活跃链霉菌nshA基因失活对那西肽发酵的影响

目的通过对活跃链霉菌那西肽生物合成基因簇中的nshA基因进行内部片段缺失,考察其对那西肽生物合成的影响。方法利用重叠延伸PCR(Gene splicing by overlap extension PCR,SOE-PCR)的方法失活nshA基因,并构建具有接合转移功能的大肠杆菌-链霉菌重组质粒pSH03,通过接合转移将重组质粒pSH03导入到活跃链霉菌细胞内,并通过同源交换形成活跃链霉菌基因组上的nshA基因失活。采用摇瓶进行发酵,采用HPLC的方法检测发酵液中诺西肽的含量。结果重组菌株的那西肽发酵产量较对照菌株有明显提高。结论 nshA基因对那西肽的生物合成有负调节作用,该基因的失活,有利于那西肽生物合成酶结构基因的表达,使诺西肽的产量有明显提高。

Gene inactivation of nshA in Streptomyces actuosus and its effect on nosiheptide fermentation

Objective To investigate the effect of gene inactivation of nshA in Streptomyces actuosus on nosiheptide fermentation.Methods nshA gene was inactivated by overlap extension PCR(Gene splicing by overlap extension PCR,SOE-PCR);an E.coli-streptomyces shuttle plasmid pSH03 containing inactive nshA gene was constructed and introduced into Streptomyces actuosus through conjugal transfer.The nshA gene in the chromosome was replaced by the inactive nshA gene in the plamid pSH03 through double crossover.Nosiheptide fermentatiom was conducted by shaking flask method and nosiheptide titer was determined by HPLC.Results The fermentation yield of nosiheptide by recombinant strain was obviously higher than the fermentation yield of control strain.Conclusions nshA gene has a negative effect on nosiheptide biosynthesis.The inactivation of nshA gene are favorable to the expression of nosiheptide biosynthesis genes,therefore the nosiheptid fermentation yield by recombinant strain is improved obviously.