关键词: 脱氧雪腐镰刀菌烯醇；枯草芽孢杆菌；表达元件；启动子；核糖体结合位点

Abstract: A deoxynivalenol-degrading enzyme DepB was successfully expressed in Bacillus subtilis RIK 1285 in this study, but the fermentation level of DepB was low, which hinders its application in food and feed processing. Thus, an integrative strategy of transcriptional and translational regulation was explored to enhance the expression level of DepB. First, nine single strong promoters were selected to replace the original promoter P43, among which the recombinant bacteria mediated by the promoter PspoVG gave the highest enzyme activity of 29.59 U/mL after fermentation. Second, four promoters (P43, PsacB, PspoVG, and PaprE) with relatively high DepB expression levels were chosen to construct a dual-promoter system. DepB mediated by the dual promoter PaprE-PspoVG reached the highest activity of 48.87 U/mL. Moreover, the DepB activity of Mutant-5 with optimized core region (-35 and -10 boxes) of PaprE-PspoVG reached 69.17 U/mL, which was 4.79 times higher than that of the original strain (14.45 U/mL). Finally, DepB expression level was further improved by optimizing the ribosome binding site (RBS) sequence of the promoter PspoVG, and the enzyme activity of RBS15 reached 115.15 U/mL, which was 7.97-fold higher than that of the original strain. The results suggest that combined transcriptional and translational regulation is an effective strategy to improve the fermentation level of recombinant proteins.

Key words: deoxynivalenol; Bacillus subtilis; expression elements; promoter; ribosome binding site