Construction of genetic engineered bacillus subtilis for riboflavin production in fed-batch fermentation

An integration plasmid for Bacillus subtilis, pRB63, which contains a deregulated riboflavin operon oi Bacillus subtilis was constructed and then transformed into B.subtilis RH13 which is a riboflavin producing strain. Engineered strains RH13:: [pRB63]n were obtained by gene amplification of the integration plasmid on the host chromosome. The gene amplification results in an enhancement of riboflavin productivity which is 6-fold higher than the corresponding values of RH13 in batch fermentation. Subsequently, B.subtilis RH33 was screened by protoplast fusion of B.subtilis RH13:: [pRB63]n and another riboflavin producing strain B.subtilis YB1. Batch fermentations were carried out with RH33 for 64 hours in a medium containing 10% glucose or sucrose and the maximum riboflavin titer is 4.2 g&middotL-1. A fed-batch culture for the strain was also conducted with a flexible adjustment of feed rate to control the glucose concentration in a low level and the fed-batch process results in an accumulation of 7-8 g&middotL-1 riboflavin in 24 h, 11-12 g&middotL-1 riboflavin in 48 h and a yield of 0.056 g riboflavin&middot(g glucose)-1.