{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T10:10:35Z","timestamp":1788862235142,"version":"build-2803163510"},"reference-count":50,"publisher":"Oxford University Press (OUP)","issue":"17","license":[{"start":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000},"content-version":"vor","delay-in-days":1,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"publisher","award":["RS-2024-00400033"],"award-info":[{"award-number":["RS-2024-00400033"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"publisher","award":["RS-2024-00453085"],"award-info":[{"award-number":["RS-2024-00453085"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"publisher","award":["2020R1A6A1A03047902"],"award-info":[{"award-number":["2020R1A6A1A03047902"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"publisher","award":["RS-2025-16070008"],"award-info":[{"award-number":["RS-2025-16070008"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100014188","name":"Ministry of Science and ICT","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100014188","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Korea Research Institute of Bioscience and Biotechnology Research Initiative Program","award":["KGM1302612"],"award-info":[{"award-number":["KGM1302612"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Conventional enzyme engineering strategies, such as directed evolution with structure-based analysis, are limited by laborious workflows and by the need to screen extensive enzyme libraries. Computational models based on multiple sequence alignment, such as SCANEER, have streamlined these engineering strategies, enabling the successful prediction of single-site mutations that enhance enzyme activity. However, when combining predicted mutations to improve enzyme performance or expand mutational space, such approaches often fail due to epistatic interactions among amino acid residues, which can lead to complex, non-additive effects. Here, we present epiSCANEER, an extended computational framework that incorporates co-evolutionary dependencies reflecting residue-level epistatic effects to identify mutation combinations with a higher likelihood of enhancing enzyme activity. By evaluating amino acid pairs at co-evolved positions across homologous sequences, epiSCANEER prioritizes mutation combinations with high evolutionary compatibility, significantly narrowing the combinatorial search space to variants more likely to exhibit activity-enhancing effects. Experimental validation demonstrated a 76% success rate for epiSCANEER prediction, compared to 30% and 38% for single-site predictions and combinatorial approaches of single-site mutants, respectively. This novel method obviates the need to construct single-mutation libraries, significantly reducing labor and costs while improving success rates. epiSCANEER has been developed as a web-server that enables researchers to access tools for rational enzyme optimization.<\/jats:p>","DOI":"10.1093\/nar\/gkag879","type":"journal-article","created":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T11:34:09Z","timestamp":1788521649000},"source":"Crossref","is-referenced-by-count":0,"title":["Enhancing prediction accuracy for enzyme activity engineering through sequence co-evolution and epistatic relationship modeling"],"prefix":"10.1093","volume":"54","author":[{"given":"Da Neub","family":"Kim","sequence":"first","affiliation":[{"name":"School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology , Pohang, Gyeongbuk, 37673 ,","place":["Korea"]},{"name":"Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology , Ulsan, 44429 ,","place":["Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-8810-8915","authenticated-orcid":false,"given":"Hyeongseop","family":"Kim","sequence":"additional","affiliation":[{"name":"School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology , Pohang, Gyeongbuk, 37673 ,","place":["Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8097-3675","authenticated-orcid":false,"given":"Donghyo","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Pohang University of Science and Technology , Pohang, Gyeongbuk, 37673 ,","place":["Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9742-3207","authenticated-orcid":false,"given":"Gyoo Yeol","family":"Jung","sequence":"additional","affiliation":[{"name":"School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology , Pohang, Gyeongbuk, 37673 ,","place":["Korea"]},{"name":"Department of Chemical Engineering, Pohang University of Science and Technology , Pohang, Gyeongbuk, 37673 ,","place":["Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3449-3814","authenticated-orcid":false,"given":"Sanguk","family":"Kim","sequence":"additional","affiliation":[{"name":"School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology , Pohang, Gyeongbuk, 37673 ,","place":["Korea"]},{"name":"Department of Life Sciences, Pohang University of Science and Technology , Pohang, Gyeongbuk, 37673 ,","place":["Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9045-7968","authenticated-orcid":false,"given":"Myung Hyun","family":"Noh","sequence":"additional","affiliation":[{"name":"Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology , Ulsan, 44429 ,","place":["Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,9,8]]},"reference":[{"key":"2026090805164884300_B1","doi-asserted-by":"publisher","first-page":"1210","DOI":"10.1021\/acscatal.9b04321","article-title":"Machine learning in enzyme engineering","volume":"10","author":"Mazurenko","year":"2020","journal-title":"ACS Catal"},{"key":"2026090805164884300_B2","doi-asserted-by":"publisher","first-page":"88","DOI":"10.1002\/anie.202006648","article-title":"Biocatalysis: enzymatic synthesis for industrial applications","volume":"60","author":"Wu","year":"2021","journal-title":"Angew Chem Int Ed"},{"key":"2026090805164884300_B3","doi-asserted-by":"publisher","first-page":"6113","DOI":"10.1007\/s00253-013-4960-4","article-title":"Developing Bacillus spp. as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology","volume":"97","author":"Liu","year":"2013","journal-title":"Appl Microbiol Biotechnol"},{"key":"2026090805164884300_B4","doi-asserted-by":"publisher","first-page":"1122","DOI":"10.1039\/C9NP00071B","article-title":"Engineering biosynthetic enzymes for industrial natural product synthesis","volume":"37","author":"Galanie","year":"2020","journal-title":"Nat Prod Rep"},{"key":"2026090805164884300_B5","doi-asserted-by":"publisher","first-page":"389","DOI":"10.1002\/bab.2117","article-title":"Enzyme engineering and its industrial applications","volume":"69","author":"Victorino\u00a0da\u00a0Silva\u00a0Amatto","year":"2022","journal-title":"Biotech App Biochem"},{"key":"2026090805164884300_B6","doi-asserted-by":"publisher","first-page":"246","DOI":"10.3109\/07388551.2014.950550","article-title":"Biocatalysts: application and engineering for industrial purposes","volume":"36","author":"Jemli","year":"2016","journal-title":"Crit Rev Biotechnol"},{"key":"2026090805164884300_B7","doi-asserted-by":"publisher","first-page":"e201209010","DOI":"10.5936\/csbj.201209010","article-title":"Recent advances in rational approaches for enzyme engineering","volume":"2","author":"Steiner","year":"2012","journal-title":"Comput Struct Biotechnol J"},{"key":"2026090805164884300_B8","doi-asserted-by":"crossref","first-page":"e16847","DOI":"10.1002\/aic.16847","article-title":"From directed evolution to computational enzyme engineering\u2014a review","volume":"66","author":"Chowdhury","year":"2020","journal-title":"AlChE J"},{"key":"2026090805164884300_B9","doi-asserted-by":"crossref","first-page":"2455","DOI":"10.1039\/D1CC04635G","article-title":"High-throughput screening, next generation sequencing and machine learning: advanced methods in enzyme engineering","volume":"58","author":"Vanella","year":"2022","journal-title":"Chem Commun"},{"key":"2026090805164884300_B10","doi-asserted-by":"publisher","first-page":"38","DOI":"10.1021\/bp0701214","article-title":"Engineering complex phenotypes in industrial strains","volume":"24","author":"Patnaik","year":"2008","journal-title":"Biotechnol Prog"},{"key":"2026090805164884300_B11","doi-asserted-by":"publisher","first-page":"313","DOI":"10.1039\/D4NP00031E","article-title":"Accelerating enzyme discovery and engineering with high-throughput screening","volume":"43","author":"Bozkurt","year":"2026","journal-title":"Nat Prod Rep"},{"key":"2026090805164884300_B12","doi-asserted-by":"publisher","first-page":"211","DOI":"10.1016\/S0022-1759(00)00236-2","article-title":"Flow cytometric screening of cell-based libraries","volume":"243","author":"Daugherty","year":"2000","journal-title":"J Immunol Methods"},{"key":"2026090805164884300_B13","doi-asserted-by":"publisher","first-page":"865","DOI":"10.1038\/s41467-024-55399-0","article-title":"Accelerated enzyme engineering by machine-learning guided cell-free expression","volume":"16","author":"Landwehr","year":"2025","journal-title":"Nat Commun"},{"key":"2026090805164884300_B14","doi-asserted-by":"publisher","first-page":"3764","DOI":"10.1073\/pnas.0900266106","article-title":"Computational structure-based redesign of enzyme activity","volume":"106","author":"Chen","year":"2009","journal-title":"Proc Natl Acad Sci USA"},{"key":"2026090805164884300_B15","doi-asserted-by":"publisher","first-page":"49","DOI":"10.1016\/j.ymben.2022.09.001","article-title":"Enzyme activity engineering based on sequence co-evolution analysis","volume":"74","author":"Kim","year":"2022","journal-title":"Metab Eng"},{"key":"2026090805164884300_B16","doi-asserted-by":"publisher","first-page":"128","DOI":"10.1038\/nbt.3769","article-title":"Mutation effects predicted from sequence co-variation","volume":"35","author":"Hopf","year":"2017","journal-title":"Nat Biotechnol"},{"key":"2026090805164884300_B17","doi-asserted-by":"publisher","first-page":"816","DOI":"10.1038\/s41592-018-0138-4","article-title":"Deep generative models of genetic variation capture the effects of mutations","volume":"15","author":"Riesselman","year":"2018","journal-title":"Nat Methods"},{"key":"2026090805164884300_B18","doi-asserted-by":"publisher","first-page":"866","DOI":"10.1038\/nrm2805","article-title":"Exploring protein fitness landscapes by directed evolution","volume":"10","author":"Romero","year":"2009","journal-title":"Nat Rev Mol Cell Biol"},{"key":"2026090805164884300_B19","doi-asserted-by":"publisher","first-page":"100","DOI":"10.1016\/S0968-0004(00)01755-2","article-title":"How enzymes adapt: lessons from directed evolution","volume":"26","author":"Arnold","year":"2001","journal-title":"Trends Biochem Sci"},{"key":"2026090805164884300_B20","doi-asserted-by":"publisher","first-page":"220","DOI":"10.1016\/j.tibtech.2025.08.007","article-title":"Semi-automated biofoundry workflows for sequence coevolution-guided isoprene synthase engineering","volume":"44","author":"Emelianov","year":"2026","journal-title":"Trends Biotechnol"},{"key":"2026090805164884300_B21","doi-asserted-by":"publisher","first-page":"492","DOI":"10.1002\/mlf2.12151","article-title":"Optimizing enzyme thermostability by combining multiple mutations using protein language model","volume":"3","author":"Bian","year":"2024","journal-title":"mLife"},{"key":"2026090805164884300_B22","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1016\/S0022-2836(02)00400-X","article-title":"Evolution of an antibiotic resistance enzyme constrained by stability and activity trade-offs","volume":"320","author":"Wang","year":"2002","journal-title":"J Mol Biol"},{"key":"2026090805164884300_B23","doi-asserted-by":"publisher","first-page":"1030","DOI":"10.1021\/acssynbio.1c00512","article-title":"Engineering strategies to overcome the stability\u2013function trade-off in proteins","volume":"11","author":"Teufl","year":"2022","journal-title":"ACS Synth Biol"},{"key":"2026090805164884300_B24","doi-asserted-by":"publisher","first-page":"535","DOI":"10.1038\/nature11510","article-title":"Epistasis as the primary factor in molecular evolution","volume":"490","author":"Breen","year":"2012","journal-title":"Nature"},{"key":"2026090805164884300_B25","doi-asserted-by":"publisher","first-page":"226","DOI":"10.1021\/acscentsci.3c01275","article-title":"Opportunities and challenges for machine learning-assisted enzyme engineering","volume":"10","author":"Yang","year":"2024","journal-title":"ACS Cent Sci"},{"key":"2026090805164884300_B26","doi-asserted-by":"publisher","first-page":"563","DOI":"10.1038\/225563a0","article-title":"Natural selection and the concept of a protein space","volume":"225","author":"Maynard\u00a0Smith","year":"1970","journal-title":"Nature"},{"key":"2026090805164884300_B27","doi-asserted-by":"publisher","first-page":"178","DOI":"10.1016\/j.molcel.2018.08.033","article-title":"Automated design of efficient and functionally diverse enzyme repertoires","volume":"72","author":"Khersonsky","year":"2018","journal-title":"Mol Cell"},{"key":"2026090805164884300_B28","doi-asserted-by":"publisher","first-page":"169011","DOI":"10.1016\/j.jmb.2025.169011","article-title":"htFuncLib: designing libraries of active-site multipoint mutants for protein optimization","volume":"437","author":"Lipsh-Sokolik","year":"2025","journal-title":"J Mol Biol"},{"key":"2026090805164884300_B29","doi-asserted-by":"crossref","first-page":"eabo5546","DOI":"10.1126\/sciadv.abo5546","article-title":"Conserved conformational dynamics determine enzyme activity","volume":"8","author":"Torgeson","year":"2022","journal-title":"Sci Adv"},{"key":"2026090805164884300_B30","doi-asserted-by":"publisher","first-page":"3389","DOI":"10.1093\/nar\/25.17.3389","article-title":"Gapped BLAST and PSI-BLAST: a new generation of protein database search programs","volume":"25","author":"Altschul","year":"1997","journal-title":"Nucleic Acids Res"},{"key":"2026090805164884300_B31","doi-asserted-by":"publisher","first-page":"1792","DOI":"10.1093\/nar\/gkh340","article-title":"MUSCLE: multiple sequence alignment with high accuracy and high throughput","volume":"32","author":"Edgar","year":"2004","journal-title":"Nucleic Acids Res"},{"key":"2026090805164884300_B32","doi-asserted-by":"publisher","first-page":"409","DOI":"10.1016\/0022-2836(71)90390-1","article-title":"Tests for comparing related amino-acid sequences. Cytochrome c and cytochrome c551","volume":"61","author":"McLachlan","year":"1971","journal-title":"J Mol Biol"},{"key":"2026090805164884300_B33","doi-asserted-by":"publisher","first-page":"3312","DOI":"10.1093\/bioinformatics\/btm515","article-title":"Co-evolving residues in membrane proteins","volume":"23","author":"Fuchs","year":"2007","journal-title":"Bioinformatics"},{"key":"2026090805164884300_B34","doi-asserted-by":"publisher","first-page":"1221","DOI":"10.1006\/jmbi.1999.3208","article-title":"Effective use of sequence correlation and conservation in fold recognition","volume":"293","author":"Olmea","year":"1999","journal-title":"J Mol Biol"},{"key":"2026090805164884300_B35","doi-asserted-by":"publisher","first-page":"e1000522","DOI":"10.1371\/journal.pcbi.1000522","article-title":"Integration of evolutionary features for the identification of functionally important residues in major facilitator superfamily transporters","volume":"5","author":"Jeon","year":"2009","journal-title":"PLoS Comput Biol"},{"key":"2026090805164884300_B36","doi-asserted-by":"publisher","first-page":"58","DOI":"10.1186\/s13068-022-02157-3","article-title":"A Vibrio-based microbial platform for accelerbrated lignocellulosic sugar conversion","volume":"15","author":"Woo","year":"2022","journal-title":"Biotechnol Biofuels"},{"key":"2026090805164884300_B37","doi-asserted-by":"publisher","first-page":"113","DOI":"10.1016\/j.ymben.2018.03.009","article-title":"Directed evolution of the 3-hydroxypropionic acid production pathway by engineering aldehyde dehydrogenase using a synthetic selection device","volume":"47","author":"Seok","year":"2018","journal-title":"Metab Eng"},{"key":"2026090805164884300_B38","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1042\/bj2370001","article-title":"Site-directed mutagenesis","volume":"237","author":"Carter","year":"1986","journal-title":"Biochem J"},{"key":"2026090805164884300_B39","doi-asserted-by":"crossref","first-page":"2486","DOI":"10.1038\/s41467-019-10371-1","article-title":"Vibrio sp. dhg as a platform for the biorefinery of brown macroalgae","volume":"10","author":"Lim","year":"2019","journal-title":"Nat Commun"},{"key":"2026090805164884300_B40","doi-asserted-by":"crossref","first-page":"314","DOI":"10.1007\/s13205-017-0966-4","article-title":"Investigation of the key factors on 3-hydroxypropionic acid production with different recombinant strains","volume":"7","author":"Niu","year":"2017","journal-title":"3 Biotech"},{"key":"2026090805164884300_B41","doi-asserted-by":"publisher","first-page":"24887","DOI":"10.1038\/srep24887","article-title":"Engineering a d-lactate dehydrogenase that can super-efficiently utilize NADPH and NADH as cofactors","volume":"6","author":"Meng","year":"2016","journal-title":"Sci Rep"},{"key":"2026090805164884300_B42","doi-asserted-by":"publisher","first-page":"2308","DOI":"10.1093\/bioinformatics\/btg299","article-title":"PDB file parser and structure class implemented in Python","volume":"19","author":"Hamelryck","year":"2003","journal-title":"Bioinformatics"},{"key":"2026090805164884300_B43","doi-asserted-by":"publisher","first-page":"1015","DOI":"10.1007\/s12257-021-0335-3","article-title":"Engineering of the substrate pocket of \u03b1-ketoglutaric semialdehyde dehydrogenase for improving the activity toward 3-hydroxypropanal","volume":"28","author":"Park","year":"2023","journal-title":"Biotechnol Bioproc E"},{"key":"2026090805164884300_B44","doi-asserted-by":"publisher","first-page":"46005","DOI":"10.1038\/srep46005","article-title":"Structural insights into the production of 3-hydroxypropionic acid by aldehyde dehydrogenase from Azospirillum brasilense","volume":"7","author":"Son","year":"2017","journal-title":"Sci Rep"},{"key":"2026090805164884300_B45","doi-asserted-by":"publisher","first-page":"W480","DOI":"10.1093\/nar\/gkq471","article-title":"DrugScorePPI webserver: fast and accurate in silico alanine scanning for scoring protein\u2013protein interactions","volume":"38","author":"Kr\u00ef\u00bf\u00bdger","year":"2010","journal-title":"Nucleic Acids Res"},{"key":"2026090805164884300_B46","doi-asserted-by":"crossref","first-page":"pl2","DOI":"10.1126\/stke.2192004pl2","article-title":"Computational alanine scanning of protein\u2013protein interfaces","volume":"2004","author":"Kortemme","year":"2004","journal-title":"Sci STKE"},{"key":"2026090805164884300_B47","doi-asserted-by":"publisher","first-page":"14243","DOI":"10.1021\/acscatal.2c01426","article-title":"Learning epistasis and residue coevolution patterns: current trends and future perspectives for advancing enzyme engineering","volume":"12","author":"Wittmund","year":"2022","journal-title":"ACS Catal"},{"key":"2026090805164884300_B48","doi-asserted-by":"publisher","first-page":"1743","DOI":"10.1089\/cmb.2011.0152","article-title":"Optimization of combinatorial mutagenesis","volume":"18","author":"Parker","year":"2011","journal-title":"J Comput Biol"},{"key":"2026090805164884300_B49","doi-asserted-by":"publisher","first-page":"3489","DOI":"10.21203\/rs.3.rs-3415956\/v1","article-title":"What makes the effect of protein mutations difficult to predict?","volume":"23","author":"Redestig","year":"2024","journal-title":"Comput Struct Biotechnol J"},{"key":"2026090805164884300_B50","doi-asserted-by":"publisher","first-page":"e7768514","DOI":"10.21203\/rs.3.rs-5354708\/v1","article-title":"Enhancing enzyme activity with mutation combinations guided by few-shot learning and causal inference","volume":"65","author":"Guo","year":"2024","journal-title":"Angew Chem Int Ed Engl"}],"container-title":["Nucleic Acids Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/54\/17\/gkag879\/70989722\/gkag879.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/54\/17\/gkag879\/70989722\/gkag879.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T09:16:54Z","timestamp":1788859014000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/nar\/article\/doi\/10.1093\/nar\/gkag879\/8787808"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,7]]},"references-count":50,"journal-issue":{"issue":"17","published-print":{"date-parts":[[2026,9,7]]}},"URL":"https:\/\/doi.org\/10.1093\/nar\/gkag879","relation":{},"ISSN":["0305-1048","1362-4962"],"issn-type":[{"value":"0305-1048","type":"print"},{"value":"1362-4962","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2026,9,23]]},"published":{"date-parts":[[2026,9,7]]},"article-number":"gkag879"}}