核黄素基因工程菌的构建及其发酵的初步研究

Construction of Genetic Engineered Bacillus subtilis for Riboflavin Production in Fed-batch Fermentation

构建整合型核黄素质粒pRB63,该质粒含有解调的B.subtilis核黄素操纵子,将其转化入B.subtilis RH13并在染色体上进行适当的扩增后得到RH13::[pRB63]n系列工程菌,其核黄素合成能力随着pRB63扩增程度的增加而增强,最终达到RH13的6～7倍。随后以RH13::[pRB63]n系列工程菌和B.subtilis YB1为亲株进行原生质体融合,筛得重组菌B.subtilis RH33。该菌在含10%葡萄糖或蔗糖的分批发酵中培养64h可产核黄素量4.2g·L-1。采用以葡萄糖为碳源的流加发酵工艺,24h可积累核黄素7～8g·L-1,48h达11～12g·L-1,核黄素对葡萄糖的得率为0.056g·g-1。

An integration plasmid for Bacillus subtilis, pRB63, which contains a deregulated riboflavin operon of 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 YBI. 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·L^-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 of7-8 g·L^-1 riboflavin in 24 h, 11-12 g·L^-1 riboflavin in 48 h and a yield of 0.056 g riboflavin.（g glucose）^-1. Key words：