Study On Riboswitch Knockout For Vitamin B₁₂ Biosynthesis In Propionibacterium Shermanii

Riboswitch is a highly structured genetic control element. It is composed of some non-coding region mRNA sequence and the sequence folds into certain conformation to response some metabolites in the body. The change of the conformation is to achieve the purpose of regulating mRNA transcription. Riboswitch metabolites ligands, sense some excess metabolites, thus inhibiting the expression of related genes.This study is proposed to destroy the inhibition of the riboswitch, corresponding to VB12 biosynthesis genes in Propionibacterium shermanii, using gene knockout technology and increase the yield of VB12. The P138 promoter and cbiB gene involving VB12 biosynthesis were cloned to construct the vector for riboswitch knockout. The results were as follows:(1) The cbiB gene in VB12 biosynthesis and the promoter P138 were cloned and fused using the PCR technology. The fused P138-cbiB gene was cloned into pMD-18T to construct vector pT-PCB.(2) The ermE from Saccharopolyspora erythraea was connected to the pT-PCB using the method of digestion and ligation. The constructed vector pT-PCBE was for the knockout of riboswitch located upstream of the cbiB gene.(3) The vector pT-PCBE was transformed into the P. shermanii by electroporation. The P. shermanii mutants losing the cbiB riboswitch regulation were confirmed by PCR.(4) The P. shermanii and the corresponding mutants were cultivated at the same conditions to induce the VB12 biosynthesis. And the yield of VB12 was measured through the high liquid chromatography. We found that the VB12 yeild of mutants were not increased compared to wild type. It proved that the simple knockout of cbiB riboswitch were not promoted the yield of VB12. We deduced that the biosynthesis of VB12 was controlled not only by riboswitch but also other elements.