Expression Of RibAH In The Site Of Gene CcpA Of B. Subtilis Which Overproduce Riboflavin

Genic reconstruction of Bacillus subtilis has been applied successfully in riboflavin production and is becoming the preponderant method. In order to construct riboflavin-producing strain, the point is how to improve the expression of the rib operon, such as using a strong promtor to express the operon, or adding the copies of the operon. Expression plasmid, integration or not, is used to express the operon in the strain. This paper invented a simple, new method to express ribAH gene of the rib operon, which use ccpA expression elements to express ribAH.At first, sequence analysis and comparison of ccpA showed strong promoter features. In the cell, ccpA gene encodes global regulator protein CcpA, which regulates almost 200 genes. That is to say, the expression element of ccpA is likely to be strong.Riboflavin producing strain B. subtilis 24 is reconstructed. RecA mutation recovered strain is named as B. subtilis 24 Y1. LipA::Cm and lipA:: Cm-recA gene was transform to B. subtilis 24 Y1, selected by chloramphenicol. No transformant of lipA::Cm was obtained, which means no homologous recombination happened; while lipA::Cm-recA homologous recombination probability is about 10-7.RibAH mutation gene fragment was obtained by overlap PCR, and was transformed to B. subtilis 24 Y1. B. subtilis 24 Y3 was gained in which ribAH contain two site mutation by homologous recombination. Riboflavin production decreased from 1.8 g/L to 0.4g/L, and the color of the colony chaged from yellow to white, which means it can be uses as selection marker in the following steps.Gene spliced ccpA promoter, ribAH CDS, and ccpA terminator. Then ligate this fragment with recA. Ligation is transformed to B. subtilis 24 Y3 to gain B. subtilis 24 Y5 and B. subtilis 24 Y6; where homologous recombination happened in the site of ccpA and both the site of ccpA and ribAH. According to the color of B. subtilis 24 Y5, we can judge that ribAH is expressed in the site of ccpA. The results indicate that our method of expression is feasible, in which we use unessential gene of the chromosome to express destination gene by homologous recombination.