作为一种潜在的蛋白激酶C抑制剂,近几十年来,真菌代谢产物balanol变得越来越有吸引力。在我们之前的工作中,我们通过在中草药真菌Tolypocladiumophiogrosoides中过表达簇定位调节基因blnR揭示了其生物合成途径。然而,关于blnR调控的信息在很大程度上仍然未知。在这项研究中,我们通过使用EMSA和RNA-seq分析的亲和结合分析进一步研究了BlnR对balanol生物合成的调节。结果表明,BlnR通过与bln基因成员的所有启动子结合,正向调节balanol的生物合成。包括自己的发起人。显微镜观察显示blnR过表达也影响孢子发育和菌丝生长。此外,RNA-seq分析表明,BlnR可以调节balanol生物合成基因簇之外的其他基因,包括参与分生孢子发育的那些。最后,通过基于响应面方法的统计优化,使用优化的培养基将balanol产量进一步提高至2187.39mg/L。
As a potential protein kinase C inhibitor, the fungus metabolite balanol has become more attractive in recent decades. In our previous work, we revealed its biosynthetic pathway through overexpression of the cluster-situated regulator gene blnR in Chinese herb fungus Tolypocladium ophioglossoides. However, information on the regulation of blnR is still largely unknown. In this study, we further investigated the regulation of balanol biosynthesis by BlnR through the analysis of affinity binding using EMSA and RNA-seq analysis. The results showed that BlnR positively regulates balanol biosynthesis through binding to all promoters of bln gene members, including its own promoter. Microscopic observation revealed blnR overexpression also affected spore development and hypha growth. Furthermore, RNA-seq analysis suggested that BlnR can regulate other genes outside of the balanol biosynthetic gene cluster, including those involved in conidiospore development. Finally, balanol production was further improved to 2187.39 mg/L using the optimized medium through statistical optimization based on response surface methodology.