Vectors for use in filamentous fungi

• US 5,503,991 A
• Filed: 05/02/1994
• Issued: 04/02/1996
• 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., Davies, Roger W., Pickett, Mark H., Scazzocchio, Claudio
• Primary Examiner(s): Fleisher, Mindy
• Assistant Examiner(s): Guzo, David
• Application Number:
  US08/237,405
• Time in Patent Office: 701 Days
• Field of Search: 435/69.1, 435/320.1, 435/172.1, 435/172.3, 435/252.3, 435/254.1, 435/254.11, 435/254.3, 435/256.1, 536/23.1, 536/24.1
• 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, said DNA construct comprising promoter DNA for promoting transcription in Aspergillus and operably linked to DNA coding for said polypeptide to enable expression thereof in said Aspergillus host, said DNA coding for said polypeptide being foreign to said promoter DNA.

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 according to claim 1, wherein said promoter DNA comprises the alcA promoter native to Aspergillus nidulans operably linked to the C. fimi endoglucanase coding region.

5. A DNA construct according to claim 1, wherein the coding DNA is foreign to said Aspergillus host.

6. A DNA construct according to claim 1, further comprising a DNA fragment encoding a promoter-inducer substance.

7. A DNA construct according to claim 6, wherein said DNA fragment encodes alcR.

8. A DNA construct according to claim 7, wherein said promoter DNA is selected from the group consisting of the alcA and the aldA promoter of Aspergillus nidulans.

9. An Aspergillus strain which upon culturing expresses a polypeptide as a result of having been transformed by a DNA construct in which DNA coding for the polypeptide is linked operably to promoter DNA for promoting transcription in Aspergillus, said DNA coding for said polypeptide being foreign to said promoter DNA.

10. A strain according to claim 9, wherein said strain is a transformed Aspergillus species selected from the group consisting of Aspergillus nidulans and Aspergillus niger.

11. A strain according to claim 9, wherein the coding DNA is foreign to said Aspergillus strain.

12. A strain according to claim 9, wherein said promoter DNA comprises the alcA promoter native to Aspergillus nidulans.

13. A strain according to claim 10, which is an Aspergillus niger transformant.

14. 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 is linked operably to promoter DNA for promoting transcription in Aspergillus, said DNA coding for said polypeptide being foreign to said promoter DNA.

15. A method according to claim 14, wherein the cultured strain is a transformed Aspergillus species selected from the group consisting of Aspergillus nidulans and Aspergillus niger.

16. A method according to claim 14, wherein the coding DNA is foreign to said Aspergillus strain.

17. A method according to claim 14, wherein said promoter DNA is selected from the alcA promoter of Aspergillus nidulans and the aldA promoter of Aspergillus nidulans.

18. A method according to claim 15, wherein said cultured strain is an Aspergillus niger transformant.