Endophytic fungus Penicillium steckii DF33 promoted tanshinones biosynthesis in Salvia miltiorrhiza by regulating the expression of CYP450 genes

Abstract

Salvia miltiorrhiza, a prominent traditional Chinese medicinal resource, has been extensively employed in the management of cardiovascular and cerebrovascular ailments. Ensuring the consistency of S. miltiorrhiza raw materials revolves around the imperative task of maintaining stable tanshinones content and composition. An effective approach in this regard involves the utilization of endophytic fungi as inducers. Within this context, our study spotlights an endophytic fungus, Penicillium steckii DF33, isolated from the roots of S. miltiorrhiza. Remarkably, this fungus has demonstrated a significant capacity to boost the biosynthesis and accumulation of tanshinones. The primary objective of this investigation is to elucidate the underlying regulatory mechanism by which DF33 enhances and regulates the biosynthesis and accumulation of tanshinones. This is achieved through its influence on the differential expression of crucial CYP450 genes within the S. miltiorrhiza hairy roots system. The results revealed that the DF33 elicitor not only promotes the growth of hairy roots but also enhances the accumulation of tanshinones. Notably, the content of cryptotanshinone was reached 1.6452 ± 0.0925 mg g−1, a fourfold increase compared to the control group. Our qRT-PCR results further demonstrate that the DF33 elicitor significantly up-regulates the expression of most key enzyme genes (GGPPS, CPS1, KSL1, CYP76AH1, CYP76AH3, CYP76AK1, CYP71D411) involved in the tanshinone biosynthesis pathway. This effect is particularly pronounced in certain critical CYP450 genes and Tanshinone ⅡA synthase (SmTⅡAS), with their expression levels peaking at 7 days or 14 days, respectively. In summary, endophytic P. steckii DF33 primarily enhances tanshinone biosynthesis by elevating the expression levels of pivotal enzyme genes associated with the modification and transformation stages within the tanshinone biosynthesis pathway. These findings underscore the potential of employing plant probiotics, specifically endophytic and root-associated microbes, to facilitate the biosynthesis and transformation of vital constituents in medicinal plants, and this approach holds promise for enhancing the quality of traditional Chinese medicinal materials.