Establishment And Application Of The New Expression And Genetic Manipulation System In Bacillus Subtilis

Bacillus subtilis is an attractive host for the production of heterologous secretory proteins for several reasons: it is non pathogenic and capable of secreting functional extracellular proteins directly to the culture medium, a great deal of vital information concerning genetic manipulation and large scale fermentation has now been acquired. Its application is restricted by secreting a variety of proteases and the unstable of recombinant expression plasmids. In the near past years, with the development of molecular biology and genetic engineering, there are many important progresses for Bacillus subtilis as the host in the genetic engineering were achieved. There is a good prospect for the application of B. subtilis expression system. This work mainly focuses on establishing the B. subtilis high-level expression and secretion system and to realize the high-level expression and secretion of methyl parathion hydrolase encoding gene mpd in B. subtilis; there are also some exploring works were performed on establishing the new homologous recombination and transformation system in B. subtilis.A facilitative and efficient promoter trapping vector, pUC-mpd, was constructed with the promoterless methyl parathion hydrolase gene as the reporter. This reporter gene can be used easily to clone promoters with different promoting strength on selective plates. Promoter regions of the ytkA and ywoF genes with strong promoting and signal peptide functions were cloned from the Bacillus subtilis 168 genomic promoter library with this vector. A new shuttle promoter-probe vector pNW33N-mpd was constrcted with the E. coli-B. subtilis shuttle vector pNW33N and the mature mpd gene without it's signal peptide-encoding sequence. The promoter fragments of B. subtilis ytkA and ywoF gene were cloned from plasmid pMPDP3 and pMPDP29 then generated the shuttle expression vector pNYTM and pNYWM. Expression vectors pNYTM and pNYWM were transformed into B. subtilis 1A751 to construct the expression strain 1A751(pNYTM) and 1A751(pNYTM), in these strains, under the control of the promoters and signal peptides of ytkA and ywoF gene, mpd gene was expressed and secreted with it's biological activity; the result showed that the promoter of ytkA gene is much stronger than that of ywoF gene. Then a new shuttle expression-secretion vector pYNMK was constructed using the ytkA...