阿维链霉菌孢子色素生物合成对阿维菌素产量的影响

Effect on Production of Avermectins of Spore Pigment Biosynthesis in Streptomyces avermitilis NRRL8165

以野生型阿维链霉菌NRRL8165为出发菌株,用PCR方法克隆孢子色素基因簇直系同源基因(whiEa)侧翼片段,并构建基因置换载体pHL643。将pHL643跨属接合转移进入阿维链霉菌NRRL8165,通过置换载体和染色体之间的同源双交换,对染色体上的whiEa基因簇进行置换,得到3株阿泊拉霉素抗性、硫链丝菌素敏感的重组菌株,均表现为孢子色素合成缺陷。通过Southern杂交分析,证明whiEa基因簇被置换。通过摇瓶发酵和HPLC检测,发现whiEa基因簇置换菌株所产阿维菌素产量明显提高,表明孢子色素与阿维菌素生物合成之间可能有竞争底物的现象。

The flanking fragments of the whiEa gene cluster was PCR amplified, cloned and used to construct the gene replacement plasmid pilL643, pilL643 was conjugated into Streptomyces avermitilis NRRL8165 followed by screening for double crossover event, fielding three apramycin resistance and thiostrepton sensitive isolates named ZJ1, ZJ2 and ZJ3, which were deficient in biosynthesis of the grey spore pigment. The whiEa gene replacement of these isolates was confirmed by Southern hybridization. Fermentation of the mutant strains in shaking flasks and HPLC analyses showed that the production of averrnectins increased by 47% compared with that of the wild type, indicating that the spore pigment biosynthesis competes with the avermectins biosynthetic pathway.