Aspergillus niger transformation system

• US 4,885,249 A
• Filed: 12/05/1984
• Issued: 12/05/1989
• Est. Priority Date: 12/05/1984
• Status: Expired due to Term

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG), NPS Pharmaceuticals Incorporated (Shire plc)
• Original Assignee: Allelix Limited
• Inventors: Davies, Roger W., Buxton, Franics P., Gwynne, David I.
• Primary Examiner(s): Martinell, James
• Application Number:
  US06/678,578
• Time in Patent Office: 1,826 Days
• Field of Search: 435/68, 435/70, 435/71, 435/91, 435/172.3, 435/254, 435/317, 435/172.1, 435/320, 536/27, 935/24, 935/14, 935/55, 935/56, 935/68
• US Class Current: 435/6.18
• CPC Class Codes: C12N 15/80 for fungi
• CPC Class Codes: Y10S 435/917 Aspergillus niger

Abstract

Transformants of Aspergillus niger and related Aspergilli, containing foreign DNA conferring modified properties of expression thereon, are prepared by use of a DNA vector which contains a selectable marker which is capable of incorporation into the DNA of the host A. niger cells, but which is not to be found in the A. niger cells prior to this transformation. The vector also contains other foreign DNA to be incorporated into the A. niger, for modified expression. The process suitably uses mutants of A. niger as hosts, the mutants lacking the selectable marker as compared with wild type A. niger, and conducts the transformation on spheroplasts of A. niger.

Claims

1. A process for transforming Aspergillus niger, said process comprising the steps of:

treating an Aspergillus niger strain with recombinant DNA under conditions permitting at least some of said Aspergillus niger to take up said recombinant DNA and form transformants wherewith; and

selecting resulting Aspergillus niger transformants.

2. The process of claim 1, wherein spheroplasts of said Aspergillus niger strain are treated with said recombinant DNA.

3. The process of claim 2, wherein said recombinant DNA is circular.

4. The process of claim 2, wherein said recombinant DNA is linear.

5. The process of claim 1, wherein said Aspergillus niger strain lacks a predetermined selectable marker and said recombinant DNA contains said predetermined selectable marker.

6. The process of claim 5, wherein said selectable marker is a gene of wild type Aspergillus niger and said strain of said Aspergillus niger which is treated with said recombinant DNA is a mutant strain thereof, deficient in said selectable marker.

7. The process of claim 6, wherein said selectable marker is able to confer antibiotic resistance to said transformants.

8. The process of claim 6, wherein said selectable marker is foreign to wild Aspergillus niger.

9. The process of claim 6, wherein said selectable marker is a gene which encodes an assayable product.

10. The process of claim 6, wherein said selectable marker is the ornithine carbamoyl transferase gene (Arg B⁺).

11. The process of claim 3 or 4, wherein said recombinant DNA comprises a replicon, permitting its replication in a bacterial host.

12. The process of claim 11, wherein said recombinant DNA comprises, as a selectable marker, ornithine carbamoyl transferase gene.

13. The process of claim 12, wherein said ornithine carbamoyl transferase gene is obtained from Aspergillus nidulans.

14. The process of claim 11, wherein said recombinant DNA is selected from the group consisting of pDG1 (ATCC 53005) and pDG3 (ATCC 53006).

15. A plasmid selected from the group consisting of pDG1 (ATCC 53005) and pDG3 (ATCC 53006).

16. Transformed Aspergillus niger comprising recombinant DNA.

17. Aspergillus niger ATCC 20739 transformed by Aspergillus nidulans argB gene.

18. A method for obtaining Aspergillus niger transformants, said method comprising the steps of:

enzymatically removing the cell wall of Aspergillus niger;

adding exogenous DNA to the resultant Aspergillus niger spheroplasts;

incubating the spheroplasts and exogenous DNA under conditions permitting some of the spheroplasts to take up exogenous DNA; and

isolating resulting Aspergillus niger transformants.

19. A method according to claim 18, wherein the exogenous DNA is linear.

20. A method according to claim 18, wherein the exogenous DNA is circular.

21. A method according to claim 18, wherein the exogenous DNA comprises the argB gene of A. nidulans.

22. A method according to claim 21, wherein the exogenous DNA comprising said argB gene is a plasmid selected from the group consisting of pDG1 (ATCC 53005) and pDG3 (ATCC 53006).