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 . • Knockout of SmCPS4 in Salvia miltiorrhiza plants via optimized Agrobacterium transformation achieved 69.4% efficiency. • Knocking out SmCPS4 boosted tanshinones, increasing total by 1.42-fold, with cryptotanshinone and dihydrotanshinone up 1.53- and 1.59-fold. • SmCPS4 suppression remodels metabolic flux and activates compensatory transcription. • Provides a robust metabolic engineering strategy to develop high-yielding cultivars of medicinal plants.