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However, limited enantioselectivity restricts the applicability of this method. In this work, a thermostable BVMO PockeMO was engineered in the active site and substrate tunnel to obtain the quadruple mutant MU4 (N541V\/N625W\/I601Y\/L226W). This mutant increased the ratio between the desired normal lactone (NL) and abnormal lactone (AL) from 1.1 to 43 (NL\/AL ratio) compared to the wild type. Structural analysis revealed that the tunnel was reshaped and the N625W\/I601Y mutation generated a gate-like structure to regulate the selectivity toward the racemic bicyclic ketone substrate. Notably, mutation N541V exerted a long-range epistatic effect on the tunnel mutations to modulate selectivity. Under a 200 mL scale, MU4 achieved an NL:AL ratio of 23 with 100 mM substrate. This study could enable the selective synthesis of chiral cyclolactone-containing natural products.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c01110","type":"journal-article","created":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T12:04:21Z","timestamp":1780488261000},"page":"18143-18155","source":"Crossref","is-referenced-by-count":0,"title":["Enantioselective\nSynthesis of Bicyclic Lactone Enabled\nby Tunnel Reshaping of Polycyclic Ketone Monooxygenase"],"prefix":"10.1021","volume":"74","author":[{"given":"Jingjing","family":"Chen","sequence":"first","affiliation":[{"name":"Fuzhou University , , ,","place":["Fuzhou, China, 350108"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2607-5230","authenticated-orcid":true,"given":"Xinqi","family":"Xu","sequence":"additional","affiliation":[{"name":"Fuzhou University , , ,","place":["Fuzhou, China, 350108"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4202-285X","authenticated-orcid":true,"given":"Juan","family":"Lin","sequence":"additional","affiliation":[{"name":"Fuzhou University , , ,","place":["Fuzhou, China, 350108"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,6,3]]},"reference":[{"key":"2026073021145578900_cit1","doi-asserted-by":"publisher","first-page":"1468","DOI":"10.1038\/s41557-023-01295-x","article-title":"Access to Unsaturated Bicyclic Lactones by Overriding Conventional\nC(sp3)-H Site Selectivity","volume":"15","author":"Das","year":"2024","journal-title":"Nat. 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Soc."},{"key":"2026073021145578900_cit31","doi-asserted-by":"publisher","DOI":"10.1016\/j.cej.2025.165291","article-title":"Boosting Amidation\nof Ortho-Substituted Anilines with Esters or Acids via Hinge and Tunnel\nEngineering of Lipase","volume":"519","author":"Wang","year":"2025","journal-title":"Chem. Eng. J."},{"key":"2026073021145578900_cit32","doi-asserted-by":"publisher","first-page":"8493","DOI":"10.1021\/acs.jafc.5c01858","article-title":"Enhancing\nthe Catalytic Efficiency of L-Rhamnose Isomerase for D-Allose Synthesis\nthrough Simultaneous Engineering of Noncatalytic and Catalytic Pockets","volume":"73","author":"Shen","year":"2025","journal-title":"J. Agric. Food Chem."},{"key":"2026073021145578900_cit33","doi-asserted-by":"publisher","first-page":"11207","DOI":"10.1021\/acscatal.9b03396","article-title":"Baeyer-Villiger\nMonooxygenases: Tunable Oxidative Biocatalysts","volume":"9","author":"Fu\u0308rst","year":"2019","journal-title":"ACS Catal."},{"key":"2026073021145578900_cit34","doi-asserted-by":"publisher","first-page":"17206","DOI":"10.1021\/acscatal.5c06238","article-title":"Reprogramming\nthe Catalytic Pocket of Baeyer-Villiger Monooxygenase for Environmentally\nCompatible Synthesis of a Chiral Sulfoxide Pharmaceutical","volume":"15","author":"Zhao","year":"2025","journal-title":"ACS Catal."},{"key":"2026073021145578900_cit35","doi-asserted-by":"publisher","first-page":"1442","DOI":"10.1126\/science.abd3623","article-title":"How Directed Evolution\nReshapes the\nEnergy Landscape in an Enzyme to Boost Catalysis","volume":"370","author":"Otten","year":"2020","journal-title":"Science"},{"key":"2026073021145578900_cit36","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1021\/ct9005745","article-title":"Relation between Free Energy Landscapes\nof Proteins and Dynamics","volume":"6","author":"Maisuradze","year":"2010","journal-title":"J. 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Bioeng."},{"key":"2026073021145578900_cit44","doi-asserted-by":"publisher","first-page":"14453","DOI":"10.1021\/acs.jafc.5c01791","article-title":"Enhancing Catalytic Activity of a Baeyer\u2013Villiger Monooxygenase\nfrom Oceanicola granulosus: Simultaneous Engineering of the Distal\nSite and Active Site","volume":"73","author":"Du","year":"2025","journal-title":"J. Agric. Food Chem."},{"key":"2026073021145578900_cit45","doi-asserted-by":"publisher","first-page":"7453","DOI":"10.1021\/acs.joc.8b00468","article-title":"Baeyer-Villiger\nMonooxygenase-Mediated Synthesis of Esomeprazole as an Alternative\nfor Kagan Sulfoxidation","volume":"83","author":"ong","year":"2018","journal-title":"J. Org. 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