{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,22]],"date-time":"2026-08-22T06:23:57Z","timestamp":1787379837143,"version":"build-2736575974"},"reference-count":34,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T00:00:00Z","timestamp":1787270400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T00:00:00Z","timestamp":1787270400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100004608","name":"Natural Science Foundation of Jiangsu Province","doi-asserted-by":"publisher","award":["BK20240202"],"award-info":[{"award-number":["BK20240202"]}],"id":[{"id":"10.13039\/501100004608","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004608","name":"Natural Science Foundation of Jiangsu Province","doi-asserted-by":"publisher","award":["BK20241619"],"award-info":[{"award-number":["BK20241619"]}],"id":[{"id":"10.13039\/501100004608","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Biotech &amp; Bioengineering"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Oxidosqualene cyclases (OSCs) catalyze the cyclization of 2,3\u2010oxidosqualene into diverse triterpenoids, yet their intrinsically low catalytic efficiency restricts biosynthetic productivity. Here, we establish a mechanism\u2010guided synergistic engineering strategy that extends beyond conventional active\u2010site engineering by integrating distal substrate access regulation with catalytic microenvironment optimization to enhance the catalytic performance of CrAS from\n                    <jats:italic>Catharanthus roseus<\/jats:italic>\n                    . Structural modeling and mechanistic analyses revealed a conserved catalytic framework involving carbocation\u2010mediated polycyclization and identified a surface\u2010exposed constriction region that regulates substrate access. Guided by these insights, distal surface engineering of the constriction region was synergistically combined with active pocket optimization. The resulting combinatorial mutant, M3 (L323A\/T327K\/N565I), exhibited a 95.2% increase in catalytic efficiency and enhanced \u03b1\u2010amyrin and \u03b2\u2010amyrin by 53.2% and 49.7%, reaching 158\u2009mg\/L and 63\u2009mg\/L, respectively. Multi\u2010scale analyses combining molecular dynamics (MD) and quantum mechanics\/molecular mechanics (QM\/MM) calculations revealed that the enhanced catalytic performance is attributable to increased flexibility of the substrate access pathway, reinforced electrostatic and cation\u2010\u03c0 interactions, and reduced reaction energy barriers. Notably, distal mutation T327K improved substrate ingress through dynamic modulation of the protein surface, while N565I optimized the catalytic microenvironment by enhancing hydrophobic packing and stabilizing key intermediates. Overall, our findings establish a generalizable framework for engineering complex cyclases and provide a foundation for the sustainable microbial production of high\u2010value triterpenoids.\n                  <\/jats:p>","DOI":"10.1002\/bit.70363","type":"journal-article","created":{"date-parts":[[2026,8,22]],"date-time":"2026-08-22T05:41:56Z","timestamp":1787377316000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Mechanism\u2010Guided Synergistic Engineering of Substrate Access and Catalytic Microenvironment in Oxidosqualene Cyclase for Enhanced Amyrin Biosynthesis"],"prefix":"10.1002","author":[{"given":"Yangyang","family":"Li","sequence":"first","affiliation":[{"name":"Science Center for Future Foods Jiangnan University Wuxi China"},{"name":"Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education Jiangnan University Wuxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ke","family":"Jin","sequence":"additional","affiliation":[{"name":"Science Center for Future Foods Jiangnan University Wuxi China"},{"name":"Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education Jiangnan University Wuxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-7153-0473","authenticated-orcid":false,"given":"Jiangong","family":"Lu","sequence":"additional","affiliation":[{"name":"Science Center for Future Foods Jiangnan University Wuxi China"},{"name":"Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education Jiangnan University Wuxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haidong","family":"Huang","sequence":"additional","affiliation":[{"name":"Science Center for Future Foods Jiangnan University Wuxi China"},{"name":"Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education Jiangnan University Wuxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xianhao","family":"Xu","sequence":"additional","affiliation":[{"name":"Science Center for Future Foods Jiangnan University Wuxi China"},{"name":"Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education Jiangnan University Wuxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3269-3476","authenticated-orcid":false,"given":"Guocheng","family":"Du","sequence":"additional","affiliation":[{"name":"Science Center for Future Foods Jiangnan University Wuxi China"},{"name":"Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education Jiangnan University Wuxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhendong","family":"Li","sequence":"additional","affiliation":[{"name":"Yixing Institute of Food Biotechnology Co., Ltd. Yixing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian","family":"Chen","sequence":"additional","affiliation":[{"name":"Science Center for Future Foods Jiangnan University Wuxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9679-9130","authenticated-orcid":false,"given":"Long","family":"Liu","sequence":"additional","affiliation":[{"name":"Science Center for Future Foods Jiangnan University Wuxi China"},{"name":"Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education Jiangnan University Wuxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3432-2166","authenticated-orcid":false,"given":"Xueqin","family":"Lv","sequence":"additional","affiliation":[{"name":"Science Center for Future Foods Jiangnan University Wuxi China"},{"name":"Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education Jiangnan University Wuxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,8,21]]},"reference":[{"issue":"7","key":"e_1_2_9_2_1","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1016\/S1875-5364(17)30076-6","article-title":"Mesua Ferrea Stem Bark Extract Induces Apoptosis and Inhibits Metastasis in Human Colorectal Carcinoma HCT 116 Cells, Through Modulation of Multiple Cell Signalling Pathways","volume":"15","author":"Asif M.","year":"2017","journal-title":"Chinese Journal of Natural Medicines"},{"issue":"11","key":"e_1_2_9_3_1","doi-asserted-by":"crossref","first-page":"1338","DOI":"10.1038\/s44160-025-00898-1","article-title":"Programmable Enzyme Catalysis Based on Multiscale Confinements","volume":"4","author":"Cao Y.","year":"2025","journal-title":"Nature Synthesis"},{"issue":"12","key":"e_1_2_9_4_1","doi-asserted-by":"crossref","first-page":"2261","DOI":"10.1039\/D1NP00015B","article-title":"Site\u2010Directed Mutagenesis and Substrate Compatibility to Reveal the Structure\u2010Function Relationships of Plant Oxidosqualene Cyclases","volume":"38","author":"Chen K.","year":"2021","journal-title":"Natural Product Reports"},{"key":"e_1_2_9_5_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.biotechadv.2023.108094","article-title":"Role of Distal Sites in Enzyme Engineering","volume":"63","author":"Gu J.","year":"2023","journal-title":"Biotechnology Advances"},{"issue":"3","key":"e_1_2_9_6_1","doi-asserted-by":"crossref","first-page":"1483","DOI":"10.1093\/plphys\/kiab545","article-title":"Reciprocal Mutations of Two Multifunctional \u03b2\u2010amyrin Synthases From Barbarea vulgaris Shift \u03b1\/\u03b2\u2010amyrin Ratios","volume":"188","author":"G\u00fcnther J.","year":"2022","journal-title":"Plant Physiology"},{"issue":"7","key":"e_1_2_9_7_1","doi-asserted-by":"crossref","DOI":"10.3390\/cimb47070545","article-title":"Genome\u2010Wide Analysis of Oxidosqualene Cyclase Genes in Artemisia annua: Evolution, Expression, and Potential Roles in Triterpenoid Biosynthesis","volume":"47","author":"Guo C.","year":"2025","journal-title":"Current Issues in Molecular Biology"},{"issue":"26","key":"e_1_2_9_8_1","doi-asserted-by":"crossref","first-page":"8075","DOI":"10.1021\/acs.jafc.2c03011","article-title":"Oxidosqualene Cyclases Involved in the Biosynthesis of Diverse Triterpenes in Camellia sasanqua","volume":"70","author":"Huang L.","year":"2022","journal-title":"Journal of Agricultural and Food Chemistry"},{"key":"e_1_2_9_9_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.biortech.2023.128819","article-title":"Combinatorial Metabolic Engineering Enables the Efficient Production of Ursolic Acid and Oleanolic Acid in Saccharomyces cerevisiae","volume":"374","author":"Jin K.","year":"2023","journal-title":"Bioresource Technology"},{"issue":"1","key":"e_1_2_9_10_1","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1186\/s12934-022-01915-0","article-title":"Enhanced Production of Amyrin in Yarrowia lipolytica Using a Combinatorial Protein and Metabolic Engineering Approach","volume":"21","author":"Kong J.","year":"2022","journal-title":"Microbial Cell Factories"},{"key":"e_1_2_9_11_1","first-page":"1","article-title":"Mining and Modification of Oryza sativa\u2010Derived Squalene Epoxidase for Improved \u03b2\u2010amyrin Production in Saccharomyces cerevisiae","volume":"375","author":"Li J. L.","year":"2023","journal-title":"Journal of Biotechnology"},{"key":"e_1_2_9_12_1","doi-asserted-by":"crossref","DOI":"10.3389\/fpls.2023.1138893","article-title":"Site\u2010Directed Mutagenesis Identified the Key Active Site Residues of 2,3\u2010oxidosqualene Cyclase HcOSC6 Responsible for Cucurbitacins Biosynthesis in Hemsleya chinensis","volume":"14","author":"Li X.","year":"2023","journal-title":"Frontiers of Plant Science"},{"key":"e_1_2_9_13_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.biotechadv.2026.108850","article-title":"Computational Dual\u2010Loop Frameworks Bridging Single\u2010Enzyme Design and Cascade Tunnel Network Engineering for Next\u2010Generation Biosynthetic Systems","volume":"89","author":"Li Y. 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