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However, whether the traditional Chinese medicinal plant H. serrata harbors such enzymes and their corresponding functions remains unexplored. In this study, we mined the transcriptome of H. serrata and identified a microbial-like terpene synthase, HsMTPSL1, which produces multiple diterpene products. Following isolation and structural elucidation, seven distinct compounds were obtained, representing three skeletal types: spatane, prenylkelsoene-type, and biflorane. Among these, compound 7 is a novel biflorane diterpene. Structural analysis and subsequent mutagenesis revealed critical residues governing the formation of distinct skeletons, uncovering the multifunctional nature of this enzyme. Notably, the S224A mutation significantly enhanced the production of spatane diterpene compound 1 by 11.6-fold, demonstrating the potential for protein engineering to improve the yield of this bioactive marine-specific diterpene. Transcriptomic profiling revealed that HsMTPSL1 is highly expressed in sporangia, and co-expression analysis with cytochrome P450s identified the CYP781 subfamily as candidates potentially involved in the downstream modification of these skeletons. Collectively, we report the first MTPSL from H. serrata and characterize it as a multifunctional diterpene synthase. Through structure-guided mutagenesis, we uncovered the molecular basis of its functional versatility, with the S224A mutation providing a powerful tool for enhancing the yields of all three diterpene skeletons, thereby laying a foundation for future protein engineering and synthetic biology applications.<\/jats:p>","DOI":"10.3390\/molecules31081329","type":"journal-article","created":{"date-parts":[[2026,4,20]],"date-time":"2026-04-20T07:15:08Z","timestamp":1776669308000},"page":"1329","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Functional Characterization and Mutagenesis Studies of a Microbial-like Diterpene Synthase from Huperzia serrata"],"prefix":"10.3390","volume":"31","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-6515-1977","authenticated-orcid":false,"given":"Ting","family":"He","sequence":"first","affiliation":[{"name":"School of Pharmaceutical Sciences, Shandong University of Traditional Chinese Medicine, Jinan 250355, China"},{"name":"Shandong Laboratory of Yantai Drug Discovery, Bohai Rim Advanced Research Institute for Drug Discovery, Yantai 264117, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-2547-3522","authenticated-orcid":false,"given":"Yao","family":"Zhao","sequence":"additional","affiliation":[{"name":"Shandong Laboratory of Yantai Drug Discovery, Bohai Rim Advanced Research Institute for Drug Discovery, Yantai 264117, China"},{"name":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"},{"name":"Nottingham Ningbo China Beacons of Excellence Research and Innovation Institute, University of Nottingham Ningbo, Ningbo 315100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-9446-0850","authenticated-orcid":false,"given":"Xin","family":"Li","sequence":"additional","affiliation":[{"name":"Shandong Laboratory of Yantai Drug Discovery, Bohai Rim Advanced Research Institute for Drug Discovery, Yantai 264117, China"},{"name":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6868-4996","authenticated-orcid":false,"given":"Bao","family":"Chen","sequence":"additional","affiliation":[{"name":"Shandong Laboratory of Yantai Drug Discovery, Bohai Rim Advanced Research Institute for Drug Discovery, Yantai 264117, China"},{"name":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-2088-3516","authenticated-orcid":false,"given":"Fangyan","family":"Chen","sequence":"additional","affiliation":[{"name":"Shandong Laboratory of Yantai Drug Discovery, Bohai Rim Advanced Research Institute for Drug Discovery, Yantai 264117, China"},{"name":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0144-2455","authenticated-orcid":false,"given":"Baofu","family":"Xu","sequence":"additional","affiliation":[{"name":"Shandong Laboratory of Yantai Drug Discovery, Bohai Rim Advanced Research Institute for Drug Discovery, Yantai 264117, China"},{"name":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Callizot, N., Campanari, M.L., Rouviere, L., Jacquemot, G., Henriques, A., Garayev, E., and Poindron, P. 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