Vector having promoter that is inducible by methanol and/or glycerol

• US 5,750,372 A
• Filed: 06/07/1995
• Issued: 05/12/1998
• Est. Priority Date: 02/28/1992
• Status: Expired due to Term

• Current Assignee: Suntory Holdings Limited (Kotobuki Realty Co., Ltd.)
• Original Assignee: Suntory Ltd (Kotobuki Realty Co., Ltd.)
• Inventors: Sakai, Yasuyoshi, Hatanaka, Haruyo, Kondo, Hiroto, Tani, Yoshiki, Shibano, Yuji
• Primary Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US08/485,284
• Time in Patent Office: 1,070 Days
• Field of Search: 435/320.1, 435/69.1, 435/71.1, 435/71.2, 435/71.3, 435/192, 435/172.3, 435/194, 435/254.2, 536/23.1, 536/23.7, 536/24.1, 536/23.2
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/815 for yeasts other than Saccharomyces
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0065 acting on hydrogen peroxide as acceptor (1.11)
• CPC Class Codes: C12N 9/12 transferring phosphorus containing groups, e.g. kinases (2.7)
• CPC Class Codes: C12Y 101/03013 Alcohol oxidase (1.1.3.13)

Abstract

The present invention relates to an expression vector which is induced by methanol and/or glycerol utilizing an alcohol oxidase gene of a methylotrophic yeast; a recombinant methylotrophic yeast containing said vector and capable of accumulating a significantly high amount of an expression product of a heterologous gene; and a method for producing useful products utilizing said recombinant.

The expression vector is constructed by utilizing the promoter and terminator of an alcohol oxidase gene of methylotrophic yeast. Further, a significantly high amount of adenylate kinase, cytochrome C552 or peroxidase is produced by using said expression vector.

Claims

1. A methanol and/or glycerol inducible expression cassette which comprises a promoter having the nucleotide sequence represented by SEQ. I.D. No. 1;

a heterologous gene downstream of said promoter; and

a terminator having the nucleotide sequence represented by SEQ. I.D. No. 2 downstream of said heterologous gene.

2. An expression vector having the expression cassette of claim 1.

3. The expression vector of claim 2, wherein said expression vector has a sequence portion homologous to a portion of the chromosomal DNA of host cells to integrate said cassette into the chromosomal DNA by a homologous recombination between the homologous sequence portions of the vector and the chromosome.

4. The expression vector of claim 2, wherein said expression vector has an autonomously replicating sequence represented by SEQ. I.D. No. 4.

5. A transformed cell which has been transformed with the expression vector as claimed in any one of claims 2 to 4.

6. A method for producing a peptide or a protein encoded by a heterologous gene comprising culturing said transformed cell of claim 5 in the presence of methanol and/or glycerol and isolating and purifying the desired peptide or protein which is an expression product of said heterologous gene from said culture.

7. The method of claim 6, wherein said heterologous gene is the gene which encodes the adenylate kinase derived from Saccharomyces cerevisiae.

8. The method of claim 6, wherein said heterologous gene is the gene which encodes the cytochrome C552 derived from Hydrogenobacter thermophilus.

9. The method of claim 6, wherein said heterologous gene is a gene which encodes the peroxidase derived from Arthromyces ramosus.