Multicopy-integration of heterologous genes and expression in methylobacterium

• US 20080026005A1
• Filed: 07/26/2006
• Published: 01/31/2008
• Est. Priority Date: 07/26/2005
• Status: Active Grant

• Current Assignee: National Research Council Canada
• Original Assignee: National Research Council Canada
• Inventors: Choi, Young-Jun, Miguez, Carlos B.
• Granted Patent: US 7,670,824 B2
• Time in Patent Office: Days
• US Class Current: 424/200.1
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora

Abstract

The integration of genes into methylobacterium, such as M. extorquens ATCC 55366 is disclosed, using a transposon system, preferably the mini-Tn7 transposon system, and under the control of a promoter, such as the strong methanol dehydrogenase promoter (P_mxaF). Multicopy integration of genes of interest is also disclosed. The unique and specific attachment site for the Tn7 attachment (attTn7) is identified for M. extorquens.

Claims

1. A vector for the integration of at least one gene into the genetic material of a Methylobacterium host cell such that the gene may be expressed by the host cell, the vector comprising a promoter operably linked to said at least one gene, and a transposon system.

2. The vector of claim 1 wherein the transposon system is a mini-Tn7 transposon system.

4. The vector of claim 1 wherein the promoter is selected from the groups consisting of a methanol dehydrogenase promoter, a lac promoter, a T5 promoter, and a λ

promoter.

5. The vector of claim 1 wherein the promoter is a methanol dehydrogenase promoter.

6. The vector of claim 1 comprising one, two, three, four or five copies of the at least one gene.

8. The vector of claim 1 wherein the at least one gene encodes a toxic protein.

9. The vector of claim 8 wherein the toxic protein is cry1Aa.

10. The vector of claim 1 wherein the at least one gene encodes a protein that is easily tracked.

11. The vector of claim 10 wherein the protein is selected from the group consisting of β

-galactosidase, esterase, and green fluorescence.

12. A Methylobacterium host cell transformed with the vector of claim 1

13. The host cell of claim 12 that is a Methylobacterium extorquens cell.

14. The host cell of claim 12 wherein integration of the vector does not cause any insertional inactivation of host genes.

15. The host cell of claim 12 wherein the vector is integrated at the attTn7 site.

16. A method for producing a polypeptide encoded by said at least one gene, comprising the step of culturing host cells as claimed in claim 14.

17. The use of a host cell as claimed in claim 14 as a biopesticide.

18. The use of a host cell as claimed in claim 14 for tracking the movement and growth of the host cell.

3. The vector of claim wherein the host cell is a Methylobacterium extorquens cell.

7. The vector of claim 3 wherein the Methylobacterium extorquens host cell is from the species designated as ATCC 55366.