Targeted knockout of SmCPS4 elevates tanshinone production in Salvia miltiorrhiza

Tanshinones are pharmaceutically active diterpenoids produced by the medicinal plant Salvia miltiorrhiza, possessing clinical bioactivities such as vasodilation and antiarrhythmic properties. In this study, we efficiently edited SmCPS4, a diterpene synthetase gene generating (ent-)13-epi-manoyl oxide in a competing branch of tanshinone biosynthesis. Building upon protoplast-optimized gene-editing elements, we developed an Agrobacterium-based transformation protocol achieving 69.4 % editing efficiency at the endogenous SmPDS locus. Targeted disruption of SmCPS4 altered the accumulation patterns of four major tanshinones, as quantified by UPLC-TQMS. Among these, cryptotanshinone levels increased substantially (1.53-fold), while dihydrotanshinone rose by 1.59-fold. The total tanshinone content in the mutant lines reached 4.09 mg/g DW, representing a 1.42-fold increase compared to the control. This was accompanied by coordinated upregulation of biosynthetic genes (SmHMGS, SmGGPPS, SmCYP76AH3, SmCYP71D373/375) and transcriptional activators (SmMYB1/9b, SmbHLH10/37, SmERF115, SmWRKY1/2, SmGRAS1/3, SmSPL7, SmNAC2). Mechanistic investigations revealed that SmCPS4 suppression relieves metabolic flux constraints in the tanshinone pathway while activating compensatory transcriptional networks. Our findings establish a robust framework for redirecting terpenoid metabolism in medicinal plants, providing actionable strategies for engineering high-tanshinone cultivars of S. miltiorrhiza.