Effect On Fermentation Performance Of Bacillus Clausii By Spo0A Genetic Deletion

Bacillus clausii,determined as a wild strain of high yields in the aspect of alkaline protease,has achieved industrial production by optimization of fermentation,and due to the formation of spore during the later fermentation in ton,in which have a momentous effect on the production of enzyme,its profit has been cut down relatively.with the development of functional genomics at present,the analysis of gene regulation mechanism of sporulation and the construction of spore deletion strain were possible.the research shows that the formation of spore is a process of multiple steps and spo0 A,kinA,kinB and ? factor play very important roles in sporulation.In this article,the essential characters of alkaline protease,gene regulation mechanism of sporulation in initial period and genetic deletion technology were discussed.And the performance of Bacillus clausii,construction of reverse selection marker of upp and deletion of spo0 A with its effect of fermentation performance were achieved during the experiment.Main work contents and research results are as follow:(1)Based on the difference between Bacillus Alcalophilus and other Bacillus,this strain was analyzed by the method of 16 rDNA in the article,and was named Bacillus clausii by contrast that the sequence amplified by PCR is similar to the sequence in genebank completely.Also the character of bacterial colony,cell staining,culture condition,production of Alkaline protease and resistance were discussed.(2)The construction of reverse selection marker of upp was finished by knocking-out gene upp of non-trance deletion technology and the constructed strain,as the initial strain,was used to transform sporulation of gene to achieve the gold that the yield of enzyme enhanced to a certain extent.(3)The key gene of sporulation,spoOA,was deleted with reverse selection marker of upp,and the new strain of fermentation performance were analyzed.In this experiment,the spo0 A was knocked-out based on the precondition of upp deletion and the plasmid,pKSU,was used to improve the probability of recombination.The 5-FU,which is similar to antibiotic,was used to select double exchange strain.The final results show that sporulation of strain was restrained and it also has an effect on production of enzyme and other fermentation performance.