Method for the positive selection of chromosomal mutations in C1 metabolizing bacteria via homologous recombination

• US 20060057726A1
• Filed: 11/24/2004
• Published: 03/16/2006
• Est. Priority Date: 12/03/2003
• Status: Abandoned Application

• Current Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Sharpe, Pamela L.
• Application Number:
  US10/997,308
• Publication Number:
  US 20060057726A1
• Time in Patent Office: Days
• US Class Current: 435/471
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• 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: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: C12N 9/0004 Oxidoreductases (1.)
• CPC Class Codes: C12N 9/001 acting on the CH-CH group of donors (1.3)
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/0083 Miscellaneous (1.14.99)
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/1085 transferring alkyl or aryl groups other than methyl groups (2.5)
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12P 23/00 Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes containing heterorings C12P17/00
• CPC Class Codes: C12R 2001/26 Methylomonas
• CPC Class Codes: C12Y 205/01029 Geranylgeranyl diphosphate synthase (2.5.1.29)

Abstract

A method for the positive selection of allelic exchange mutants is provided for C1 metabolizing bacteria. The chromosomal integration vectors, based on the pGP704 suicide vector, comprise at least one genetic selectable marker and the sacB gene, encoding levansucrase. A one- and two-step selection strategy is provided for the facile identification of double-crossover mutations in C1 metabolizing bacteria. This methodology enables production of “markerless” transformants, such that multiple rounds of mutation can be performed. Optimized conditions for conjugal transfer, homologous recombination, transformant purification, and screening are also presented.

Claims

1. A method for the positive selection of double-crossover events in a C1 metabolizing bacterial host cell comprising:

a) providing a C1 metabolizing bacteria selected from the group consisting of methanotrophs and methylotrophs;

b) providing a positive selection vector comprising;

(i) a first genetic selectable marker;

(ii) an origin of transfer for a C1 metabolizing bacteria;

(iii) plasmid mobilization genes;

(iv) a sacB coding region encoding a levansucrase enzyme, under the control of a suitable promoter;

(v) a replacement nucleotide sequence of interest having at least two regions homology to a chromosomal nucleotide sequence of interest in the C1 metabolizing bacteria, said replacement nucleotide sequence of interest being disrupted by a second genetic selectable marker;

wherein the first and second genetic selectable markers are different and the vector is unable to replicate in the C1 metabolizing bacteria;

c) transforming the C1 metabolizing bacteria of (a) with the vector of (b)

d) selecting the transformants of (c) on the basis of the genetic selectable markers and sacB gene expression wherein, the transformants which are positive for the second selectable marker and grow in the presence of sucrose, but are negative for the first selectable marker have undergone double-crossover events; and

e) recovering the selected transformants of (d) which have undergone double-crossover events.

3. A method according to either of claims 1 or 2 wherein the regions of homology are at least about 50 bp in length.

5. A method according to either of claims 1 or 2, wherein the C1 metabolizing bacteria is selected from the group consisting of Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocyctis, Methylomicrobium, Methanomonas, Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, and Pseudomonas.

6. A method according to either of claims 1 or 2, wherein the suitable promoter is a npr promoter.

7. A method according to either of claims 1 or 2, wherein said sacB coding region is derived from the group consisting of Bacillus subtilis, Bacillus pumilus, Bacillus loceniformis, Bacillus amyloliquifaciens, Bacillus thurgiensis, Bacillus stearothermophilus, and Bacillus sphaericus.

8. A method according to either of claims 1 or 2, wherein the positive selection vector is pGP704:

;

sacB.

9. A method according to either of claims 1 or 2 wherein any of the genetic selectable markers are independently selected from the group consisting of antibiotic resistance markers, metal resistance markers, substrate-utilization markers, genes encoding fluorescent and bioluminescent proteins, lacZ, gfp, cat, galK, inaZ, luc, luxAB, bgaB, nptll, phoA, uidA, and xylE.

10. A C1 metabolizing bacteria transformant selected by the process of either claim 1 or claim 2 employing a positive selection vector.

11. A recombinant C1 metabolizing bacteria transformant of claim 10 wherein the genome of said transformant lacks any sequence of the positive selection vector, other than the replacement nucleotide sequence of interest.

12. A recombinant C1 metabolizing bacteria transformant of claim 10 wherein the genome of said transformant lacks any genetic selectable marker sequence.

2. A method for the positive selection of double-crossover events in C1 metabolizing bacterial host cells comprising:

a) providing a C1 metabolizing bacteria selected from the group consisting of methanotrophs and methylotrophs;

b) providing a positive selection vector comprising;

(i) a genetic selectable marker;

(ii) an origin of transfer for a C1 metabolizing bacteria;

(iii) plasmid mobilization genes;

(iv) a sacB coding region encoding a levansucrase enzyme under the control of a suitable promoter;

(v) a mutant replacement nucleotide sequence of interest having at least two regions of homology to a chromosomal nucleotide sequence of interest in the C1 metabolizing bacteria;

the vector being unable to replicate in the C1 metabolizing bacteria chromosome;

c) transforming the C1 metabolizing bacteria of (a) with the vector of (b);

d) isolating the transformants of (c) on the basis of the genetic selectable marker; and

e) isolating the transformants of (d) which grow in the presence of sucrose, wherein said transformants contain double-crossover events.

4. A method according to claim 2 wherein the mutation in the mutant nucleotide of interest is at least about 50 bp downstream of the 5′

end of the sequence.

13. A positive selection vector for the positive selection of double-crossover events in a C1 metabolizing bacterial host cell comprising:

(i) at least one genetic selectable marker;

(ii) an origin of transfer for a C1 metabolizing bacteria;

(iii) plasmid mobilization genes;

(iv) a sacB coding region encoding a levansucrase enzyme under the control of a suitable promoter;

(v) a replacement nucleotide sequence of interest having at least two regions of homology to a chromosomal nucleotide sequence of interest in the C1 metabolizing bacteria;

the vector being unable to replicate in the C1 metabolizing bacteria chromosome.

14. A vector according to claim 13 wherein said sacB coding region is derived from the group consisting of Bacillus subtilis, Bacillus pumilus, Bacillus loceniformis, Bacillus amyloliquifaciens, Bacillus thurgiensis, Bacillus stearothermophilus, and Bacillus sphearicus.

15. A vector according to claim 13 wherein the suitable promoter is the npr promoter.

16. A vector according to claim 13 wherein the replacement nucleotide of interest is disrupted by at least one second genetic selectable marker.

17. A vector according to claim 13 or 16 wherein the genetic selectable marker is selected independently from the group consisting of antibiotic resistance markers, metal resistance markers, substrate-utilization markers, genes encoding fluorescent and bioluminescent proteins, lacZ, gfp, cat, galK, inaZ, luc, luxAB, bgaB, nptll, phoA, uidA, and xylE.

18. A vector according to claim 13 wherein the regions of homology are at least about 50 bp in length.

19. A vector according to claim 13 wherein the replacement nucleotide sequence of interest is a mutant replacement nucleotide sequence of interest.

20. A vector according to claim 13 wherein the vector is pGP704:

;

sacB.

21. A transformed C1 metabolizing bacteria comprising the vector of claim 13.

22. A transformed C1 metabolizing bacteria of claim 21 selected from the group consisting of methanotrophs and methylotrophs.

23. A methylotroph of claim 22 selected from the group consisting of Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocyctis, Methylomicrobium, Methanomonas, Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, and Pseudomonas.