Construction Of Novel Gene Knockout And Expression Systems Of Bacillus Subtilis And Cloning Of Nattokinase Gene

Nattokinase is one kind of alkaline serine protease which was discovered in natto by Japanese scientist Sumi H in early 1987. Natto is fermented by Bacillus subtilis, which is well known as "super healthy food" in the 21st century. The value of the product in the field of food and medicine is very prominent. Nattokinase can alleviate cardiac and cerebral vascular diseases, decrease blood viscosity, blood pressure and blood fat, prevent osteoporosis and diabetes. In addition, it can also be used for the degradation of wood fiber and fur, etc. Therefore, it is of great significance to further research it for the development of health products and health foods. But to our knowledge, although nattokinase is highly valuable and well know, its expression level still needs to be improved, and substrate specificity and activity need to be further optimized. In this study, the protease deficient WB800 strain of B. subtilis was used for expression of nattokinase, which can effectively alleviate the shortages of protein degradation caused by protease of other B. subtilis expression strain. To realize efficient expression of nattokinase by integrating target gene into B. subtilis genome, a novel plasmid vector was constructed which can be used for gene knockout and knockin in B. subtilis. With this vector, homologous recombination can be realized by ligation of PCR products with the linearized plasmid followed by transformation into B. subtilis, which can effectively avoid the time consuming cloning steps whenever traditional knockout or knockin vectors are used. In addition, the currently used Escherichia coli-B. subtilis shuttle plasmid can only be replicated in E. coli, and then expressed in B. subtilis. This system is very inefficient when construction of mutation libraries and high throughput screening are performed. Therefor, if the plasmid can be expressed in both E. coli and B. subtilis, the workload could be significantly reduced, which should be particularly important for directed evolution of the enzymes. Thus, we tried to construct a novel plasmid vector pSHUTTLE-1-NATTO which may be used for this purpose. The plasmid has duplicons of E. coli and B. subtilis, and also has promoters suitable for E. coli and B. subtilis. In addition, ribosome recognition site (RBS) of the plasmid was optimized for protein expression in both E. coli and B. subtilis. Based on the above features, the plasmid should be used for efficient expression of proteins in E. coli and B. subtilis. In addtion, the bioinformatic analysis of nattokinase was performed for further understanding of its catalysis characters. To sum up, this study would lay the foundation of efficient protein expression, directed evolution and further studies of nattokinase gene.In addition, we have developed an improved method for convenient and rapid isolation of genomic DNA from B. subtilis. In order to detect the range of use of this method, we also apply this method to extract DNA from spinach, fish and other microbes. As a result, it was found that the method is also effective in these cases. The extracted DNA has the advantages of high yield, high quality, no pollution and so on, and the extraction speed and simplicity are significantly better than other reported methods. To our knowledge, there is no universal method of DNA isolation from plant, animal and microbial cells. Therefore, this method can not only lay a foundation for this study, but also provide a useful reference for related studies.