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We developed the combined pocket-tunnel diagnosis (CPTD) framework to identify the rate-limiting step. It integrates static structural analysis with molecular dynamics to diagnose geometric and energetic barriers in the substrate transport and binding. Applying this to glycosyltransferase BsYjiC for the synthesis of bulky sterol glycosides revealed a steric tunnel bottleneck. A compact 44 mutant library achieved 5.5-fold, 9.4-fold, and 4.3-fold activity enhancements for DHEA, pregnenolone, and diosgenin, respectively. Subsequent cavity engineering boosted diosgenin activity 9.29-fold and expanded the substrate scope to cholesterol. Comparative validation confirmed diagnostic accuracy, as a pocket-first strategy failed for both DHEA and pregnenolone and yielded only marginal gains for diosgenin. In contrast, the same pocket mutations produced substantially greater enhancements after the tunnel bottleneck was removed.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c07673","type":"journal-article","created":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T22:01:26Z","timestamp":1788213686000},"page":"27743-27756","source":"Crossref","is-referenced-by-count":1,"title":["Combined Pocket-Tunnel\nDiagnosis Guides Engineering\nof UGTs for Enhanced Sterol Glycosylation"],"prefix":"10.1021","volume":"74","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-8445-0563","authenticated-orcid":true,"given":"Jiawei","family":"Dai","sequence":"first","affiliation":[{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Institute of Fermentation Engineering, Zhejiang University of Technology , ,","place":["Hangzhou, China, 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310014"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuhao","family":"Wang","sequence":"additional","affiliation":[{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Institute of Fermentation Engineering, Zhejiang University of Technology , ,","place":["Hangzhou, China, 310014"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yazhou","family":"Lu","sequence":"additional","affiliation":[{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Institute of Fermentation Engineering, Zhejiang University of Technology , ,","place":["Hangzhou, China, 310014"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingling","family":"Bao","sequence":"additional","affiliation":[{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Institute of Fermentation Engineering, Zhejiang University of Technology , ,","place":["Hangzhou, China, 310014"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7077-160X","authenticated-orcid":true,"given":"Linjiang","family":"Zhu","sequence":"additional","affiliation":[{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Institute of Fermentation Engineering, Zhejiang University of Technology , ,","place":["Hangzhou, China, 310014"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0505-1010","authenticated-orcid":true,"given":"Hanchi","family":"Chen","sequence":"additional","affiliation":[{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Institute of Fermentation Engineering, Zhejiang University of Technology , ,","place":["Hangzhou, China, 310014"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3869-352X","authenticated-orcid":true,"given":"Xiaolong","family":"Chen","sequence":"additional","affiliation":[{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Institute of Fermentation Engineering, Zhejiang University of Technology , ,","place":["Hangzhou, 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