Expression Of Structural Genes Of Rib Operon In The Site Of CcpA In Bacillus Subtilis

Riboflavin is an important fine chemical product, which has broad applications in the fields of medicine, food and feed industries. At present, the main method to produce riboflavin commercially is fermentation with recombinant Bacillus subtilis. After relieving the feedback repression of purine synthesis of Bacillus subtils, the expression level of the rib operon is the key factor which influences the synthetic rate and the final output of riboflavin. Free or integrated expression plasmids are usually used to express the rib operon excessively. But, the inherent instability of free expression plasmids and the environmental safety issues caused by the use of antibiotics to choose integrated plasmids make these techniques defective. In Bacillus subtilis, CcpA is a global regulator, which mediates carbon catabolite repression in the cell.The knockout of ccpA gene can relieve carbon catabolite repression, which is in favor of the synthesis of riboflavin.Judging from the function of the protein, we think that the expression of ccpA is constitutive, and the promoter of ccpA should belong to strong promoter.Expressing the structural genes of rib operon with the expression elements of ccpA, not only express the rib operon excessively, but also relieve carbon catabolite repression,which indicate that it is a simple and pleiotropic method.At first, riboflavin producing strain B. subtilis 24 is reconstructed. By the method of homologous recombination, recA mutation recovered strain is constructed and named as B. subtilis 24X1.The growth rate and biomass of B. subtilis 24X1 are grater than those of B. subtilis 24.The yield of riboflavin improves by 25%.Then, sequence analysis and comparison of ccpA show that it has strong promoter features, the SD sequence and the terminator sequence are similar with the conservative sequence.Then, ccpA promoter, ribDEAH and ccpA terminator are spliced by overlapping PCR. The spliced fragment is transformed to B. subtilis 24CS1 which is defective of riboflavin.to gain B. subtilis 24X2. According to the color of B. subtilis 24X2, we can judge that ribDEAH is expressed in the site of ccpA.