Use of clostridial methyltransferases for generating novel strains
View Patent ↗This invention provides isolated polynucleotides encoding DNA Type I methyltransferase and uses thereof for improving transformation efficiencies of exogenous and endogenous plasmid DNA into Clostridial hosts.
1. A method of generating a bacterial recombinant cell expressing a DNA Type I methyltransferase having biological activities, comprising:
(a) introducing a methylation vector into a bacterial host cell wherein said methylation vector expresses a DNA Type I methyltransferase polynucleotide, wherein the DNA Type I methyltransferase polynucleotide is selected from the group consisting of a polynucleotide comprising a nucleotide sequence having at least 98% sequence identity with SEQ ID NO: 1, a polynucleotide encoding a polypeptide having an amino acid sequence at least 97% sequence identity with the polypeptide SEQ ID NO: 2, a polynucleotide comprising a nucleotide sequence having at least 97% sequence identity with SEQ ID NO: 3, and a polynucleotide encoding a polypeptide having an amino acid sequence at least 98% sequence identity with the polypeptide of SEQ ID NO: 4;
(b) introducing a transforming vector into the bacterial host cell wherein said transforming vector comprises plasmid DNA that is methylated by said methylation vector; and
(c) purifying and transferring the methylated plasmid DNA from the bacterial host cell to a second bacterial recipient cell that can degrade the plasmid DNA but which cannot degrade the methylated DNA.
2. The method of claim 1 wherein the DNA Type I methyltransferase polynucleotide is expressed from an operably-linked promoter.
3. The method of claim 2 wherein the operably-linked promoter is phosphotransacetylase-acetate kinase operon.
4. The method of claim 1 wherein the bacterial host cell is Escherichia coli.
5. The method of claim 1 wherein the second bacterial recipient cell is Clostridia.
6. The method of claim 1 wherein the methylated plasmid DNA is transferred from the bacterial host cell to a second bacterial recipient cell by transformation.
7. The method of claim 6 wherein transformation is electroporation or conjugation.
8. The method of claim 1 further comprising isolating stable bacterial transformants containing the methylated plasmid DNA.
9. The method of claim 1 further comprising introducing a shuttle vector into the bacterial host cell.
10. The method of claim 9 wherein the shuttle vector is pACR-1.
11. The method of claim 1 further comprising introducing a conjugation vector into the bacterial host cell.
12. The method of claim 11 wherein the conjugation vector is pACC-1.