透明颤菌血红蛋白基因的克隆及其在林可链霉菌中的表达

目的通过将透明颤菌血红蛋白基因vgb克隆到林可链霉菌染色体黑色素生物合成基因m elC位点,使m elC基因被破坏失活,vgb基因实现表达同时提高林可霉素产量。方法构建大肠杆菌-链霉菌重组质粒pYHT04,通过接合转移实验将重叠延伸PCR得到的具有红霉素抗性基因启动子的透明颤菌血红蛋白基因,以双交换的方式整合进林可链霉菌黑色素生物合成基因m elC中。采用不同装瓶量摇瓶发酵,HPLC检测发酵液中林可霉素含量。结果重组菌株颜色变浅,CO结合差光谱显示在420 nm处有明显吸收峰,随着装瓶量的增加重组菌株发酵液中林可霉素效价与对照菌株相比降低幅度较小。结论重组菌株m elC基因失活不能继续合成黑色素,vgb基因得到表达并表现出生物学功能,限氧条件下重组菌株发酵液中林可霉素效价高于对照菌株,有希望应用于工业发酵生产。

Cloning of Vitreoscilla hemoglobin gene and its expression in Streptomyces lincolnensis

Objective To increase lincomycin production associated with melanin biosynthesis gene (melC) disruption and Vitreoscilla hemoglobin gene (vgb) expression through introducing vgb gene into melC gene in chromosome of Streptomyces lincolnensis. Methods Vgb gene was obtained by PCR under control of promotor of erythromycin resistance gene (PermE) via splicing by overlap extension PCR (SOE-PCR). An E. coli-Streptomyces shuttle plasmid pYHT04 containing PermE-vgb gene and melC gene fragment was constructed and was introduced into Streptomyces lincolnensis through conjugal transfer. PermE-vgb gene was integrated into the melC gene in the chromosome of Streptomyces lincolnensis. HPLC was used to determine the titer of lincomycin in fermentation broth after shaking flask fermentation in different media amount. Results The pigment produced by the recombinant strain changed into a light color. There is a remarkable absorption peak at the wavelength of 420 nm in CO binding difference spectrum. The titer of lincomycin produced by recombinant strain degraded slower than the control strain along with the increase of media amount in shaking flask fermentation. Conclusion There is no melanin synthesis due to melC gene inactivation and that vgb gene is expressed successfully in recombinant Streptomyces lincolnensis. The lincomycin yield of recombinant strain is higher than the control strain at low concentration of dissolved oxygen. Therefore, it shows a good prospect of applying to industrial fermentation.