/71.1
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/56 IFN-alpha
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/036 targeting to the medium outside of the cell, e.g. type III secretion
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C12N 15/80 for fungi
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/2422 from plant source
• CPC Class Codes: C12N 9/2428 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12N 9/2434 acting on beta-1,4-glucosidic bonds
• CPC Class Codes: C12Y 101/01001 Alcohol dehydrogenase (1.1.1.1)
• CPC Class Codes: C12Y 102/01003 Aldehyde dehydrogenase (NAD+) (1.2.1.3)
• CPC Class Codes: C12Y 302/01003 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Abstract

Novel vectors are disclosed for use in filamentous fungi such as Aspergillus sp. in particular, whereby protein coding regions may be inserted therein to achieve expression or expression followed by secretion of the coded protein from the host. Signal peptide sequences and promoter sequences valuable for this purpose are disclosed as are expression vectors containing coding regions native or foreign to the fungal host. In accordance with the invention, a filamentous fungus such as Aspergillus may be provided with foreign or natural coding regions associated with foreign or natural promoter sequences and optionally signal peptide sequences which can be used to control the expression and/or secretion of the proteins encoded by these coding regions.

Claims

1. A DNA construct for use in transforming an Aspergillus host to obtain expression therein of a polypeptide foreign thereto, said DNA construct comprising promoter DNA corresponding in sequence to the promoter region of the glucoamylase gene of Aspergillus niger operably linked to DNA coding for said polypeptide to enable expression thereof in said Aspergillus host.

2. A DNA construct according to claim 1, wherein said Aspergillus host is Aspergillus niger.

3. A DNA construct according to claim 1, wherein said Aspergillus host is Aspergillus nidulans.

4. A DNA construct for use in transforming an Aspergillus nidulans host to obtain expression therein of a polypeptide foreign thereto, said DNA construct comprising promoter DNA corresponding in sequence to the promoter region of an Aspergillus nidulans gene selected from the group consisting of the alcohol dehydrogenase 1 gene and the aldehyde dehydrogenase gene operably linked to DNA coding for said polypeptide to enable expression thereof in said Aspergillus nidulans host.

5. A DNA construct according to claim 4, wherein said promoter DNA corresponds in sequence to the promoter region of the alcohol dehydrogenase 1 gene of Aspergillus nidulans.

6. A DNA construct according to claim 4, wherein said promoter DNA corresponds in sequence to the promoter region of the aldehyde dehydrogenase gene of Aspergillus nidulans.

7. An Aspergillus strain which upon culturing expresses a polypeptide foreign thereto as a result of having been transformed by a DNA construct in which DNA coding for the foreign polypeptide is linked operably to promoter DNA corresponding in sequence to the promoter region of the glucoamylase gene of Aspergillus niger.

8. A strain according to claim 7, wherein said strain is a transformed Aspergillus species selected from the group consisting of Aspergillus nidulans and Aspergillus niger.

9. A strain according to claim 8, which is an Aspergillus niger transformant.

10. An Aspergillus nidulans strain which upon culturing expresses a polypeptide foreign thereto as a result of having been transformed by a DNA construct in which DNA coding for the foreign polypeptide is linked operably to promoter DNA corresponding in sequence to the promoter region of an Aspergillus nidulans gene selected from the group consisting of the alcohol dehydrogenase 1 gene and the aldehyde dehydrogenase gene.

11. An Aspergillus nidulans strain according to claim 10, wherein said promoter DNA corresponds in sequence to the promoter region of the alcohol dehydrogenase 1 gene of Aspergillus nidulans.

12. A method for producing a polypeptide, said method comprising the step of culturing an Aspergillus strain transformed by a DNA construct in which DNA coding for a polypeptide foreign to said Aspergillus strain is linked operably to promoter DNA corresponding in sequence to the promoter region of the glucoamylase gene of Aspergillus niger.

13. A method according to claim 12, wherein the cultured strain is a transformed Aspergillus species selected from the group consisting of Aspergillus nidulans and Aspergillus niger.

14. A method according to claim 13, wherein said cultured strain in an Aspergillus niger transformant.

15. A method for producing a polypeptide, said method comprising the step of culturing an Aspergillus nidulans strain transformed by a DNA construct in which DNA coding for a polypeptide foreign to said Aspergillus nidulans is linked operably to promoter DNA corresponding in sequence with the promoter region of an Aspergillus nidulans gene selected from the group consisting of the alcohol dehydrogenase 1 gene and the aldehyde dehydrogenase gene.

16. A method according to claim 15, wherein said promoter DNA corresponds in sequence to the promoter region of the alcohol dehydrogenase 1 gene of Aspergillus nidulans.

17. A method according to claim 16, wherein said strain is cultured in a culturing medium containing an expression-inducing amount of threonine.