Application Of Recombineering In Zymomonas.mobilis

Recombineering is a new genetic engineering biotechnology that has recently been developed. It can efficiently boost the recombination between linear DNA molecule and linear DNA molecule, linear DNA molecules and chromosome or plasmid DNA. It is one of the most forceful new implement in gene manipulation and genomics research in E.coli, it has also been used for several other kinds of bacteria.Zymomonas mobilis has unique biology characteristics and can highly effectively ferment sugars to alcohol. It is considered as a potential alcohol producer and has been researched widely. But the lack of highly effective,simple and speedy genetic manipulation method has embarrassed researcher for further studying its biology characteristics, and genetic manipulation using recombineering in Z.mobilis will greatly accelerate research for it.This study is the first attempt to introduce recombineering system into Z. mobilis and use it for recombination. Firstly, plasmid pKD46(araC,Para-red,pSC101 ori,amp),being widely used in E.coli recombineering, was directly introduced into Z. mobilis, resulting in strain ZM4(pKD46), and was initially proved to duplicate and exist in ZM4 by antibiotic resistance check, PCR identification and plasmid enzymatic digest analysis of ZM4(pKD46).Secondly, pZB21-red (E.coli ori,Z.mobilis ori,amp,cml,tet) was constructed from shuttle vector pZB21(E.coli ori,Z.mobilis ori,amp,cml,tet)and pKD46, and further transformed into ZM4, leading to strain ZM4(pZB21-red).Lastly, targeting experiment was carried out for ZM4(pKD46) and ZM4(pZB21-red),under the same targeting conditions as used for E.coli strains with pKD46, and the targeting fragment, with both 79bp long homologous arms and cml gene, was prepared by PCR. It was convenient to get the desired recombinants from ZM4(pZB21-red) in which genomic DNA ldh gene was deleted and replaced by targeting fragment .Moreover, it was also easy to obtain the recombinants losing pZB21-red by continuous inoculation and incubation .But no recombinant was found to result from ZM4(pKD46).So this study has initially proved that recombineering could be used in Z.mobilis and layed the good foundations for next step to optimize the recombination system and improve recombination effiency.