摘要
通过PCR扩增得到变铅青链霉菌 (Streptomyceslividans)TK2 4secE基因上游 4 96bp的片段 ,其序列与S .coelicolorsecE启动子序列同源性为 99 8%。将该序列克隆到以儿茶酚加氧酶基因 (xylE)为报告基因的链霉菌启动子探测质粒pIJ4 0 83上 ,并转化S .lividansTK2 4原生质体 ,获得了重组菌株S .lividans[pIJ4 0 83 secE]。S .lividans[pIJ4 0 83 secE]菌株发酵结果表明 ,secE启动子为强启动子 ,活性与vsi基因启动子相当。secE启动子的表达在对数生长期达到高峰 ,平台期下降 ;2 8℃发酵培养时secE启动子活性远高于 37℃发酵培养 ;比较了不同发酵培养基Phage ,NB和CM中secE启动子的活性 ;实验结果还表明培养基中葡萄糖含量对secE启动子的表达有抑制作用。
Streptomyces are Gram positive,filamentous soil bacteria,which produce a wide variety of metabolites that are currently being exploited in both medicine and agriculture.Moreover, Streptomyces lividans is used as a host strain to effectively express and secrete heterologous proteins to culture media.Genes encoding Sec proteins responsible for the translocation of preproteins have been identified in S.lividans. SecYEG,a complex of integral membrane proteins SecY,SecE,and SecG in the cytoplasmic membrane,constitutes a pathway for polypeptide movement.SecA plays a central role in translocation as it is the site of perprotein entry into the translocase and it is the only ATPase essential for preprotein translocation.The role of SecD and SecF is to regulate the movement of the translocating protein.A better understanding of their regulatory mechanism could help to develop S.lividans strains with hyper secretory capacity.In this study,a reporter system was used to investigate the regulatory mechanism of secE promoter.Upstream sequence (496 bp) of secE of S.lividans TK24 was cloned and characterized in a promoter probe vector pIJ4083 upstream of promoterless xylE .Sequencing analysis revealed that secE upstream sequence of S.lividans shares 99 8% homology with the secE promoter of S.coelicolor .The transcriptive activity of this fragment approximates vsi promoter in CMI medium,a strong promoter from S.venezuelae .The activity of secE promoter was observed in different growth phase,culture temperature and culture media.The expression level of secE was higher during the log phase,but decreased at the beginning of the stationary phase.At growth temperature of 28℃,the expression level was much higher than that at 37℃.When the bacteria were incubated in NB,Phage,and CM medium respectively,the expression level of secE promoter was different at a certain fermentation time,but all reached the highest level at log phase.Further analysis revealed that glucose may repress secE promoter expression.
基金
国家自然科学基金 (3 0 0 70 0 0 9)~~
