Regulation of heterologous recombinant protein expression in methylotrophic and methanotrophic bacteria

• US 8,216,821 B2
• Filed: 08/21/2006
• Issued: 07/10/2012
• Est. Priority Date: 08/23/2005
• Status: Expired due to Fees

• Current Assignee: National Research Council Canada
• Original Assignee: National Research Council Canada
• Inventors: Miguez, Carlos B., Choi, Young-Jun, Mullick, Alaka, Massie, Bernard
• Primary Examiner(s): Navarro, Albert
• Assistant Examiner(s): Portner, Ginny
• Application Number:
  US11/990,779
• Publication Number:
  US 20100221813A1
• Time in Patent Office: 2,150 Days
• Field of Search: None
• US Class Current: 435/252.34
• 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 methylotrophic or methanotrophic prokaryotic host cell comprising two copies, integrated into the chromosome of the host cell, of a polynucleotide sequence that encodes a cysteine metabolism repressor (cymR repressor) protein exhibiting repressor activity which is regulatable by addition of cumate, 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 a cymR repressor protein encoded by the polynucleotide sequence binds to the cymR operator sequence and wherein the addition of cumate upregulates expression of the at least one gene of interest.

2. A host cell as claimed in claim 1 which belongs to the genus Methylobacterium.

3. A host cell as claimed in claim 2 which belongs to the species Methylobacterium extorquens.

4. A host cell as claimed in claim 3 which belongs to the species identified as Methylobacterium extorquens ATCC55366.

5. A host cell as claimed in claim 1 wherein the cymR repressor protein is derived from Pseudomonas putida.

6. A host cell as claimed in claim 5 wherein the cymR operator sequence is at least a portion of the cmt operon of Pseudomonas putida.

7. A host cell as claimed in claim 1 wherein the promoter is P_mxaF.

8. A host cell as claimed in claim 1 wherein the at least one gene of interest comprises a gene that encodes a reporter protein.

9. A host cell as claimed in claim 8 wherein the reporter protein is selected from the group consisting of β

-galactosidase, esterase and green fluorescence protein.

10. A host cell as claimed in claim 1 wherein the at least one gene of interest comprises a gene that encodes a recombinant protein.

11. A host cell as claimed in claim 1, wherein the two copies of the polynucleotide sequence that encodes the cymR repressor protein are inserted into the host cell chromosome at a mini-Tn7 transposon site.

12. 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 1 wherein expression of the at least one gene of interest is repressed due to the repressor activity of the cymR repressor protein;

b) culturing the host cell under conditions for expressing the cymR repressor protein; and

c) exposing the host cell to cumate which inhibits the repressor activity and allows for expression of the at least one gene of interest.