Regulation of heterologous recombinant protein expression in methylotrophic and methanotrophic bacteria

• US 20100221813A1
• Filed: 08/21/2006
• Published: 09/02/2010
• Est. Priority Date: 08/23/2005
• Status: Active Grant

• Current Assignee: National Research Council Canada
• Original Assignee: National Research Council Canada
• Inventors: Choi, Young-Jun, Massie, Bernard, Miguez, Carlos B., Mullick, Alaka
• Granted Patent: US 8,216,821 B2
• Time in Patent Office: Days
• US Class Current: 435/252.340
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

Methylotrophic or methanotrophic bacteria such as Methylobacterium are transformed with a gene of interest, and expression of the gene is regulated by means of a cumate repressor protein and an operator sequence which is operatively linked to the gene of interest, and the addition of an external agent. Specifically, the cymR repressor and cmt operator from Pseudomonas putida may serve to regulate gene expression in methylotrophic or methanotrophic bacteria with the addition of cumate.

Claims

1. A polynucleotide construct for use in association with a methylotrophic car methanotrophic host cell, the host cell being capable or expressing a cymR repressor protein exhibiting repressor activity which is regulatable by the addition of an external agent, and the polynucleotide construct comprising a constitutive promoter native to the host cell, wherein the promoter is fused to a cymR operator sequence which is operatively linked to at least one gene of interest, wherein the cymR operator sequence is in operative association with the cymR operator binding protein and wherein addition of the external agent upregulates expression of the at least one gene of interest.

2. A polynucleotide construct as claimed in claim 1 wherein the cymR repressor protein is derived from Pseudomonas putida.

3. A polynucleotide construct as claimed in claim 2 wherein the cymR operator sequence is at least a portion of the cmt operon of Pseudomonas putida.

4. A polynucleotide construct as claimed in claim 1 wherein the external agent is cumate.

5. A polynucleotide construct as claimed in claim 1 wherein the host cell comprises a chromosome, and wherein at least one polynucleotide sequence encoding the operator binding protein is integrated into the chromosome.

6. A polynucleotide construct as claimed in claim 1 wherein the promoter is λ

or P_mxaF.

7. A polynucleotide construct as claimed in claim 1 wherein the at least one gene of interest encodes a reporter protein.

8. A polynucleotide construct as claimed in claim 1 wherein the at least one gene of interest encodes a protein that is toxic or difficult to express in the host cell.

9. A polynucleotide construct as claimed in claim 7 wherein the reporter protein is selected from the group consisting of β

-galactosidase, esterase and green fluorescence protein.

10. A polynucleotide construct as claimed in claim 1, further defined as a vector.

11. The polynucleotide construct of claim 10, wherein the vector is a viral vector or a plasmid vector.

12. A methylotrophic or methanotrophic prokaryotic host cell comprising at least one polynucleotide sequence that encodes a cymR repressor protein exhibiting repressor activity which is regulatable by the addition of an external agent and further comprising a polynucleotide construct which comprises a constitutive promoter native to the host cell, wherein the promoter is fused to a cymR operator sequence which is operatively linked to at least one gene of interest, wherein the cymR operator sequence is in operative association with the cymR repressor protein and wherein addition of the external agent upregulates expression of the at least one gene of interest.

13. A host cell as claimed in claim 12 which belongs to the genus Methylobacterium.

14. A host cell as claimed in claim 13 which belongs to the species Methylobacterium extorquens.

15. A host cell as claimed in claim 14 which belongs to the species identified as Methylobacterium extorquens ATCC55366.

16. A host cell as dunned in claim 12 wherein the cymR repressor protein is derived from Pseudomonas putida.

17. A host cell as claimed in claim 16 wherein the cymR operator sequence is at least a portion of the cmt operon or Pseudomonas putida.

18. A host cell as claimed in claim 12 wherein the external agent is cumate.

19. A host cell as claimed in claim 12 wherein the promoter is P_mxaF.

20. A host cell as claimed in claim 12 wherein the at least one gene of interest comprises a gene that encodes a reporter protein.

21. A host cell as claimed in claim 20 wherein the reporter protein is selected from the group consisting of β

-galactosidase, esterase and green fluorescence protein.

22. A host cell as claimed in claim 12 wherein the at least one gene of interest comprises a gene that encodes a recombinant protein.

23. A host cell as claimed in claim 12 having a chromosome, wherein the at leak one polynucleotide sequence that encodes the operator binding protein is integrated into the chromosome.

24. A host cell as claimed in claim 23, wherein the at least one polynucleotide sequence that encodes the operator binding protein is inserted into the host cell chromosome at the mini-Tn7 transposon site.

25. A gene expression system comprising a host cell as claimed in claim 12.

26. A method for selectively controlling the expression of at least one gene of interest comprising the steps of a) obtaining a host cell as claimed in claim 12 wherein expression of the at least one gene of interest is repressed clue to the repressor activity of the cymR repressor protein;

and b) exposing the host coil to an external agent which inhibits the repressor activity and allows for expression of the at least one gene of interest.