透明颤菌血红蛋白基因在弗氏链霉菌中的表达及其对菌体生长和泰乐星合成的影响

Expression of Vitreoscilla hemoglobin gene in Streptomyces fradiae and its effect on cell growth and synthesis of tylosin

质粒 p IJ4 0 2 6和 p RK4 0 4 - vhb分别含有红霉素抗性基因启动子 (Perm E)和透明颤菌血红蛋白基因 (vhb) ,先用 PCR分别扩增二者 ,再用 SOE- PCR(Splicing by overlap extension PCR)将不含自身启动子的vhb基因置于 Perm E之下 ;将 SOE- PCR产物克隆到链霉菌整合性载体 p SET15 2上 ,构建成 vhb基因表达载体 p WQ2 0 0 4。通过大肠埃希氏菌 -链霉菌属间接合转移的方式将 p WQ2 0 0 4导入弗氏链霉菌 (Streptomycesfradiae)中 ,利用 p WQ2 0 0 4上 Φ C31整合性位点使 vhb基因整合到弗氏链霉菌染色体上。PCR验证表明 ,vhb基因已整合到染色体上 ,CO结合差光谱分析则表明 vhb基因在弗氏链霉菌中成功表达。摇瓶与上罐发酵显示

The ermE promoter and vhb gene fragments were generated by PCR from plasmids pIJ4026 and pRK404-vhb containing promoter of erythromycin resistance gene (PermE) and Vitreoscilla hemoglobin (vhb) gene respectively. vhb gene(no native promoter) was placed under control of PermE via SOE-PCR (Splicing by overlap extension PCR). An expression vector, pWQ2004, was constructed by cloning the SOE-PCR product into pSET152, an intergrative vector for Streptomyces. pWQ2004 was introduced into Streptomyces fradiae from E.coli ET12567 (pUZ8002) through conjugal transfer, and it was intergrated into chromosome of Streptomyces fradiae by its ΦC31 attachment site. PCR analysis indicated vhb gene has been integrated into chromosome of Streptomyces fradiae. CO binding difference spectrum revealed that vhb gene has been (expressed) successfully in Streptomyces fradiae, and the fermentation results showed that expression of vhb gene could dramatically promote cell growth and biosynthesis of tylosin.