Ziting Lan

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) serves as a key rate-limiting enzyme in the mevalonate pathway and plays a central regulatory role in the biosynthesis of tanshinones. To date, four HMGR family members (SmHMGR1–4) have been identified in Salvia miltiorrhiza. Here, we cloned and identified a novel member, SmHMGR5, by integrating multiple genomic datasets. Genomically, SmHMGR5 formed an inverted repeat with SmHMGR3 (98.04% homology) and phylogenetically clustered with SmHMGR2. Based on the expression patterns of the five HMGR genes, we further generated SmHMGR2 and SmHMGR5 knockout mutants using CRISPR/Cas9 technology and compared their effects on the accumulation of 12 tanshinones and 4 phenolic acids via UPLC-MS-based metabolomic analysis. Knockout of SmHMGR2 significantly suppressed the accumulation of seven tanshinones, whereas SmHMGR5 knockout downregulated only three tanshinones, and neither mutation affected phenolic acids. Notably, the major compound tanshinone IIA remained stable across different mutants, but tanshinone IIB was markedly reduced upon SmHMGR2 knockout, suggesting complex regulatory mechanisms in tanshinone biosynthesis. These findings provide new insights into the biosynthetic network of tanshinones and establish a theoretical foundation for metabolic engineering strategies aimed at enhancing the production of bioactive constituents in S. miltiorrhiza.