Integration Plasmid Construction Of Strong Promotor Riboflavin Operon And Expression In Bacillus Subtilis

The main work presented in this study is as follows:Riboflavin-producing strain B.subtilis 24, a recA4 mutant, was transformed with a DNA fragment containing whole recA gene by an approach of protoplast transformation with DNA fragments, then the ability of homologous recombination in B.subtilis 24 was recovered. This process has no negative effect on riboflavin yield of the strain, which is about 1.3g/L. This strain is an idea host for engineering strain. Three integration plasmids of B.subtilis were constructed by genetic engineering.The plasmids have riboflavin operon under the control of the original promoter and two strong promoters SPO2 and P43, respectively. All the rib plasmids can be expressed in E.coli. The yield of riboflavin in E.coli containing riboflavin operon under the control of P43 is higher than the other two promoters, which has similar effect on riboflavin synthesis. Then, the expression level of riboflavin operon in B.subtilis was studied by inserting the integration plasmid in the chromosome of B.subtilis D3 at the amyE locus and a random locus. The riboflavin yield of the strain which had riboflavin operon under the control of the original promoter inserted in by a double-cross event at the amyE locus is higher than the other two promoters, and under the control of P43 is higher than that of SPO2. Comparatively, we have filtrated out a high riboflavin yield strain of P43 which was inserted in by a single-cross event, and the yield is about 3.1g/L. The result indicated the expression level by a random insert is higher.In order to open out the reason why the riboflavin operon of promoteless can accumulate riboflavin when it was cloned in E.coli Top10, we have done this work. A structural gene of the riboflavin operon, ribA, was isolated by PCR from B.subtilis. The gene was cloned in E.coli and made the strain accumulate some riboflavin in the medium. The result made the reason of promoteless rib accumulating riboflavin clear and also indicated that riboflavin yield can be increased by only enhancing ribA gene.