Novel promoter of gene expression

• US 20030003525A1
• Filed: 11/05/2001
• Published: 01/02/2003
• Est. Priority Date: 05/05/1999
• Status: Abandoned Application

• Current Assignee: Leiv Eiriksson Nyfotek AS
• Original Assignee: Leiv Eiriksson Nyfotek AS
• Inventors: Winther-Larsen, Hanne, Valla, Svein
• Application Number:
  US10/012,898
• Publication Number:
  US 20030003525A1
• Time in Patent Office: Days
• US Class Current: 435/32
• CPC Class Codes: C12N 15/78 for Pseudomonas

Abstract

The invention provides an isolated DNA molecule comprising a promoter sequence, said promoter sequence being a Pm or a Pu promoter of a TOL plasmid, said promoter sequence having sequence modifications in its 10 region together with DNA constructs, expression vectors and transformed cells containing such molecules. Also provided by the invention is a method for assaying promoter activity, comprising expressing in an antibiotic-susceptible (i.e. sensitive) host cell, an antibiotic resistance gene under the control of the promoter to be assayed, and assessing the growth of said cell in the presence of said antibiotic.

Claims

1. An isolated DNA molecule comprising a promoter sequence, said promoter sequence being a Pm or a Pu promoter of a TOL plasmid, said promoter sequence having sequence modifications in its −

10 region.

2. A DNA construct comprising a modified Pm or Pu promoter as defined in claim 1 together with a corresponding regulatory gene xylS or xylR.

3. The DNA construct as defined in claim 2 which is an expression cassette.

4. The molecule or construct as defined in any one of claims 1 to 3 wherein said sequence modifications are present in the region spanning the nucleotide sequence from −

1 to −

25 nucleotides upstream of the transcriptional start site of said promoter sequence.

5. The molecule or construct as defined in any one of claims 1 to 4 wherein said sequence modifications comprise 1 to 6 base changes which may be contiguous or non-contiguous.

6. The molecule or construct as defined in claim 4 wherein said sequence modifications in the −

10 region are as set out in FIG. 2, or as in any one of SEQ ID NO. 1 to SEQ ID NO. 8.

7. The molecule or construct as defined in any one of claims 1 to 6, wherein the mutant promoters have reduced or enhanced expression levels.

8. The molecule or construct as defined in any one of claims 1 to 7, wherein the mutant promoters result in reduced leakage or background levels of expression.

9. A process for preparing a mutant Pm or Pu promoter as defined in claim 1 comprising the step of addition, insertion, deletion or substitution of single or multiple nucleotides and/or inversion or repeat of two or more nucleotides in the −

10 region thereof.

10. An expression vector comprising a Pm or Pu promoter mutant which exhibits a modified nucleotide sequence as defined in any one of claims 1 or claims 4 to 8.

11. The expression vector as defined in claim 8 wherein said vector is selected from the group consisting of a plasmid, virus, transposon, phagemid or phage-derived vector, or any other replicon.

12. The expression vector as defined in claim 10 or claim 11 wherein said vector exists or functions extrachromosomally in an autologously replicating form or is integrated into a chromosome.

13. The expression vector as defined in claim 10 wherein said vector is based on the RK2-based minimum replicon.

14. The expression vector as defined in any one of claims 10-13 wherein said vector comprises a transcriptional terminator inserted upstream of the modified Pm/Pu promoter.

15. A cell transformed with a vector as defined in any one of claims 10 to 14.

16. A method of expressing a desired gene within a host cell, comprising the steps of;

i) introducing into said cell an expression vector as defined in any one of claims 10 to 14, and containing said desired gene, and;

ii) culturing said cell under conditions in which said desired gene is expressed.

17. A method of preparing a desired polypeptide product encoded by a desired gene comprising the steps of;

i) culturing a host cell containing an expression vector as defined in any one of claims 10 to 14 into which the desired gene has been introduced under the control of the mutant Pm or Pu promoter, under conditions whereby said polypeptide is expressed, and;

ii) recovering said polypeptide thus produced.

18. Use of a mutant Pm or Pu promoter as defined in any one of claim 1 or claims 4 to 8, in the control of a biosynthetic pathway, wherein at least one structural gene in said pathway is placed under the regulatory control of the mutant Pm or Pu promoter.

19. A method for assaying promoter activity, said method comprising expressing in an antibiotic-susceptible (i.e. sensitive) host cell, an antibiotic resistance gene under the control of the promoter to be assayed, and assessing the growth of said cell in the presence of said antibiotic.