Optimization Of Transformation Methods Of Bacillus Subtilis WB800 And Effects Of Different Promoters On The Expression Of Target Protein

Bacillus subtilis is a gram-positive bacterium.Because of its simple culture,non-pathogenicity and good secretory ability,Bacillus subtilis is an important object of industrial production and scientific research.As a highly expressed host strain,Bacillus subtilis has a wide application prospect and great potential for development.However,there are still some problems in the research and application of Bacillus subtilis expression system,such as the lack of universality of transformation methods,low transformation efficiency,serious degradation of secretory proteins and so on,which seriously restrict the development of its industrial application.The purpose of this experiment is to further study Bacillus subtilis by optimizing the transformation conditions of Bacillus subtilis WB800 and the effects of different promoters P43,veg and arg C on the secretory expression of Man A,est A and amy E,so as to provide some feasible basis for its application in industrial production In this study,Bacillus subtilis WB800 was used as the expression host strain,which was a protease gene deficient strain.Eight protease genes with strong secretion were knocked out,which overcame the main defect of the exuberant protease secretion of original Bacillus subtilis,which led to the degradation of foreign proteins.Then,the optimum resistance concentration and transformation method were optimized,and it was concluded that the most suitable resistance concentration of plasmid p HT315used in this experiment was 8?g/m L.There are generally three transformation methods of subtilis,namely,electro-transformation,chemical transformation and xylose induction transformation,to prepare the competent state under different transformation methods for transformation,to explore the transformation conditions,and to choose the most suitable and concise transformation method.The results of transformation optimization experiment showed that the optimum voltage and plasmid addition of subtilis WB800 were 2.0 kv and 300 ng,respectively,and the optimum plasmid addition of chemical transformation method was 250 ng,and the best incubation condition was incubation at 37?for 30 min and 200 rpm for 1 h.Xylose induction transformation method was not suitable for the strain,and many experiments were unsuccessful.Among the three transformation methods,the chemical transformation method had the highest transformation efficiency,and the transformation rate reached 1.5×10~4CFU/?g DNA when the plasmid was incubated at37?for 30 min and cultured for 1 h with 200 rpm.After exploring the optimal transformation conditions,the promoter of the vector was replaced.The promoter is the element that controls the initiation of transcription,and different promoters have different effects on the expression of the target protein.Therefore,the screening of high-intensity promoters is very important for the expression system of Bacillus subtilis.In this study,three constitutive promoters P43,veg and arg C,were cloned from the genome of Bacillus subtilis and ligated to the vector by means of genetic engineering,and three different new vectors were constructed.Three different target gene proteins Man A,est A and amy E were used to verify the strength and universality of the three promoters.The enzyme activity of the recombinant strain was determined by fermentation.The intensity of P43 in the three promoters was higher than that of the other two promoters,which significantly promoted the expression of the target protein,and the results were consistent in the three target proteins.When the cells were broken,it was found that part of the intracellular enzyme activity was retained,and the intracellular retained enzyme activity of Man A was as high as 70%.This result also verified the prediction of the signal peptide of the target gene.The predicted results showed that est A and amy E had signal peptide,and Man A had no signal peptide.It is known that the signal peptide also has an effect on the exocrine secretion of the target protein,which provides a feasible idea for follow-up research.