Vectors for transformation by ascomycetes

• US 4,816,405 A
• Filed: 10/16/1987
• Issued: 03/28/1989
• Est. Priority Date: 10/24/1984
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Timberlake, William E., Hamer, John E., Yelton, M. Melanie
• Primary Examiner(s): Wiseman, Thomas G.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US07/111,091
• Time in Patent Office: 529 Days
• Field of Search: 435/68, 435/172.3, 536/320, 536/27, 935/22, 935/24, 935/26, 935/28, 935/56, 935/68, 935/69
• US Class Current: 435/243
• CPC Class Codes: C07K 14/39 from yeasts
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 15/80 for fungi
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 9/0061 Laccase (1.10.3.2)
• CPC Class Codes: C12Y 110/03002 Laccase (1.10.3.2)
• CPC Class Codes: Y10S 435/849 Escherichia coli
• CPC Class Codes: Y10S 435/913 Aspergillus

Abstract

Vectors and procedures are provided that enable genetic manipulation of the filamentous ascomycetes such as Aspergillus nidulans and Aspergillus niger. The systems of the invention permit transformation of various Aspergillus strains as well as the production and secretion of desired foreign proteins. Also provided are cosmid vectors which enable the isolation, cloning, sequencing and modifications of genes from the filamentous ascomycetes.

Claims

1. A vector effective in transformation of a bacterial or filamentous ascomycete host which vector comprises:

(a) a DNA sequence comprising the Aspergillus trpC gene; and

(b) replication and marker sequences from a pBR329 or derivatives thereof.

2. The transformation vector of claim 1 which is pHY201 or a derivative thereof.

3. A recombinant expression vector for a desired coding sequence placed under the control of said trpC gene of claim 1, and wherein said coding sequence is operable in a species of an ascomycete.

4. A recombinant expression vector for a desired coding sequence placed under the control of said trpC gene of claim 1, and wherein said coding sequence is mutagenically derived and operable in a species of ascomycete.

5. A recombinant bacterial or ascomycete host transformed with the vector of claim 1.

6. A recombinant bacterial or ascomycete host transformed with the vector of claim 3.

7. A recombinant bacterial or ascomycete host transformed with the vector of claim 4.

19. The sequence of claim 1 which is a 4.1 bp XhoI restriction fragment of λ

AnTrpC12.

8. A cosmid vector or a derivative thereof which comprises:

(a) a DNA sequence comprising the Aspergillus trpC gene,

(b) replication and marker sequences operable in a bacterial host; and

(c) a unique restriction cleavage site external to the gene of (a).

9. The vector or derivative of claim 8 wherein the restriction cleavage site of (c) is complementary to cleavage products of a restriction enzyme which randomly cuts filamentous ascomycete DNA at intervals of approximately 100-500 bp.

10. The vector or derivative of claim 8 which is pKBY2.

11. The vector or derivative of claim 8 which further includes a 30-45 kb fragment of genomic DNA derived from a species of filamentous ascomycete.

12. A recombinant bacterial or ascomycete host transformed with the vector or derivative of claim 11.

13. A genomic library from a filamentous ascomycete which comprises 30-45 kb fragments of the genome of said ascomycete inserted into the cosmid vector of claim 8.

14. The library of claim 13 transformed into a recombinant bacterial or ascomycete host.

15. The vector or derivative of claim 11 wherein the fragment of genomic DNA includes the yA2 gene.

16. The vector or derivative of claim 11 wherein the fragment of genomic DNA includes the pabaA gene.

17. The vector or derivative of claim 11 which is Cos YAI or Cos yAII.

18. A filamentous ascomycete gene derived from the vector of claim 11.

20. The vector or derivative of claim 8 wherein the bacterial host is E. coli.

21. The host of claim 12 which is E. coli.

22. The host of claim 12 which is an Aspergillus strain.

23. The host of claim 22 which is A. nidulans.