{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T15:55:52Z","timestamp":1791388552993,"version":"4.3.3"},"reference-count":57,"publisher":"American Chemical Society (ACS)","issue":"39","license":[{"start":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T00:00:00Z","timestamp":1790121600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T00:00:00Z","timestamp":1790121600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T00:00:00Z","timestamp":1790121600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32101902"],"award-info":[{"award-number":["32101902"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32572509"],"award-info":[{"award-number":["32572509"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>S-Allyl-l-cysteine (SAC) is a bioactive organosulfur compound from aged garlic that has attracted considerable interest for its health-promoting properties. Here, we developed a channel-guided engineering strategy to enhance SAC biosynthesis using the \u03b2-subunit of tryptophan synthase (TrpB). Genome mining guided by channel similarity-identified a thermostable Thermotoga maritima TrpB (TmTrpB) as a suitable scaffold with intrinsic activity toward allyl mercaptan. Engineering the tunnel-lining residues yielded a beneficial mutation, TmTrpB4T, which increased SAC production by 2.5-fold compared with the wild-type enzyme. Molecular simulations indicated that this mutation improved substrate accessibility by increasing channel hydrophobicity and optimizing tunnel geometry. The engineered enzyme also exhibited broad substrate promiscuity toward diverse thiols. Under optimized whole-cell biocatalytic conditions, SAC reached 87 g\u00b7L\u20131 with 90% serine conversion. This work demonstrates the utility of channel-guided engineering for improving TrpB catalysis and provides an efficient biocatalytic platform for the synthesis of pharmaceutically valuable S-substituted l-cysteines.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c09353","type":"journal-article","created":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T19:01:45Z","timestamp":1790190105000},"page":"30999-31009","source":"Crossref","is-referenced-by-count":0,"title":["Channel-Guided\nEngineering of Tryptophan Synthase\n\u03b2-Subunit for High-Level Biosynthesis of the Garlic Bioactive\nCompound S-Allyl-\n                    <scp>l<\/scp>\n                    -Cysteine"],"prefix":"10.1021","volume":"74","author":[{"given":"Zhaolin","family":"Song","sequence":"first","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, P. R. China, 300457"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dequan","family":"Liu","sequence":"additional","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, P. R. China, 300457"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhao","family":"Zhang","sequence":"additional","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, China, 300457"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fangyue","family":"Bi","sequence":"additional","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, P. R. China, 300457"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xueying","family":"Liu","sequence":"additional","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, P. R. China, 300457"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8546-6090","authenticated-orcid":true,"given":"Wei","family":"Cao","sequence":"additional","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, P. R. China, 300457"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-5689-932X","authenticated-orcid":true,"given":"Qinggang","family":"Li","sequence":"additional","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, P. R. China, 300457"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu","family":"Li","sequence":"additional","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, P. R. China, 300457"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yihan","family":"Liu","sequence":"additional","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, P. R. China, 300457"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0610-8827","authenticated-orcid":true,"given":"Fuping","family":"Lu","sequence":"additional","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, P. R. China, 300457"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-3160-3600","authenticated-orcid":true,"given":"Fenghua","family":"Wang","sequence":"additional","affiliation":[{"name":"Tianjin University of Science and Technology , , ,","place":["Tianjin, P. R. China, 300457"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,23]]},"reference":[{"issue":"1","key":"2026100711065184700_cit1","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1002\/cbic.201600537","article-title":"Design of S-allylcysteine in situ production and incorporation\nbased on a novel pyrrolysyl-tRNA synthetase variant","volume":"18","author":"Exner","year":"2017","journal-title":"ChemBioChem"},{"issue":"3","key":"2026100711065184700_cit2","doi-asserted-by":"publisher","first-page":"716S","DOI":"10.1093\/jn\/136.3.716S","article-title":"Clarifying\nthe real bioactive constituents of garlic1","volume":"136","author":"Amagase","year":"2006","journal-title":"J. Nutr."},{"issue":"10","key":"2026100711065184700_cit3","doi-asserted-by":"publisher","first-page":"1701","DOI":"10.1248\/bpb.b16-00449","article-title":"Evaluation of the effects of S-allyl-L-cysteine,\nS-methyl-L-cysteine, trans-S-1-propenyl-L-cysteine, and their N-acetylated\nand S-oxidized metabolites on human CYP activities","volume":"39","author":"Amano","year":"2016","journal-title":"Biol. Pharm. Bull."},{"key":"2026100711065184700_cit4","doi-asserted-by":"publisher","first-page":"105657","DOI":"10.1016\/j.jff.2023.105657","article-title":"S-Allyl-L-cysteine \u2500a garlic bioactive:\nphysicochemical nature, mechanism, pharmacokinetics, and health promoting\nactivities","volume":"107","author":"Rais","year":"2023","journal-title":"J. Funct. Foods"},{"issue":"23","key":"2026100711065184700_cit5","doi-asserted-by":"publisher","first-page":"9227","DOI":"10.1039\/D5FO03746H","article-title":"Mechanistic insights into S-allyl cysteine\u2019s\ninsulin-mimetic role: glucose uptake, receptor kinase interaction,\nand sensitivity recovery in skeletal myotubes","volume":"16","author":"Geddo","year":"2025","journal-title":"Food Funct."},{"key":"2026100711065184700_cit6","doi-asserted-by":"publisher","first-page":"189","DOI":"10.1007\/BF02536728","article-title":"Garlic reduces plasma lipids by inhibiting hepatic\ncholesterol and triacylglycerol synthesis","volume":"29","author":"Yeh","year":"1994","journal-title":"Lipids"},{"issue":"5","key":"2026100711065184700_cit7","doi-asserted-by":"publisher","first-page":"1089","DOI":"10.1007\/s43440-024-00625-1","article-title":"Therapeutic effects\nof S-allyl-L-cysteine in a mouse endometriosis model and its immunomodulatory\neffects via regulation of T cell subsets and cytokine expression","volume":"76","author":"Park","year":"2024","journal-title":"Pharmacol Rep."},{"key":"2026100711065184700_cit8","doi-asserted-by":"publisher","first-page":"188","DOI":"10.3390\/biom14020188","article-title":"S-allyl-L-cysteine\naffects cell proliferation and expression\nof H2S-synthetizing enzymes in MCF-7 and MDA-MB-231 adenocarcinoma\ncell Lines","volume":"14","author":"Bentke-Imiolek","year":"2024","journal-title":"Biomolecules"},{"key":"2026100711065184700_cit9","doi-asserted-by":"publisher","first-page":"1565","DOI":"10.3892\/etm.2019.8391","article-title":"Neuroprotective\nmechanisms of S-allyl-L-cysteine in neurological disease (Review)","volume":"19","author":"Kosuge","year":"2019","journal-title":"Exp. Ther. Med."},{"issue":"11","key":"2026100711065184700_cit10","doi-asserted-by":"publisher","first-page":"6319","DOI":"10.1021\/jf201160a","article-title":"s-Allyl cysteine,\ns-ethyl cysteine, and s-propyl cysteine alleviate \u03b2-amyloid,\nglycative, and oxidative injury in brain of mice treated by d-galactose","volume":"59","author":"Tsai","year":"2011","journal-title":"J. Agric. Food Chem."},{"key":"2026100711065184700_cit11","doi-asserted-by":"publisher","first-page":"758","DOI":"10.3389\/fpls.2014.00758","article-title":"Garlic \u03b3-glutamyl\ntranspeptidases that catalyze deglutamylation of biosynthetic intermediate\nof alliin","volume":"5","author":"Yoshimoto","year":"2015","journal-title":"Front. Plant Sci."},{"key":"2026100711065184700_cit12","doi-asserted-by":"publisher","first-page":"1574","DOI":"10.3892\/etm.2019.8393","article-title":"Chemistry\nof aged garlic: Diversity of constituents in aged garlic extract and\ntheir production mechanisms via the combination of chemical and enzymatic\nreactions (Review)","volume":"19","author":"Kodera","year":"2019","journal-title":"Exp. Ther. Med."},{"key":"2026100711065184700_cit13","doi-asserted-by":"publisher","first-page":"146","DOI":"10.1021\/jf00037a026","article-title":"Volatile\ncompounds generated from thermal degradation of alliin and deoxyalliin\nin an aqueous solution","volume":"42","author":"Yu","year":"1994","journal-title":"J. Agric. Food Chem."},{"issue":"26","key":"2026100711065184700_cit14","doi-asserted-by":"publisher","first-page":"16505","DOI":"10.1021\/acs.jafc.5c03490","article-title":"Highly efficient one-pot\nmultienzyme cascades for the synthesis of\nS-allyl-l-cysteine from l-serine and allyl mercaptan","volume":"73","author":"Zhu","year":"2025","journal-title":"J. Agric. Food Chem."},{"issue":"3","key":"2026100711065184700_cit15","doi-asserted-by":"publisher","first-page":"182","DOI":"10.1016\/j.jbiosc.2022.06.001","article-title":"Identification\nof tryptophanase from Escherichia coli for the synthesis of S-allyl-l-cysteine and related S-substituted\ncysteine derivatives","volume":"134","author":"Mizutani","year":"2022","journal-title":"J. Biosci. Bioeng."},{"issue":"12","key":"2026100711065184700_cit16","doi-asserted-by":"publisher","first-page":"2861","DOI":"10.1080\/00021369.1983.10866047","article-title":"Enzymatic synthesis\nof s-substituted l-cysteines with tryptophan synthase of Escherichia coli","volume":"47","author":"Esaki","year":"1983","journal-title":"Agric. Biol.\nChem."},{"issue":"5","key":"2026100711065184700_cit17","doi-asserted-by":"publisher","first-page":"593","DOI":"10.1016\/j.cbpa.2008.07.011","article-title":"Tryptophan\nsynthase, an allosteric\nmolecular factory","volume":"12","author":"Barends","year":"2008","journal-title":"Curr. Opin. Chem. Biol."},{"issue":"6","key":"2026100711065184700_cit18","doi-asserted-by":"publisher","first-page":"254","DOI":"10.1016\/j.tibs.2008.04.008","article-title":"Tryptophan\nsynthase: the workings of a channeling nanomachine","volume":"33","author":"Dunn","year":"2008","journal-title":"Trends Biochem. Sci."},{"key":"2026100711065184700_cit19","doi-asserted-by":"publisher","first-page":"233","DOI":"10.1016\/j.copbio.2021.08.014","article-title":"Microenvironmental\neffects can masquerade as substrate channelling in cascade biocatalysis","volume":"73","author":"Abdallah","year":"2022","journal-title":"Curr. Opin. Biotechnol."},{"key":"2026100711065184700_cit20","doi-asserted-by":"publisher","first-page":"923042","DOI":"10.3389\/fmolb.2022.923042","article-title":"Allosteric\nregulation of substrate channeling: Salmonellatyphimurium tryptophan synthase","volume":"9","author":"Ghosh","year":"2022","journal-title":"Front. Mol. Biosci."},{"key":"2026100711065184700_cit21","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1007\/978-3-319-50413-1_1","article-title":"Directed evolution of an allosteric tryptophan synthase to create\na platform for synthesis of noncanonical amino acids","volume-title":"Directed enzyme evolution: advances and applications","author":"Murciano-Calles","year":"2017"},{"issue":"23","key":"2026100711065184700_cit22","doi-asserted-by":"publisher","first-page":"7256","DOI":"10.1021\/jacs.8b03490","article-title":"Directed evolution mimics allosteric\nactivation by\nstepwise tuning of the conformational ensemble","volume":"140","author":"Buller","year":"2018","journal-title":"J. Am. Chem. Soc."},{"issue":"47","key":"2026100711065184700_cit23","doi-asserted-by":"publisher","first-page":"14599","DOI":"10.1073\/pnas.1516401112","article-title":"Directed evolution of the tryptophan synthase \u03b2-subunit\nfor stand-alone function recapitulates allosteric activation","volume":"112","author":"Buller","year":"2015","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"45","key":"2026100711065184700_cit24","doi-asserted-by":"publisher","first-page":"14764","DOI":"10.1002\/anie.201807998","article-title":"Engineered biosynthesis\nof \u03b2-alkyl\ntryptophan analogues","volume":"57","author":"Boville","year":"2018","journal-title":"Angew. Chem., Int. Ed."},{"issue":"31","key":"2026100711065184700_cit25","doi-asserted-by":"publisher","first-page":"10769","DOI":"10.1021\/jacs.7b05007","article-title":"Unlocking\nreactivity of TrpB: a general biocatalytic platform for synthesis\nof tryptophan analogues","volume":"139","author":"Romney","year":"2017","journal-title":"J. Am. Chem. Soc."},{"issue":"2","key":"2026100711065184700_cit26","doi-asserted-by":"publisher","first-page":"154","DOI":"10.1016\/j.abb.2012.01.016","article-title":"Allosteric\nregulation of substrate channeling and catalysis in the tryptophan\nsynthase bienzyme complex","volume":"519","author":"Dunn","year":"2012","journal-title":"Arch. Biochem. Biophys."},{"issue":"1","key":"2026100711065184700_cit27","doi-asserted-by":"publisher","first-page":"4248","DOI":"10.1038\/s41467-019-12247-w","article-title":"Modular enzyme assembly for enhanced cascade biocatalysis and metabolic\nflux","volume":"10","author":"Kang","year":"2019","journal-title":"Nat. Commun."},{"issue":"10","key":"2026100711065184700_cit28","doi-asserted-by":"publisher","first-page":"6686","DOI":"10.1021\/jacs.3c12876","article-title":"Dynamic\nmetabolons using stimuli-responsive protein cages","volume":"146","author":"Kang","year":"2024","journal-title":"J. Am. Chem. Soc."},{"issue":"48","key":"2026100711065184700_cit29","doi-asserted-by":"publisher","first-page":"15506","DOI":"10.1039\/D5TB01129A","article-title":"A modular two-component protein cage\nfor spatially organized enzymatic assembly and enhanced metabolic\nflux","volume":"13","author":"Wang","year":"2025","journal-title":"J. Mater. Chem. B"},{"issue":"n\/a","key":"2026100711065184700_cit30","doi-asserted-by":"publisher","DOI":"10.1002\/advs.76328","article-title":"Computation-guided tunnel\nengineering enhances O2 transport\nand H2O2 resistance of Fe(II)\/\u03b1-ketoglutarate-dependent\ndioxygenases","volume":"n\/a","author":"Liu","year":"2026","journal-title":"Adv. Sci."},{"issue":"23","key":"2026100711065184700_cit31","doi-asserted-by":"publisher","first-page":"20108","DOI":"10.1021\/acscatal.5c03170","article-title":"ESTEEM strategy: revolutionizing enzymatic catalysis through the\nevolutionary gateway of enzyme tunnels","volume":"15","author":"Dong","year":"2025","journal-title":"ACS\nCatal."},{"issue":"1","key":"2026100711065184700_cit32","doi-asserted-by":"publisher","first-page":"1312","DOI":"10.1021\/acs.jafc.5c14467","article-title":"Structure-guided engineering\nof tryptophan hydroxylase relieves tunnel congestion and substrate\ntrapping to enhance 5hydroxytryptophan biosynthesis","volume":"74","author":"Han","year":"2026","journal-title":"J. Agric. Food Chem."},{"issue":"13","key":"2026100711065184700_cit33","doi-asserted-by":"publisher","first-page":"1658","DOI":"10.1093\/bioinformatics\/btl158","article-title":"Cd-hit: a fast program\nfor clustering and comparing\nlarge sets of protein or nucleotide sequences","volume":"22","author":"Li","year":"2006","journal-title":"Bioinformatics"},{"issue":"1","key":"2026100711065184700_cit34","doi-asserted-by":"publisher","first-page":"2736","DOI":"10.1038\/s41467-025-58038-4","article-title":"Robust enzyme discovery\nand engineering with deep learning using CataPro","volume":"16","author":"Wang","year":"2025","journal-title":"Nat. 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Natl. Acad. Sci. U. S. A."},{"key":"2026100711065184700_cit40","doi-asserted-by":"publisher","first-page":"173","DOI":"10.1016\/j.aca.2012.03.056","article-title":"Determination of biothiols\nby a novel on-line HPLC-DTNB assay with post-column detection","volume":"750","author":"O\u0308zyu\u0308rek","year":"2012","journal-title":"Anal. Chim. Acta"},{"key":"2026100711065184700_cit41","doi-asserted-by":"publisher","first-page":"3891","DOI":"10.1021\/acs.jcim.1c00203","article-title":"AutoDock Vina\n1.2.0:\nnew docking methods, expanded force field, and python bindings","volume":"61","author":"Eberhardt","year":"2021","journal-title":"J. Chem. Inf. Model."},{"issue":"13","key":"2026100711065184700_cit42","doi-asserted-by":"publisher","first-page":"134110","DOI":"10.1063\/5.0018516","article-title":"Heterogeneous\nparallelization\nand acceleration of molecular dynamics simulations in GROMACS","volume":"153","author":"Pa\u0301ll","year":"2020","journal-title":"J. Chem. Phys."},{"key":"2026100711065184700_cit43","doi-asserted-by":"publisher","first-page":"3314","DOI":"10.1021\/ct300418h","article-title":"MMPBSA.py: an efficient program for end-state free\nenergy calculations","volume":"8","author":"Miller","year":"2012","journal-title":"J. Chem. Theory Comput."},{"key":"2026100711065184700_cit44","doi-asserted-by":"publisher","first-page":"6183","DOI":"10.1021\/acs.jcim.3c01153","article-title":"AmberTools","volume":"63","author":"Case","year":"2023","journal-title":"J. Chem. Inf. 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Commun."},{"key":"2026100711065184700_cit48","doi-asserted-by":"publisher","first-page":"130248","DOI":"10.1016\/j.biortech.2023.130248","article-title":"Tunnel engineering\nof gas-converting enzymes for inhibitor retardation and substrate\nacceleration","volume":"394","author":"Kim","year":"2024","journal-title":"Bioresour. Technol."},{"issue":"12","key":"2026100711065184700_cit49","doi-asserted-by":"publisher","first-page":"8183","DOI":"10.1021\/acscatal.3c01426","article-title":"Rational design\nof the substrate\ntunnel of \u03b2ketothiolase reveals a local cationic domain modulated\nrule that improves the efficiency of Claisen condensation","volume":"13","author":"Liu","year":"2023","journal-title":"ACS Catal."},{"issue":"9","key":"2026100711065184700_cit50","doi-asserted-by":"publisher","first-page":"807","DOI":"10.1038\/s41929-022-00834-y","article-title":"O2-tolerant CO dehydrogenase\nvia tunnel redesign for the removal of CO from industrial flue gas","volume":"5","author":"Kim","year":"2022","journal-title":"Nat. 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