Process for producing/secreting a protein by a transformed mould using expression/secretion regulating regions derived from a aspergillus endoxylanase II gene

• US 5,705,358 A
• Filed: 06/07/1994
• Issued: 01/06/1998
• Est. Priority Date: 12/09/1991
• Status: Expired due to Fees

• Current Assignee: Unilever Patent Holdings BV (Unilever PLC)
• Original Assignee: Unilever Patent Holdings BV (Unilever PLC)
• Inventors: Stam, Hein, Gouka, Robertus Johannes, Verbakel, Johannes Maria, van den Hondel, Cornelis Antonius, Musters, Wouter
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Bugaisky, Gabriele E.
• Application Number:
  US08/244,686
• Time in Patent Office: 1,309 Days
• Field of Search: 435/69.1, 435/68.1, 435/9.4, 435/172.3
• US Class Current: 435/69.1
• 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 9/20 Triglyceride splitting, e.g. by means of lipase
• CPC Class Codes: C12N 9/2482 Endo-1,4-beta-xylanase (3.2.1.8)
• CPC Class Codes: C12Y 302/01008 Endo-1,4-beta-xylanase (3.2.1.8)
• CPC Class Codes: C12Y 302/01031 Beta-glucuronidase (3.2.1.31)
• CPC Class Codes: C12Y 302/01032 Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
• CPC Class Codes: C12Y 302/01136 Glucuronoarabinoxylan endo-1,4-beta-xylanase (3.2.1.136), i.e. feraxanase or feraxan-endoxylanase

Abstract

Methods are described for the isolation and characterization of DNA sequences from Aspergillus niger var. awamori which are involved in the expression and secretion of endoxylanase II (exlA) by said Aspergillus mould. A process using these expression and/or secretion regulating regions to direct the production and optionally the secretion of proteins other than endoxylanase II by transformed moulds is provided.

Claims

1. A process for production of a protein which comprises culturing cells of a mould transformed with an expression vector comprising a gene encoding said protein operatively linked to at least one regulating region selected from the group consisting of

a) expression and secretion regulating regions of the endoxylanase II gene (exlA gene) of Aspergillus niger var. awamori present on plasmid pAW14B (FIG. 3), which is present in a transformed E. coli strain JM109 deposited at the Centraalbureau voor Schimmelcultures in Baarn, The Netherlands, under N°

CBS 237.90 on 31 May 1990, and

b) functional derivatives of a) having at least one activity selected from the group consisting of expression and secretion regulating activity, wherein said protein is not endoxylanase type II ex Aspergillus niger var. awamori;

and wherein the gene encoding said protein is in frame with any secretion regulating region present.

2. The process according to claim 1 wherein the selected expression regulating region is a promoter and said vector comprises a gene encoding said protein under control of said promoter, the latter being selected from the group consisting of

a) the promoter of the endoxylanase II gene (exlA gene) of Aspergillus niger var. awamori present on plasmid pAW14B (FIG. 3), which is present in a transformed E. coli strain JM109 deposited at the Centraalbureau voor Schimmelcultures in Baarn, The Netherlands, under N°

CBS 237.90 on 31 May 1990, and

b) functional derivatives of a) also having promoter activity.

3. The process according to claim 2, in which said promoter corresponds with the promoter present on the 5'"'"' part upstream of the exlA gene having a size of about 2.5 kb located between the SalI restriction site at position 0 and the start codon ATG of the exlA gene in plasmid pAW14B.

4. The process according to claim 2, in which said promoter comprises at least the polynucleotide sequence 1-350 according to FIG. 1 (SEQ ID NO:

1).

5. The process according to claim 2, in which said promoter is induced by wheat bran, xylan, or xylose, or a mixture of any combination thereof, present in a medium in which the transformed mould is incubated.

6. The process according to claim 5, in which said promoter is induced by xylose present in the medium in which the transformed mould is incubated.

7. The process according to claim 1 wherein the selected expression regulating region is a terminator and said vector comprises a gene encoding said protein followed by said terminator, the latter being selected from the group consisting of

a) the terminator of the endoxylanase II gene (exlA gene) of Aspergillus niger var. awamori present on plasmid pAW14B (FIG. 3), which is present in a transformed E. coli strain JM109 deposited at the Centraalbureau voor Schimmelcultures in Baarn, The Netherlands, under N°

CBS 237.90 on 31 May 1990, and

b) functional derivatives of a) also having terminator activity.

8. The process according to claim 7, in which said terminator corresponds with the terminator present on the 3'"'"' part downstream of the exlA gene having a size of about 1.0 kb located right downstream of the stop codon (TAA) of the exlA gene in plasmid pAW14B.

9. The process according to claim 7, in which said vector also comprises a promoter and said vector comprises a gene encoding said protein under control of said promoter, the latter being selected from the group consisting of

a) the promoter of the endoxylanase II gene (exlA gene) of Aspergillus niger var. awamori present on plasmid pAW14B (FIG. 3), which is present in a transformed E. coli strain JM109 deposited at the Centraalbureau voor Schimmelcultures in Baarn, The Netherlands, under N°

CBS 237.90 on 31 May 1990, and

b) functional derivatives of a) also having promoter activity.

10. A process according to claim 1 for the production and secretion of a protein by means of a transformed mould, wherein the selected secretion regulating region is a DNA sequence encoding a signal sequence and said vector comprises a gene encoding said protein preceded by said DNA sequence encoding a signal sequence, the latter being selected from the group consisting of

a) the DNA sequence encoding the signal sequence of the endoxylanase II gene (exlA gene) of Aspergillus niger var. awamori present on plasmid pAW14B (FIG. 3), which is present in a transformed E. coli strain JM109 deposited at the Centraalbureau voor Schimmelcultures in Baarn, The Netherlands, under N°

CBS 237.90 on 31 May 1990, and

b) functional derivatives of a) also directing secretion of the protein;

and wherein the gene encoding said protein is in frame with said DNA encoding the signal sequence.

11. The process according to claim 10, in which the gene encoding said protein is also preceded by at least an essential part of a DNA sequence encoding the mature endoxylanase II protein, whereby said essential part of said DNA sequence is present between said DNA sequence encoding a signal sequence and the gene, and wherein the gene encoding said protein is in frame with both said DNA encoding a signal sequence and the essential part of said DNA sequence encoding the mature endoxylanase II protein.

12. The process according to claim 10, in which said signal sequence is the signal sequence encoded by polynucleotide 351-431 of the DNA sequence given in FIG. 1 (SEQ ID NO:

1), which polynucleotide precedes the exlA gene in plasmid pAW14B.

13. The process according to claim 10, in which said vector also comprises at least one of

a) a promoter and said vector comprises a gene encoding said protein under control of said promoter, the latter being selected from the group consisting of the promoter of the endoxylanase II gene (exlA gene) of Aspergillus niger var. awamori present on plasmid pAW14B (FIG. 3), which is present in a transformed E. coli strain JM109 deposited at the Centraalbureau voor Schimmelcultures in Baarn, The Netherlands, under N°

CBS 237.90 on 31 May 1990, and functional derivatives thereof also having promoter activity and

b) a terminator and said vector comprises a gene encoding said protein followed by said terminator, the latter being selected from the terminator of the endoxylanase II gene (exlA gene) of Aspergillus niger var. awamori present on plasmid pAW14B (FIG.

     3), which is present in a transformed E. coli strain JM109 deposited at the Centraalbureau voor Schimmelcultures in Baarn, The Netherlands, under N°

CBS 237.90 on 31 May 1990, and functional derivatives thereof also having terminator activity.