关键词: 枯草芽孢杆菌, 海藻糖合成酶, 麦芽糖启动子, 诱导表达

Abstract: This work aims to construct a safe expression system of maltose-induced trehalose synthase while applying the maltose inducible promoter in Bacillus subtilis,and by which the prepared trehalose can be widely used in food and medical industry. The trehalose synthase gene TreS originated from the Pseudomonas putida KT2440 was selected as a reporter gene. The maltose-induced promoter Pglv from B. subtilis was utilized as a regulatory element after its cre sequence was optimized by site directed mutagenesis（AT base instead of CG base）. The shuttle plasmid PHT01 between Escherichia coli and B. subtilis was as the skeleton of the vector. By digestion and replacement of restriction enzyme BamH I and AatII,the highly efficient expression vector Pglv-PHT01-TreS of the trehalose synthase was constructed and transformed into B. subtilis WB800n,and the expression result was verified. The results from the optimization of fermentation conditions using the basic fermentation medium showed that the enzyme activity of trehalose synthase reached 18.9 U/mL when adding maltose with the final mass fraction of 4.5% while the OD₆₀₀ of the fermentation broth reached 1.2,and inducing 18 h at 37℃. In order to increase the expression of trehalose synthase,this experiment also constructed recombinant strain B. subtilis WB800n（ΔamyE）by single crossover method of knock the gene of α-amylase（amyE）,reducing the degradation of maltose into glucose by extracellular α-amylase and improving the maltose-induced expression and reducing the feedback inhibition of glucose. Therefore,the enzymatic activity of trehalose synthase rose to 29.2 U/mL when the expression plasmid was induced to express in this strain. This work is the first report that the highly efficient expression of trehalose synthase in B. subtilis was achieved,and lays the foundation for the safe and efficient expression system of trehalose synthase.

Key words: Bacillus subtilis, trehalose synthase, maltose promoter, induced expression