Divergent Roles of SmHMGR2 and a Novel SmHMGR5 in Tanshinone Biosynthesis Revealed by CRISPR/Cas9-Mediated Knockout in Salvia miltiorrhiza

Lan, Ziting; Tian, Mei; Liu, Jianing; Shi, Wenlong; Chen, Tong; Ma, Qing; Jin, Baolong; Zhao, Yujun; Zhang, Haiyan; Lai, Chang-Jiang-Sheng; Cui, Guanghong

doi:10.3390/ijms27083485

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Divergent Roles of SmHMGR2 and a Novel SmHMGR5 in Tanshinone Biosynthesis Revealed by CRISPR/Cas9-Mediated Knockout in Salvia miltiorrhiza

by

Ziting Lan

^1,†,

Mei Tian

^2,†,

Jianing Liu

¹,

Wenlong Shi

¹

,

Tong Chen

¹,

Qing Ma

¹,

Baolong Jin

¹,

Yujun Zhao

¹,

Haiyan Zhang

¹,

Chang-Jiang-Sheng Lai

^1,* and

Guanghong Cui

^1,*

¹

State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China

²

Dalian Institute of Marine Traditional Chinese Medicine, China Academy of Chinese Medical Sciences, Dalian University, Dalian 116622, China

^*

Authors to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Int. J. Mol. Sci. 2026, 27(8), 3485; https://doi.org/10.3390/ijms27083485

Submission received: 6 March 2026 / Revised: 3 April 2026 / Accepted: 10 April 2026 / Published: 13 April 2026

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants: 3rd Edition)

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Abstract

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.

Keywords: Salvia miltiorrhiza; HMGR; CRISPR/Cas9; tanshinone biosynthesis; functional divergence

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MDPI and ACS Style

Lan, Z.; Tian, M.; Liu, J.; Shi, W.; Chen, T.; Ma, Q.; Jin, B.; Zhao, Y.; Zhang, H.; Lai, C.-J.-S.; et al. Divergent Roles of SmHMGR2 and a Novel SmHMGR5 in Tanshinone Biosynthesis Revealed by CRISPR/Cas9-Mediated Knockout in Salvia miltiorrhiza. Int. J. Mol. Sci. 2026, 27, 3485. https://doi.org/10.3390/ijms27083485

AMA Style

Lan Z, Tian M, Liu J, Shi W, Chen T, Ma Q, Jin B, Zhao Y, Zhang H, Lai C-J-S, et al. Divergent Roles of SmHMGR2 and a Novel SmHMGR5 in Tanshinone Biosynthesis Revealed by CRISPR/Cas9-Mediated Knockout in Salvia miltiorrhiza. International Journal of Molecular Sciences. 2026; 27(8):3485. https://doi.org/10.3390/ijms27083485

Chicago/Turabian Style

Lan, Ziting, Mei Tian, Jianing Liu, Wenlong Shi, Tong Chen, Qing Ma, Baolong Jin, Yujun Zhao, Haiyan Zhang, Chang-Jiang-Sheng Lai, and et al. 2026. "Divergent Roles of SmHMGR2 and a Novel SmHMGR5 in Tanshinone Biosynthesis Revealed by CRISPR/Cas9-Mediated Knockout in Salvia miltiorrhiza" International Journal of Molecular Sciences 27, no. 8: 3485. https://doi.org/10.3390/ijms27083485

APA Style

Lan, Z., Tian, M., Liu, J., Shi, W., Chen, T., Ma, Q., Jin, B., Zhao, Y., Zhang, H., Lai, C.-J.-S., & Cui, G. (2026). Divergent Roles of SmHMGR2 and a Novel SmHMGR5 in Tanshinone Biosynthesis Revealed by CRISPR/Cas9-Mediated Knockout in Salvia miltiorrhiza. International Journal of Molecular Sciences, 27(8), 3485. https://doi.org/10.3390/ijms27083485

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Divergent Roles of SmHMGR2 and a Novel SmHMGR5 in Tanshinone Biosynthesis Revealed by CRISPR/Cas9-Mediated Knockout in Salvia miltiorrhiza

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