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

Abstract:

[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.