Cloning And Optimization Of New Promoters In Bacillus Species

Bacillus is a Gram-positive bacterium with good ability to secrete extracellular proteins.It is a production strain of various important industrial enzymes such as protease,amylase and lipase.With the rapid development of molecular biology and genetic engineering,Bacillus has been continuously improved as a gene expression system and has shown good application prospects.Promoters play an important role in the expression system,and the existing promoters are insufficient to meet the industrial requirements for the large-scale expression of foreign genes.So,a new type of strong promoter is very necessary for protein expression in Bacillus.This study aims to clone new promoters,improve and optimize the Bacillus expression system,provide a basic operating platform for the improvement of strains of industrial enzyme preparations,and also lay a theoretical foundation for the highly efficient expression system of Bacillus with great application potential.The main research contents are as follows:(1)A variety of recombinant protease expression vectors were constructed in Bacillus subtilis by using the alkaline protease gene Alk as the reporter gene and various reported Bacillus promoters as the expression elements.The results showed that the activity of the eight promoters were significantly different,and the expression activity of the constitutive promoters were significantly higher than those of the inducible promoters.Among them,Pshuttle-09 has higher activity than P43,reaching 345.93 U/mL.(2)Established a promoter trapping system in B.subtilis by using partial filling-in reaction to construct a Bacillus licheniformis promoter library.From the screening,16 new promoters were obtained.Compared with a strong promoter P43,expression activity of P4,P5 and P12 are almost the same.The promoters are not very strong,and the transformation efficiency is low.(3)The E.coli-Bacillus shuttle vector pWHA 1520 was constructed to express alkaline protease and improve the conversion efficiency.The promoters obtained from transcriptome analysis were constructed to expression vectors,and 22 recombinant expression vectors were successfully constructed.A bidirectional promoter pLA/pLB with high expression level was obtained,with the expression activity of pLA reaching 1.43 times more than that of Pshuttle09.(4)The expression vectors were used to express the alkaline protease in Bacillus amyloliquefaciens and B.licheniformis,and the efficient expression of alkaline protease was achieved.In B.amyloliquefaciens,the expression level of P4129,P4130,P3197,P1758 and P3035 were significantly higher than that of the other 17 promoters,and the expression activity of P4129 was the highest,reaching 528.45 U/mL.The amount of protein secreted by B.amyloliquefaciens was significantly higher than that of B.subtilis.The enzyme production level of B.licheniformis reached 1.24×104 U/mL in shaking flask fermentation,nearly 40%higher than that of the original strain 2709.