Process for the production of protein products in aspergillus

• US 5,536,661 A
• Filed: 03/07/1994
• Issued: 07/16/1996
• Est. Priority Date: 03/10/1987
• Status: Expired due to Term

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novo Nordisk A/S
• Inventors: Woldike, Helle, Boel, Esper, Christensen, Tove
• Primary Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US08/208,092
• Time in Patent Office: 862 Days
• Field of Search: 935/69.1, 935/71.1, 935/172.3, 935/91, 935/254.3, 935/320.1, 935/198, 935/6, 935/9, 935/22, 935/24, 935/33, 935/34, 935/59, 935/60, 935/61, 935/66, 935/68, 536/24.1
• US Class Current: 435/254.3
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 15/80 for fungi
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase
• CPC Class Codes: C12N 9/2417 from microbiological source
• CPC Class Codes: C12N 9/242 Fungal source
• CPC Class Codes: C12N 9/6481 Pepsins (3.4.23.1; 3.4.23.2; 3.4.23.3)
• CPC Class Codes: C12N 9/6483 Chymosin (3.4.23.4), i.e. rennin
• CPC Class Codes: C12Y 304/23004 Chymosin (3.4.23.4), i.e. rennin

Abstract

A process for expression of a protein product in Aspergillus oryzae is disclosed. The process comprises transforming Aspergillus oryzae with a vector system comprising DNA-sequences encoding functions facilitating gene expression, a suitable marker for selection of transformants, and a DNA-sequence encoding the desired protein product. The process enables industrial production of many different polypeptides and proteins in A. oryzae. Examples of such products are chymosin or prochymosin and other rennets, proteases, lipases and amylases. Also disclosed is an effective promoter for expression of a protein in Aspergillus. A preferred promoter is the TAKA-amylase promoter or functional parts thereof. There is also provided a process for the production of a recombinant Humicola lipase. The recombinant Humicola lipase from A. oryzae differs from the native lipase in having a greater glycosylation and in exhibiting an improved thermostability.

Claims

1. A promoter and upstream activating sequences suitable for expression of a protein product in Aspergillus, characterized by being the Aspergillus oryzae TAKA-amylase promoter preceded by its upstream activating sequences.

2. A promoter and upstream activating sequences according to claim 1 having the following sequence ##STR16##

3. A promoter and upstream activating sequences, wherein the promoter and upstream activating sequences according to claim 2 are preceded by the 1.05 kb unsequenced upstream region from plasmic pTAKA17, wherein said region extends from position 0 to 1.05 in FIG. 2.

4. A promoter and upstream activating sequences according to claim 1 having the following sequence ##STR17##

5. An Aspergillus which has been transformed with a recombinant DNA cloning vector system capable of integration into the genome of the Aspergillus in one or more copies, the vector system comprising:

(a) DNA sequences encoding functions facilitating gene expression comprising;

an Aspergillus oryzae TAKA amylase promoter preceded by its upstream activating sequences;

a transcription initiation site; and

transcription terminator and polyadenylation functions; and

(b) a DNA sequence encoding a desired protein product operatively linked to the DNA sequences encoding functions facilitating gene expression.

6. The Aspergillus of claim 5 which is an Aspergillus oryzae strain.

7. The Aspergillus of claim 5 wherein the protein product is a Humicola lanuginosa lipase.