Production of heterologous proteins in filamentous fungi

• US 5,674,707 A
• Filed: 06/06/1995
• Issued: 10/07/1997
• Est. Priority Date: 12/10/1992
• Status: Expired due to Term

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: Gist-Brocades N.V. (DSM-Firmenich AG)
• Inventors: Hintz, William E., Lagosky, Peter A.
• Primary Examiner(s): Chambers, Jasemine C.
• Assistant Examiner(s): PRIEBE, SCOTT DAVID
• Application Number:
  US08/470,958
• Time in Patent Office: 854 Days
• Field of Search: 435/320.1, 435/69.1, 435/172.3, 435/254.3, 435/254.11, 536/24.1
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/80 for fungi

Abstract

Expression of proteins in filamentous fungal hosts is achieved in the presence of glucose using promoter variants in which creA binding sites are functionally disrupted.

Claims

1. A recombinant DNA construct in which protein-encoding DNA is linked operably with a promoter variant of an Aspergillus promoter, wherein said Aspergillus promoter comprises a creA binding site, which is at least six contiguous guanine or cytosine nucleotides and mediates carbon-catabolite repression of said promoter, and other regulatory sequences;

and wherein said promoter variant lacks the creA binding site, comprises the other regulatory sequences, and mediates expression of said protein-encoding DNA in the presence of glucose.

2. The recombinant DNA construct according to claim 1, wherein said creA binding site of said Aspergillus promoter is disrupted by replacement of nucleotide residues in the corresponding site of the promoter variant.

3. The recombinant DNA construct according to claim 2, wherein the nucleotide replacements are selected from adenine and thymidine nucleotides.

4. The recombinant DNA construct according to claim 1, wherein said Aspergillus promoter is selected from the group consisting of alcA, aldA, and AlcR.

5. A filamentous fungal strain having incorporated therein a recombinant DNA construct as defined in claim 1.

6. The filamentous fungal strain according to claim 5, of the genus Aspergillus.

7. The filamentous fungal strain according to claim 6, of the species Aspergillus nidulans.

8. A method for producing a desired protein, comprising the step of culturing in the presence of glucose a filamentous fungus strain as defined in claim 5, thereby producing the desired protein.

9. An Aspergillus nidulans strain that expresses the alcR gene product in the presence of glucose and has incorporated therein a recombinant DNA construct in which protein-encoding DNA is linked operably with a promoter variant of the alcA promoter, wherein said alcA promoter comprises creA binding sites at positions -302 and -167 each with at least six contiguous guanosine or cytosine nucleotides and mediates carbon-catabolite repression of said promoter, and other regulatory sequences;

and wherein said promoter variant lacks the creA binding sites, comprises the other regulatory sequences, and mediates expression of said protein encoding DNA in the presence of glucose, whereby the protein-encoding DNA is expressed when said strain is cultured in the presence of glucose and inducer.

10. A method for producing a desired protein, comprising the step of culturing an Aspergillus nidulans strain as defined in claim 9 in the presence of glucose and inducer, thereby producing the desired protein.