枯草芽孢杆菌核黄素操纵子与ribC基因的修饰与遗传效应

The genetic modification effect of rib operon and ribC gene in Bacillus subtilis

【目的】研究核黄素操纵子(rib)组成型高表达,以及rib C基因低水平表达对枯草芽孢杆菌过量合成核黄素的影响。【方法】在染色体原位修饰启动子,用m RNA稳定子替换m RNA前导区,使rib操纵子组成型高表达;修饰rib C基因的启动子,降低rib C基因的表达水平。采用q RT-PCR方法,表征基因的相对表达水平;通过摇瓶发酵,测定重组菌的生物量和核黄素产量,表征相关基因修饰所表现的遗传效应。【结果】用gsi B m RNA稳定子替换核黄素操纵子的m RNA前导区,使其相对表达水平提高了约1 500倍。rib C基因启动子-35区的首个碱基由"T"突变为"C",使rib C基因的表达水平下降了97%以上。得到的重组菌株LX34在补加蔗糖20 g/L的LB培养基上摇瓶发酵36 h,可积累核黄素2.1 g/L,同时生物量没有明显下降。【结论】使用gsi B m RNA稳定子,能够有效地提高目标基因或操纵子的表达水平;启动子-35区首个碱基的点突变,能够有效降低rib C基因的表达水平;rib操纵子过量表达和rib C基因低水平表达,使细胞能够过量合成并积累核黄素。

[Objective] This research focus on the influence of rib operon constitutively over-expression and ribC gene low levels expression on the synthesis and accumulation of riboflavin in Bacillus subtilis. [Methods] For the constitutively over-expression of rib operon, its promoter was modified in situ, and the mRNA leader region was replaced by mRNA stabilizer. Using point mutations of its promoter -35 region, ribC gene transcription level was reduced. The transcription levels of the target genes were analysed by qRT-PCR method. The genetic effects of the modified genes were assessed by measuring the biomass and riboflavin production of the recombinant in shaking flask fermentation. [Results] The relative transcription levels of gsiB stabilizer-modified rib operon have increased about 1 500 times. The first base mutations of ribC promoter -35 region can lead to its expression levels decreased by more than 97%. With the LB medium supplemented with sucrose 20 g/L, fermentation was completed in 36 h and the resulting recombinant strains LX34 can accumulate riboflavin 2.1 g/L, meanwhile no significant decline in the biomass. [Conclusion] The gsiB mRNA stabilizer can effectively improve transcription levels of the target gene or operon. A point mutation in the first base of promoter -35 region can effectively reduce the transcription of ribC gene. The over-expression of rib operon and the expression reduced significantly of ribC gene result in the accumulation of riboflavin.