Study On The Select And Functional Analysis Of Bacillus Promoter

Bacillus is a gram-positive bacterium which widely exist in nature.It is an important engineering strain which is widely used in industrial production of lipase,amylase and protease.Bacillus subtilis is a kind of model strain of Bacillus.It has the characteristics of non-pathogenicity,clear genetic background,secreting protein strongly,easy separation and culture.Thereforelt it is an ideal host for the secretion and expression of heterologous proteins.The current Bacillus subtilis promoters are not sufficient to meet the requirements for the industrial demand of Bacillus subtilis to express a large number of foreign genes.Therefore,it is very necessary to find new strong promoters that can be expressed in Bacillus subtilis.The purpose of this project is to explore a new method for screening promoters.By the means of selecting promoters in Bacillus by SDS-PAGE and biological software analysis.we have successfully obtained a constitutive promoter BSP3 and a inducible promoter BSP5B.We use maltose a-amylase in Saccharomonosra viridis as a reporter gene to construct the recombinant plasmid pHCMC04-BSP3-sva and pHCMC04-BSP5B-sva,and then to introduce into B.subtilis 1A857 for expressing respectively.The results show that both BSP3 and BSP5B can express SVA gene in Bacillus subtilis.The highest crude enzyme activity of extracellular fermentation broth measured by DNS method was 16.248 U/mL and 21.758 U/mL,respectively.In this research,we replace the existing promoter P43 with constructive promoter BSP3 which is obtained by cloning and accomplish hybridization in the core conserved region.The hybrid vectors were named as BSP3D2-sva,BSP3D3-sva,XL3-sva,XL4-sva,XL5-sva,and XL6-sva.Then the hybrid plasmids were transferred to B.subtilis 1A857.And measure datas of sva enzyme activity and OD600 every 12 hours.According to the result the highest crude enzyme activities of extracellular fermentation broth are 19.924 U/mL,19.063 U/mL,14.982 U/mL,20.578 U/mL,26.142 U/mL,and 17.538 U/mL,respectively;The OD600 values are 4.308,3.592,and 3.35,respectively.3.35,6.418,and 4.672;Crude enzyme activity units per unit biomass were 4.624 U/mL,5.307 U/mL,4.470 U/mL,5.036 U/mL,4.073 U/mL,and 3.754 U/mL,respectively.The BSP3 promoter in BSP3D3-sva has a better effect,it is 1.11 times that of the original promoter BSP3.the P43 promoter in hybrid XL4-sva is 1.44 times activity compared with theoriginal P43 promoter.Experimental results show that the combination of advantages of different promoters in the core conserved regions can optimize the structure of the promoter that to increase the transcription efficiency of the promoter.Based on the successfully constructed expression vector pHCMC04-BSP5B-sva,we site-directed mutant the conserved core region of the inducible promoter BSP5B and obtain the mutation vectors BSP5B-35A-sva,BSP5B-10A-sva,BSP5B-10B-sva,BSP5B-35C-sva,BSP5B-10C-sva.Then transferred them to B.subtilis 1A857.The highest crude enzyme activities datas of sva fermentation broth were 21.991 U/mL,23.595 U/mL,31.156 U/mL,43.228 U/In mL and 35.981 U/mL,.The OD600 values were 5.115,4.992,5.28,5.318,and 4.592,respectively.The crude enzyme activity units per unit biomass were 4.299 U/mL,4.793 U/mL,5.899 U/mL,and 8.130 U/mL,7.836 U/mL,respectively.The mutations of BSP5B-35C-svfa and BSP5B-10C-sva have obvious advantages.The enzyme activity was 1.75times and 1.69 times higher than the original BSP5B promoter.The experimental results show that mutant the promoter's conserved region will change the promoter greening activity,in other words site-directed mutant the promoter's conserved core region into the classical promoter conserved core region will increase the transcriptional activity of the promoter.