基于表达元件优化提高脱氧雪腐镰刀菌烯醇解毒酶DepB在枯草芽孢杆菌中的表达水平

Enhanced Expression of Deoxynivalenol-Degrading Enzyme DepB in Bacillus subtilis by Optimizing Expression Elements

本研究实现了脱氧雪腐镰刀菌烯醇解毒酶DepB在枯草芽孢杆菌(Bacillus subtilis)RIK 1285中的异源表达,但DepB较低的发酵水平限制了其在食品加工和饲料中的应用,可采用转录和翻译相结合的策略提高DepB在枯草芽孢杆菌中的表达水平。首先,选择9个单启动子替换原始启动子P43,其中单启动子P_(spoVG)介导的重组菌经发酵后酶活力最高,酶活力为29.59 U/mL。其次,选择4个具有较高DepB表达水平的启动子(P43、PsacB、P_(spoVG)和P_(aprE))构建16个双启动子系统,其中,DepB在双启动子P_(aprE^(-))P_(spoVG)的介导下活力达到最高,为48.87 U/mL。此外,通过对启动子P_(aprE^(-))P_(spoVG)的核心区域(-35和-10区)进行优化,Mutant-5酶活力高达69.17 U/mL,是原始菌株(14.45 U/mL)的4.79倍。在此基础上,通过优化启动子P_(spoVG)的核糖体结合位点序列,进一步提高了DepB的表达水平,RBS15酶活力为115.15 U/mL,是原始菌株的7.97倍。研究结果表明,转录和翻译相结合策略是提高重组蛋白发酵水平的有效手段。

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 P_(spoVG) gave the highest enzyme activity of 29.59 U/mL after fermentation.Second,four promoters(P43,PsacB,P_(spoVG),and P_(aprE))with relatively high DepB expression levels were chosen to construct a dual-promoter system.DepB mediated by the dual promoter P_(aprE^(-))P_(spoVG) 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 P_(aprE^(-))P_(spoVG) 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 P_(spoVG),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.