{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T17:14:58Z","timestamp":1788455698632,"version":"build-2803163510"},"reference-count":41,"publisher":"American Chemical Society (ACS)","issue":"34","license":[{"start":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T00:00:00Z","timestamp":1787097600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T00:00:00Z","timestamp":1787097600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T00:00:00Z","timestamp":1787097600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/na","name":"National Basic Research Program of China","doi-asserted-by":"publisher","award":["2025YFA0923700"],"award-info":[{"award-number":["2025YFA0923700"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Natural Science Foundation of Tianjin City","doi-asserted-by":"publisher","award":["25JCZDJC00550"],"award-info":[{"award-number":["25JCZDJC00550"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32572514"],"award-info":[{"award-number":["32572514"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,2]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Rhamnosyltransferases are remarkable biocatalysts for the synthesis of rhamnosylated natural products with valuable physicochemical properties and bioactivities. However, their application is hindered by poor stability and low catalytic efficiency. Here, we achieved simultaneous enhancement of catalytic efficiency and stability of a 1,2-rhamnosyltransferase by a distal mutational engineering strategy. The variant M9 exhibited a 589.43-fold extension in half-life, a 2.5 \u00b0C increase in Tm, a 13.6 \u00b0C increase in T50, and an 8- to 763-fold increase in activity toward diverse flavonoids compared with the wild type. Molecular dynamics simulations provided insights into enhanced thermostability and catalytic efficiency. To demonstrate its synthetic utility, a whole-cell biocatalytic system was constructed in E. coli by coexpressing M9 and UDP-rhamnose synthase, enabling a neohesperidin titer of 1.14 g L\u20131 without exogenous sugar donor supplementation. This study presents a practical enzyme engineering strategy for simultaneous activity\u2013stability enhancement in glycosyltransferases and provides a promising biocatalyst for rhamnosylated natural product biosynthesis.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c07461","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T00:01:16Z","timestamp":1787184076000},"page":"27168-27178","source":"Crossref","is-referenced-by-count":0,"title":["Reshaping the Activity\u2013Stability\nLandscape\nof a 1,2-Rhamnosyltransferase via Distal Mutational Engineering"],"prefix":"10.1021","volume":"74","author":[{"given":"Wenjuan","family":"Dai","sequence":"first","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, China, 300457"]},{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, China, 300308"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chaorong","family":"Guo","sequence":"additional","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, China, 300457"]},{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, China, 300308"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongyan","family":"Yang","sequence":"additional","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, China, 300457"]},{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, China, 300308"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taiyan","family":"Chen","sequence":"additional","affiliation":[{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, China, 300308"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Qu","sequence":"additional","affiliation":[{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, China, 300308"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenwen","family":"Pei","sequence":"additional","affiliation":[{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, China, 300308"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Zeng","sequence":"additional","affiliation":[{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, China, 300308"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6592-3733","authenticated-orcid":true,"given":"Yueming","family":"Zhu","sequence":"additional","affiliation":[{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, China, 300308"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2588-3634","authenticated-orcid":true,"given":"Hui-Min","family":"Qin","sequence":"additional","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, China, 300457"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4392-4003","authenticated-orcid":true,"given":"Jiao","family":"Li","sequence":"additional","affiliation":[{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, China, 300308"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peng","family":"Chen","sequence":"additional","affiliation":[{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, China, 300308"]},{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, China, 300308"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8160-4564","authenticated-orcid":true,"given":"Yuanxia","family":"Sun","sequence":"additional","affiliation":[{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, China, 300308"]},{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, China, 300308"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,19]]},"reference":[{"key":"2026090311503116600_cit1","doi-asserted-by":"publisher","first-page":"5315","DOI":"10.3390\/molecules27165315","article-title":"Rhamnose-containing compounds: Biosynthesis and applications","volume":"27","author":"Li","year":"2022","journal-title":"Molecules"},{"key":"2026090311503116600_cit2","doi-asserted-by":"publisher","first-page":"110","DOI":"10.1021\/acs.bioconjchem.5b00528","article-title":"Synthesis of a liposomal\nMUC1 glycopeptide-based immunotherapeutic and evaluation of the effect\nof L-rhamnose targeting on cellular immune responses","volume":"27","author":"Karmakar","year":"2016","journal-title":"Bioconjugate Chem"},{"key":"2026090311503116600_cit3","doi-asserted-by":"publisher","first-page":"5172","DOI":"10.3892\/ol.2018.9254","article-title":"D Rhamnose\n\u03b2-Hederin against\nhuman breast cancer by reducing tumor-derived exosomes","volume":"16","author":"Chen","year":"2018","journal-title":"Oncol Lett"},{"key":"2026090311503116600_cit4","doi-asserted-by":"publisher","first-page":"639","DOI":"10.1055\/s-0032-1328401","article-title":"Antiproliferative\nand apoptotic activities\nof triterpenoid saponins from the roots of Platycodon grandiflorum and their structure-activity relationships","volume":"79","author":"Chun","year":"2013","journal-title":"Planta Med"},{"key":"2026090311503116600_cit5","doi-asserted-by":"publisher","first-page":"2559","DOI":"10.1002\/jssc.201100326","article-title":"Preparative\nseparation of minor saponins from Platycodi Radix by high-speed counter-current chromatography","volume":"34","author":"Ha","year":"2011","journal-title":"J. Sep. Sci"},{"key":"2026090311503116600_cit6","doi-asserted-by":"publisher","first-page":"110656","DOI":"10.1016\/j.enzmictec.2025.110656","article-title":"Rhamnosyltransferases: Biochemical\nactivities, potential biotechnology for production of natural products\nand their applications","volume":"189","author":"Thuan","year":"2025","journal-title":"Enzyme Microb. Technol"},{"key":"2026090311503116600_cit7","doi-asserted-by":"publisher","first-page":"16","DOI":"10.1002\/cbic.200300620","article-title":"Structure-based enzyme engineering\nand its impact on\nin vitro glycorandomization","volume":"5","author":"Thorson","year":"2004","journal-title":"ChemBiochem"},{"key":"2026090311503116600_cit8","doi-asserted-by":"publisher","first-page":"9814","DOI":"10.1002\/anie.200801204","article-title":"Natural-product\nsugar biosynthesis and enzymatic glycodiversification","volume":"47","author":"Thibodeaux","year":"2008","journal-title":"Angew. Chem., Int. 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Sci"},{"key":"2026090311503116600_cit17","doi-asserted-by":"publisher","first-page":"7407","DOI":"10.1021\/acscatal.3c00503","article-title":"Enhanced\nthermostability of Candida ketoreductase by computation-based\ncross-regional combinatorial\nmutagenesis","volume":"13","author":"Chen","year":"2023","journal-title":"ACS Catal"},{"key":"2026090311503116600_cit18","doi-asserted-by":"publisher","first-page":"202","DOI":"10.1186\/s13068-021-02052-3","article-title":"Improvement\nof thermostability and catalytic efficiency\nof glucoamylase from Talaromyces leycettanus JCM12802\nvia site-directed mutagenesis to enhance industrial saccharification\napplications","volume":"14","author":"Tong","year":"2021","journal-title":"Biotechnol. 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Biotechnol"},{"key":"2026090311503116600_cit27","doi-asserted-by":"publisher","first-page":"393","DOI":"10.1016\/j.chembiol.2008.02.017","article-title":"Optimizing\nglycosyltransferase\nspecificity via \u201chot spot\u201d saturation mutagenesis presents\na catalyst for novobiocin glycorandomization","volume":"15","author":"Williams","year":"2008","journal-title":"Chem. 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Bioeng. Biotechnol"},{"key":"2026090311503116600_cit32","doi-asserted-by":"publisher","first-page":"112898","DOI":"10.1016\/j.mcat.2022.112898","article-title":"Improving the thermostability\nof glycosyltransferase YojK by targeting\nmutagenesis for highly efficient biosynthesis of rebaudioside D","volume":"535","author":"Yang","year":"2023","journal-title":"Mol. Catal"},{"key":"2026090311503116600_cit33","doi-asserted-by":"publisher","first-page":"105867","DOI":"10.1016\/j.fbio.2025.105867","article-title":"Conformational adjustment\nof 1,2-rhamnosyltransferase by prepeptides:\nExperimental and dynamic analysis","volume":"64","author":"Wang","year":"2025","journal-title":"Food Biosci"},{"key":"2026090311503116600_cit34","doi-asserted-by":"publisher","first-page":"14483","DOI":"10.1021\/acs.jafc.6c01848","article-title":"Structure-Guided\nTunnel Engineering of Rhamnosyltransferase Cm1,2RhaT: Mechanistic\nInsights and Enhanced Catalytic Efficiency for Neohesperidin Synthesis","volume":"74","author":"Chen","year":"2026","journal-title":"J. Agric. Food Chem"},{"key":"2026090311503116600_cit35","doi-asserted-by":"publisher","first-page":"101539","DOI":"10.1016\/j.xplc.2025.101539","article-title":"Structural insights\ninto the catalytic mechanism of the phenylethanoid glycoside rhamnosyltransferase\nUGT79G15 from Rehmannia glutinosa","volume":"6","author":"Ma","year":"2025","journal-title":"Plant Commun"},{"key":"2026090311503116600_cit36","doi-asserted-by":"publisher","first-page":"100047","DOI":"10.1016\/j.bidere.2025.100047","article-title":"Identification\nand engineering of a UDP-rhamnosyltransferase\nfrom Trillium tschonoskii for heterologous biosynthesis\nof polyphyllin II in yeast","volume":"7","author":"Yang","year":"2025","journal-title":"Biodes Res"},{"key":"2026090311503116600_cit37","doi-asserted-by":"publisher","first-page":"110203","DOI":"10.1016\/j.bej.2026.110203","article-title":"Combinatorial\nmetabolic\nengineering enables efficient bioconversion of hesperidin into neohesperidin\nin Bacillus subtilis","volume":"232","author":"Wang","year":"2026","journal-title":"Biochem.\nEng. J"},{"key":"2026090311503116600_cit38","doi-asserted-by":"publisher","first-page":"100190","DOI":"10.59717\/j.xinn-life.2026.100190","article-title":"Industrial\nproduction of natural low-calorie sweetener neohesperidin dihydrochalcone","volume":"4","author":"Zhu","year":"2026","journal-title":"Innovation Life"},{"key":"2026090311503116600_cit39","doi-asserted-by":"publisher","first-page":"3629","DOI":"10.1021\/acscatal.9b05232","article-title":"Efficient O-glycosylation of triterpenes enabled by protein engineering\nof plant glycosyltransferase UGT74AC1","volume":"10","author":"Li","year":"2020","journal-title":"ACS Catal"},{"key":"2026090311503116600_cit40","doi-asserted-by":"publisher","first-page":"1739","DOI":"10.1021\/jm0306430","article-title":"Glide:\nA new approach for rapid, accurate docking and scoring. 1. Method\nand assessment of docking accuracy","volume":"47","author":"Friesner","year":"2004","journal-title":"J. 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Chem.\nTheory Comput"}],"container-title":["Journal of Agricultural\nand Food Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jafc.6c07461","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jafcau\/article-pdf\/74\/34\/27168\/66886790\/acs.jafc.6c07461.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jafcau\/article-pdf\/74\/34\/27168\/66886790\/acs.jafc.6c07461.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T16:20:12Z","timestamp":1788452412000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jafcau\/article\/74\/34\/27168\/5277209\/Reshaping-the-Activity-Stability-Landscape-of-a-1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,19]]},"references-count":41,"journal-issue":{"issue":"34","published-online":{"date-parts":[[2026,8,19]]},"published-print":{"date-parts":[[2026,9,2]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jafc.6c07461","relation":{},"ISSN":["0021-8561","1520-5118"],"issn-type":[{"value":"0021-8561","type":"print"},{"value":"1520-5118","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,19]]}}}