The Modification Of The Expression Element Of Rib Operon In B. Subtilis

Genetic reconstructed Bacillus subtilis has been applied successfully in riboflavin production and is the most advanced method. In order to construct riboflavin-producing strain, the key point is to improving the expression level of the rib operon.Traditionally, the rib operon is multiplied within integration plasmid or maintained within the dissociative plasmid by putting antibiotic pressure for the overexpression of rib operon. The strategy indeed enhance the expression level of riboflavin, however, it lead to a potential environmental harmness and production pollution because of the antibiotic pressure mentioned above.This paper use the natural constituted strong operon and mRNA leading sequence of geng gInR in B. subtilis chromosome to replace the original promoter region of rib operon, which accomplish the modification right on the site of rib operon.A gene sequence with anti chloramphenicol A-cat-R is created by multiple PCR.By homologous recombination the rib operon promoter region of source bacterial ,RecA+ B.subtilis 24 X1 was replace by A-cat-R.In this way , B.subtilis24 CS1 with anti chloramphenicol and rib operon mutation is created.A gene sequence with promoter and SD sequence of gInR is created.By multiple PCR, recombinative A'-prm-R'sequence is created.By homologous recombination A'-prm-R'to B. subtilis 24 CS1, the rib operon promoter region is deregulated .In this way , we get B.subtilis24 CS2 .