Cloning And Molecular Modification Of The Promoters Of Bacillus Subtilis

Bacillus subtilis is a kind of Bacillus,belonging to aerobic Gram-positive bacteria.It has the advantages of clear genetic background,easy isolation and culture,strong extracellular secretion and non-pathogenicity.It is very suitable as a host strain for expressing and secreting heterologous proteins and widely used in industrial production as a model strain of Bacillus.The results of industrial production show that the existing Bacillus subtilis vector system can not meet the increasing expression of more and more foreign genes,Therefore,attempts to start from the promoter of the expression systerrm,screening and transformiation to obtain a new strong promoter that can meet the requirements of as many exogenous gene expression as possible,thereby improving the expression of Bacillus subtilis system.In this study,we constructed a probe-type vector to screen a fragment with promoter ability,and constructed a two-way promoter probe vector pKT2 with kanamycin resistance gene and tetracycline resistance gene,Then the soil bacteria DNA was extracted for screening promoter,However,due to the low efficiency of transformed Bacillus subtilis,sufficient transformants could not be obtained to screen for efficient promoters.Finally,try to mutate constitutive promoter P43 and the inducible promoter Pglv.After site-directed mutagenesis and key sequence substitution hybridization,new promoters P43-35,P43-35quan,P43-35-10,P43-Pglv-35,Pgdv-10,Pglv-35 were screened according to the database alignment analysis.The promoter was constructed by using the maltose a-amylase gene sva and the alkaline phosphatase gene phoA as reporter genes to detect the function of the promoters,the recombinant vectors pHS-P43?pHS-P4335?pHS-P4335quan?pHS-P433510?pHCMC04-sva-PP35? pHS-Pglv?pHS-Pglv10?pHS-Pglv35?pHS-PP 10 were constructed using the maltose?-amylase gene sva as the reporter gene.After successful construction,the vector was transformed into the host strain B.subtilis 1A857 for expression.,the highest crude enzyme activity units of the fermentation broth were 1.214 U/mL?1.334 U/mL?1.042 U/mL?1.1U/mL?1.184 U/mL?15.284 U/mL?14.635 U/mL?18.849 U/mL?7.651 U/mL,the highest OD595 of the bacterial liquid were 2.553?2.188?2.485?2.338?2.57?5.433?5.847?5.29?7.103,,and 7.1.Among them,only the mutant enzymepHS-P4335 and pHS-Pglv35,which was mutated in the-35 region of the promoter P43 and Pglv,had a certain increase,which was 1.099 and 1.233 times that before the mutation,and the expression of the other mutants showed decreasing states.The recombinant vector pHP-Pglv and pHP-Pglv35 were constructed with the alkaline phosphatase gene phoA as the reporter gene.After successful construction,they were transformed into the host strain B.subtilis 1A857 for fermentation and expression.The highest crude enzyme activity units of the fermentation broth were 10.176 U/mL and 6.992 U/mL,respectively,which was 31%lower.Studies have shown that mutations at the core conserved sequence of the promoters may have a positive effect and increase the transcriptional activity of the promoters.