Metabolic Engineering Of Bacillus Subtilis For Riboflavin Production

In this paper, a riboflavin producing strain(Bacillus subtilis) was used to construct an efficient marker- free genetic manipulation system. Using this system, we detected the influence of pentose phosp hate pathway, purine pathways, respiratory chain and key enzymes to the production of riboflavin. And we used single factor optimization method to optimize the component of culture media.First of all, upp gene was ultilized as the negative selection marker in the riboflavin producing B.subtilis to construct a single crossover manipulation system. The system was simple and easy in transformation, but low in the screening efficiency and high negative rate. In order to improved the positive clones, araR gene was used as a negative selection marker to construct a double crossover marker- free manipulation system. The system no longer needed to use vector transformation, it could simply ultilize the homologous fusion segment constructed by PCR technique. Compared to the single crossover system, the double crossover system had higher positive efficiency. Then, this system was improved on the fusion PCR homologous segment.In order to reduce consumption of 5-ribose phosphate of the branch channel, increase the accumulation of 5-ribose phosphate and GTP as the riboflavin precursors, we used a strong promoter P43 to overexpress gene ndk and gene gmk by intergrating into the rbsk locus, which was named strain FRX1. Through shake flask fermentation, the yield of riboflavin increased by 55.5% compared to the original strain FRX0, the yield was 557.6 mg/L.With the promoter P43 to overexpressed the gene ywlF of the pentose phosphate pathway, and integrated it in the locus of gene ypaA which encoded one of the riboflavin intake transport protein, but riboflavin yield presented an evident decline, probably because the YpaA was a bidirectional transport protein and also responsible for the efflux of riboflavin.Phosphoribosyl pyrophosphate(PRPP) kinase encoded by the prs gene is a key enzyme in the pathway of riboflavin synthesis. Integrating prs into xyl R sites can increase the concentration of PRPP. The shaking flask fermentation results showed that riboflavin production was increased by 34.4%, the yield was 481.3 mg/L.The cyd encoding cytochrome bd oxidase was deleted in order to improve the efficiency of energy production in cell, and strain was achieved. The fermentation results showed that riboflavin production was increased by 22.9%, the yield was 440.1 mg/L.Through knocking out the genes of branch pathway, Ru5 P can be saved from the branch consumption, the production of riboflavin increased by 11.8%, the yield was 400.3 mg/L. Finally, through the single factor optimization for the fermentation culture media, we chosen yeast extract as the best nitrogen source and added(NH4)2HPO4 and urea as the addictive. In addition, serine, sodium formate and folic acid could promote the synthesis of riboflavin at certain degree.