Study On The Cloning And Functional Analysis Of Bacillus Subtilis Promoter

Bacillus subtilis,as a class of widespread gram-positive bacteria,has some characteristics which are secreted protein strong,non-pathogen iceasy to isolate and culture,clear genetic background and so on.Bacillus subtilis is the ideal host for heterologous gene expression.And it has become an important industrial strain that widely used in the industrial production of protease,amylase and lipase.However,the practice has shown that the frequent promoter of Bacillus subtilis expression system is not commonly used for all genes expressed efficiently.So the screening of new strong promoter as well as transformation promoter has great significance for realizing the efficient expression of heterologous gene.In this study,a probe-type plasmid vector PUC-kana was constructed to directly screen the strong promoter in Escherichia coli.The PK3 fragment with promoter activity was obtained,and the recombinant plasmid of pHCMC04-Pk3-sva was constructed,then introduced into B.subtilis 1A857 for expression.The results showed that sva gene was successfully expressed in Bacillus subtilis.The OD600 of recombinant strains was 8.6.The crude enzyme activity of the extracorporeal fermentation was 27.3 U/mL in 96 h.The crude enzyme activity was 3.17 U/mL perunit biomass.The promoter PaprE was cloned from Bacillus subtilis secreting alkaline protease,and promoter P43 of the gene coding cytidine deaminase was also cloned.The recombinant plasmid pHCMC04-PaprE-sva,pHCMC04-P43-sva,pHCMC04-P43-PaprE-sva and pHCMC04-PaprE-p43-sva were constructed,respectively.Subsequently,the recombinant plasmid was both transformed into B.subtilis 1A857.The recombinant plasmid was both constructed and introduced into B.subtilis 1A857 for expression.The recombinant strain was cultured with 5%inoculation amount at 100 mL LB medium.The amylase activity was determined by the DNS method.The crude enzyme activity of pHCMC04-PaprE-sva,pHCMC04-P43-sva,pHCMC04-P43-PaprE-sva,pHCMC04-PaprE-p43-sva were 69.4 U/mL,79.7 U/mL,97.6 U/mL and 121.8U/mL,respectively.The OD600 of the recombinant strains was 8.5,10.3,9.5 and 9.03,respectively.So the crude enzyme activity were respectively 8.06 U/mL,8.8 U/mL,10.02 U/mL,13.09 U/mL per unit biomass.P43-PaprE was increased by 1.2 times compared with single promoter,and PaprE-p43 activity was 1.6 times higher than that of single promoter.Therefore the activity of tadem promoter was better than that of single promoter.The combination of PaprE-p43 was among the best.Compared with the constitutive promoter,the inducible promoter has a higher application value because of its controllability.Based on the previous experimental results,we intended to obtain a more efficient induction expression vector.The recombinant plasmids pHCMC04-P43-pglv-sva and pHCMC04-Pglv-p43-sva were constructed by using pHCMC04-pglv-sva as the backbone.The recombinant plasmids pHCMC04-pglv-sva pHCMC04-P43-pglv-sva and pHCMC04-Pglv-p43-sva were respectively transformed into B.subtilis 1A857 competent cells for fermentation.The results showed that in the case of 1.5%maltose induction,the activity of pHCMC04-pglv-sva,pHCMC04-P43-pglv-sva,pHCMC04-Pglv-p43-sva was 112.4 U/mL,123.8 U/mL and 52.3 U/mL,respectively.The double promoter showed no advantage of the single promoter in the highest activity,but the expression of the enzyme was prolonged after the Pglv promoter was linked to the P43 promoter.