Genetic Analyses Of The Reasons For Riboflavin Over-production In Bacillus Subtilis 368 And Primary Construction Of Engineered Strain

The research mainly focused on Bacillus subtilis368 which can over produce riboflavin. In order to finalize the reason which caused the riboflavin over-producing, three kinds of genes were cloned, which directly relate to riboflavin production. The results showed that the reason lied in a point mutation which resides in the ribC gene. RibC is the kinase involved in conversion of riboflavin to the active cofactor FMN and FAD. The mutation resulted in reduced activity of RibC and then decreased the rate of conversion of riboflavin to the flavin coenzymes, which increased the accumulation of riboflavin. In addition, the mutation also reduces the repressive effects of FMN and FAD on rib operon and ypaA(a riboflavin transporter), which also contribute to the riboflavin over-production. On the other hand the rib operon of the Bacillus subtilis was also modified : eliminating the rfn box, replacing the rib promoter by constructive bacteriophage promoter spol and taking the tetracycline resistance gene as a selectable marker etc. Then we transferred these modified rib operon into various kinds of Escherichia coli and compared the riboflavin production of these strains. The results showed that the expression of the rib genes was drastically increased by replacing the first rib promoter and rfn box by strong constructive promoter spol, and when the ampicillin(100μg/ml) was used as a selective pressure, the riboflavin production of the host strain JM109 could reached 86μg/ml, but when the tetracycline resistance gene was ligated into the vector and the tetracycline(20μg/ml) was used as the selective pressure, an even higher riboflavin production was reached (approximately 108μg/ml about ten times higher than that of unmodified rib operon). Finally, we also developed a protocol for transformation of Bacillus subtilis, which could greatly increase the transformation efficiency. When the Bacillus subtilis ISW1214 was used as the host strain the transformation efficiency could reach 1.5 ×10~4 ,evidently higher than other protocols. In addition, we also constructed an integration vector, which have the optimized rib operon and made it integrate into the genome of the ISW1214. Then the integration point was examined and the expression of the optimized rib operon was analyzed. These researches would be utilized to generate engineered strains, which greatly overproduce riboflavin..