摘要
Staurosporine, belonging to indolocarbazole compounds, is regarded as an excellent lead compound for synthesizing antitumor agents as a potent inhibitor against various protein kinases. In this study, two separate clusters(cluster A and cluster B),corresponding to biosyntheses of K-252 c(staurosporine aglycone) and sugar moiety, were identified in Streptomyces fradiae CGMCC 4.576 and heterologously expressed in Streptomyces coelicolor M1146 separately or together. Sta R, a cluster-situated LAL family regulator, activates staurosporine biosynthesis by binding to the promoter regions of sta O-sta C and sta G-sta N. The conserved sequences GGGGG and GCGCG were found through gradually truncating promoters of sta O and sta G, and further determined by mutational experiments. Overexpression of sta R with the supplementation of 0.01 g L^–1 Fe SO4 increased staurosporine production to 5.2-fold compared with that of the parental strain Streptomyces fradiae CGMCC 4.576 in GYM medium. Our results provided an approach for improvement of staurosporine production mediated by a positive regulator and established the basis for dissecting the regulatory mechanisms of other indolocarbazole compounds with clinical application value.
Staurosporine, belonging to indolocarbazole compounds, is regarded as an excellent lead compound for synthesizing antitumor agents as a potent inhibitor against various protein kinases. In this study, two separate clusters(cluster A and cluster B),corresponding to biosyntheses of K-252 c(staurosporine aglycone) and sugar moiety, were identified in Streptomyces fradiae CGMCC 4.576 and heterologously expressed in Streptomyces coelicolor M1146 separately or together. Sta R, a cluster-situated LAL family regulator, activates staurosporine biosynthesis by binding to the promoter regions of sta O-sta C and sta G-sta N. The conserved sequences GGGGG and GCGCG were found through gradually truncating promoters of sta O and sta G, and further determined by mutational experiments. Overexpression of sta R with the supplementation of 0.01 g L–1 Fe SO4 increased staurosporine production to 5.2-fold compared with that of the parental strain Streptomyces fradiae CGMCC 4.576 in GYM medium. Our results provided an approach for improvement of staurosporine production mediated by a positive regulator and established the basis for dissecting the regulatory mechanisms of other indolocarbazole compounds with clinical application value.
基金
supported by the National Natural Science Foundation of China (31800029 and 31771378)
Beijing Natural Science Foundation (5184034)
the National Key Research and Development Program of China (2018YFA0901904)
