The Modification Of Genes Involved In Riboflavin Synthesis Pathway In B. Subtilis

In the riboflavin biosynthesis pathway of B. subtilis, the expression level of rib operon and the activity of riboflavin kinase/FAD synthase, is the key to influence riboflavin excessive synthesis. This paper studies the influence of rib promoter modification, mRNA leader region replacement by stabilizer, and ribC point mutation on riboflavin excessive synthesis.First, DNA fragments and plasmids were used to protoplast transformation, in order to evaluate the recombinant ability of B. subtilis24A1, and found that it can’t be homologous recombination in the state of protoplasts, or recombination rate is less than4X10-3, discomfort for use as genetically modified strain.Using B. subtilis L30as initial strain, riboflavin-deficient strain B. subtilis LX31was constructed by double crossover recombination of chloramphenicol with the promoter region of rib operon, this strain used as the initial strain for further modified rib operon expression element. Strain B. subtilis LX32and LX33were constructed by homologous recombination, whose rib operon-35region was modified to consensus, and mRNA leader region is replaced by gsiB and asnH mRNA stabilizer respectively. qRT-PCR was used to detect recombinant strains rib operon transcribed mRNA levels, found using gsiB and asnH mRNA stabilizer modified rib operon, the mRNA levels increased1531-fold and9-fold respectively relative to departure. The result shows that, rib operon-35region was modified to consensus, and mRNA leader region is replaced by gsiB mRNA stabilizer, the rib operon can be constitutive overexpression.The recombinant plasmids which contain mutation of B.subtilis24A1in ribC ind erythromycin resistance marker, transformed strain LX32, resistance plates were screened for the yellow colonies LX34. Verified by sequencing and found LX34having a mutation Tâ†'C at the first base of ribC promoter. Shake fermentation display, after culture36h, LX34can produce riboflavin2.13g/L, higher than the24A1strain1.94g/L, and the growth ability of strain LX34is better than24A1. The results showes that, the mutation of ribC promoter-35region Tâ†'C, the genetic effect is considerable with G596A coding sequence mutation, allows excess riboflavin synthesis.