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However, its biosynthesis is limited by a single rate-limiting glycosylation step converting mogroside V, catalyzed by the inherently low-activity plant glycosyltransferase UGT73\u2013327\u20132. In this study, we applied a structure-guided engineering strategy to overcome this catalytic bottleneck. By combining substrate-channel expansion with catalytic pocket remodeling, the double mutant W192F\/K206E was generated, showing a 22.2-fold increase in catalytic activity. Molecular dynamics simulations and kinetic analyses indicated that the enhanced performance results from an enlarged substrate-access channel, improved substrate-binding stability, and a more favorable active-site geometry that reduces key catalytic distances. Furthermore, coupling the engineered UGT with Arabidopsis thaliana sucrose synthase enabled an in situ UDP-glucose regeneration system, achieving a Mog VI titer of 5.6 g\u00b7L\u20131 with a 76.8% molar conversion. This work establishes an efficient biocatalytic route for Mog VI production and highlights the potential of structure-based glycosyltransferase engineering for the synthesis of rare natural glycosides.<\/jats:p>","DOI":"10.1021\/acs.jafc.5c17370","type":"journal-article","created":{"date-parts":[[2026,8,8]],"date-time":"2026-08-08T15:02:22Z","timestamp":1786201342000},"page":"25369-25379","source":"Crossref","is-referenced-by-count":1,"title":["Structure-Guided\nEngineering of Glycosyltransferase\nUGT73\u2013327\u20132 Coupled with UDP-Glucose Regeneration Enables\nHighly Efficient Biosynthesis of Mogroside VI"],"prefix":"10.1021","volume":"74","author":[{"given":"Dong","family":"Guo","sequence":"first","affiliation":[{"name":"Jiangnan University , , ,","place":["Wuxi, P. R. China, 214122"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4941-8622","authenticated-orcid":true,"given":"Yan","family":"Zhang","sequence":"additional","affiliation":[{"name":"Jiangnan University , , ,","place":["Wuxi, P. R. China, 214122"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xupeng","family":"Guo","sequence":"additional","affiliation":[{"name":"Jiangnan University , , ,","place":["Wuxi, P. R. China, 214122"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2479-6050","authenticated-orcid":true,"given":"Zhiwei","family":"Deng","sequence":"additional","affiliation":[{"name":"Jiangnan University , , ,","place":["Wuxi, P. R. China, 214122"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yilin","family":"Chen","sequence":"additional","affiliation":[{"name":"Jiangnan University , , ,","place":["Wuxi, P. R. 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China, 214122"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3087-5663","authenticated-orcid":true,"given":"Zhengshan","family":"Luo","sequence":"additional","affiliation":[{"name":"Jiangnan University , , ,","place":["Wuxi, P. R. China, 214122"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,8]]},"reference":[{"key":"2026081910143360500_cit1","doi-asserted-by":"publisher","DOI":"10.3389\/fphar.2019.01400","article-title":"The fruits\nof Siraitia grosvenorii: a review of\na chinese food-medicine","volume":"10","author":"Gong","year":"2019","journal-title":"Front. Pharmacol."},{"issue":"13","key":"2026081910143360500_cit2","doi-asserted-by":"publisher","first-page":"6850","DOI":"10.1021\/acs.jafc.4c00018","article-title":"Siraitia grosvenorii As a homologue of food and medicine: A review of biological activity,\nmechanisms of action, synthetic biology, and applications in future\nfood","volume":"72","author":"Guo","year":"2024","journal-title":"J. Agric. Food. Chem."},{"issue":"2","key":"2026081910143360500_cit3","doi-asserted-by":"publisher","first-page":"89","DOI":"10.1016\/S1875-5364(14)60015-7","article-title":"Chemistry\nand pharmacology of Siraitia grosvenorii: A review","volume":"12","author":"Li","year":"2014","journal-title":"Chin.\nJ. Nat. 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