{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,18]],"date-time":"2026-06-18T17:23:47Z","timestamp":1781803427941,"version":"3.54.5"},"reference-count":22,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2021,2,2]],"date-time":"2021-02-02T00:00:00Z","timestamp":1612224000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"the C1 Gas Refinery Program funded by the Ministry of Science and ICT","award":["2018M3D3A1A01055732"],"award-info":[{"award-number":["2018M3D3A1A01055732"]}]},{"name":"the C1 Gas Refinery Program funded by the Ministry of Science and ICT","award":["2018M3D3A1A01056181"],"award-info":[{"award-number":["2018M3D3A1A01056181"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Methanol dehydrogenase (Mdh), is a crucial enzyme for utilizing methane and methanol as carbon and energy sources in methylotrophy and synthetic methylotrophy. Engineering of Mdh, especially NAD-dependent Mdh, has thus been actively investigated to enhance methanol conversion. However, its poor catalytic activity and low methanol affinity limit its wider application. In this study, we applied a transcriptional factor-based biosensor for the direct evolution of Mdh from Lysinibacillus xylanilyticus (Lxmdh), which has a relatively high turnover rate and low KM value compared to other wild-type NAD-dependent Mdhs. A random mutant library of Lxmdh was constructed in Escherichia coli and was screened using formaldehyde-detectable biosensors by incubation with low methanol concentrations. Positive clones showing higher fluorescence were selected by fluorescence-activated cell sorting (FACS) system, and their catalytic activities toward methanol were evaluated. The successfully isolated mutants E396V, K318N, and K46E showed high activity, particularly at very low methanol concentrations. In kinetic analysis, mutant E396V, K318N, and K46E had superior methanol conversion efficiency, with 79-, 23-, and 3-fold improvements compared to the wild-type, respectively. These mutant enzymes could thus be useful for engineering synthetic methylotrophy and for enhancing methanol conversion to various useful products.<\/jats:p>","DOI":"10.3390\/ijms22031471","type":"journal-article","created":{"date-parts":[[2021,2,2]],"date-time":"2021-02-02T05:44:42Z","timestamp":1612244682000},"page":"1471","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":25,"title":["Biosensor-Based Directed Evolution of Methanol Dehydrogenase from Lysinibacillus xylanilyticus"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5239-4943","authenticated-orcid":false,"given":"Thien-Kim","family":"Le","sequence":"first","affiliation":[{"name":"School of Biological Sciences and Biotechnology, Graduate School, Chonnam National University, Yongbong-ro 77, Gwangju 61186, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Su-Bin","family":"Ju","sequence":"additional","affiliation":[{"name":"School of Biological Sciences and Biotechnology, Graduate School, Chonnam National University, Yongbong-ro 77, Gwangju 61186, Korea"},{"name":"School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2255-3418","authenticated-orcid":false,"given":"Hyewon","family":"Lee","sequence":"additional","affiliation":[{"name":"Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jin-Young","family":"Lee","sequence":"additional","affiliation":[{"name":"Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"So-Hyung","family":"Oh","sequence":"additional","affiliation":[{"name":"Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea"},{"name":"Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science &amp; Technology, Daejeon 34113, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kil-Koang","family":"Kwon","sequence":"additional","affiliation":[{"name":"Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bong-Hyun","family":"Sung","sequence":"additional","affiliation":[{"name":"Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea"},{"name":"Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science &amp; Technology, Daejeon 34113, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4539-9812","authenticated-orcid":false,"given":"Seung-Goo","family":"Lee","sequence":"additional","affiliation":[{"name":"Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea"},{"name":"Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science &amp; Technology, Daejeon 34113, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7307-237X","authenticated-orcid":false,"given":"Soo-Jin","family":"Yeom","sequence":"additional","affiliation":[{"name":"School of Biological Sciences and Biotechnology, Graduate School, Chonnam National University, Yongbong-ro 77, Gwangju 61186, Korea"},{"name":"School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,2,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1128\/mr.42.2.251-273.1978","article-title":"Evolutionary aspects of autotrophy","volume":"42","author":"Quayle","year":"1978","journal-title":"Microbiol. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4083","DOI":"10.1039\/C6RA27038G","article-title":"Guidance for engineering of synthetic methylotrophy based on methanol metabolism in methylotrophy","volume":"7","author":"Zhang","year":"2017","journal-title":"RSC Adv."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1016\/j.ymben.2014.12.008","article-title":"Engineering Escherichia coli for methanol conversion","volume":"28","author":"Meyer","year":"2015","journal-title":"Metab. Eng."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"4969","DOI":"10.1007\/s00253-016-7320-3","article-title":"Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1","volume":"100","author":"Wu","year":"2016","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.ymben.2016.10.015","article-title":"Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli","volume":"39","author":"Whitaker","year":"2017","journal-title":"Metab. Eng."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"46966","DOI":"10.1074\/jbc.M207547200","article-title":"Identification of a Magnesium-dependent NAD(P)(H)-binding Domain in the Nicotinoprotein Methanol Dehydrogenase from Bacillus methanolicus","volume":"277","author":"Hektor","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Krog, A., Heggeset, T.M.B., M\u00fcller, J.E.N., Kupper, C.E., Schneider, O., Vorholt, J.A., Ellingsen, T.E., and Brautaset, T. (2013). Methylotrophic Bacillus methanolicus Encodes Two Chromosomal and One Plasmid Born NAD+ Dependent Methanol Dehydrogenase Paralogs with Different Catalytic and Biochemical Properties. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0059188"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"67","DOI":"10.3389\/fbioe.2020.00067","article-title":"Discovery and Biochemical Characterization of a Methanol Dehydrogenase from Lysinibacillus xylanilyticus","volume":"8","author":"Lee","year":"2020","journal-title":"Front. Bioeng. Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2659","DOI":"10.1038\/s41598-018-20943-8","article-title":"Evolution of enzymes with new specificity by high-throughput screening using DmpR-based genetic circuits and multiple flow cytometry rounds","volume":"8","author":"Kwon","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1021\/sb400112u","article-title":"Toward a Generalized and High-throughput Enzyme Screening System Based on Artificial Genetic Circuits","volume":"3","author":"Choi","year":"2014","journal-title":"ACS Synth. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1021\/acssynbio.5b00287","article-title":"A Cell\u2013Cell Communication-Based Screening System for Novel Microbes with Target Enzyme Activities","volume":"5","author":"Kim","year":"2016","journal-title":"ACS Synth. Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1002\/bit.26455","article-title":"Development of a formaldehyde biosensor with application to synthetic methylotrophy","volume":"115","author":"Woolston","year":"2018","journal-title":"Biotechnol. Bioeng."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-018-07488-0","article-title":"A synthetic microbial biosensor for high-throughput screening of lactam biocatalysts","volume":"9","author":"Yeom","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2776","DOI":"10.1002\/1873-3468.13206","article-title":"Structural and functional analyses of the cellulase transcription regulator CelR","volume":"592","author":"Fu","year":"2018","journal-title":"FEBS Lett."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1328","DOI":"10.1016\/j.bbrc.2017.11.157","article-title":"Development of a novel cellulase biosensor that detects crystalline cellulose hydrolysis using a transcriptional regulator","volume":"495","author":"Kwon","year":"2018","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_16","first-page":"682","article-title":"Temperature-Dependent Expression of a CFP-YFP FRET Diacylglycerol Sensor Enables Multiple-Read Screening for Compounds That Affect C1 Domains","volume":"24","author":"Yang","year":"2019","journal-title":"SLAS Discov. Adv. Life Sci. RD"},{"key":"ref_17","first-page":"7208","article-title":"Single-Cell Nanometric Coating Towards Whole-Cell-Based Biodevices and Biosensors","volume":"3","author":"Dai","year":"2018","journal-title":"Chem. Sel."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1106","DOI":"10.4014\/jmb.1611.11070","article-title":"Construction of Methanol-Sensing Escherichia coli by the Introduction of a Paracoccus denitrificans MxaY-Based Chimeric Two-Component System","volume":"27","author":"Ganesh","year":"2017","journal-title":"J. Microbiol. Biotechnol."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Lee, J.-Y., Sung, B.H., Oh, S.-H., Kwon, K.K., Lee, H., Kim, H., Lee, D.-H., Yeom, S.-J., and Lee, S.-G. (2019). C1 Compound Biosensors: Design, Functional Study, and Applications. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20092253"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2993","DOI":"10.1016\/j.febslet.2014.06.008","article-title":"In vitro activation of NAD-dependent alcohol dehydrogenases by Nudix hydrolases is more widespread than assumed","volume":"588","author":"Ochsner","year":"2014","journal-title":"FEBS Lett."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1002\/jobm.201000479","article-title":"Comparative analysis of two types of methanol dehydrogenase from Methylophaga aminisulfidivorans MPT grown on methanol","volume":"52","author":"Kim","year":"2012","journal-title":"J. Basic Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1707","DOI":"10.1021\/acscatal.6b03181","article-title":"Combining Aldolases and Transaminases for the Synthesis of 2-Amino-4-hydroxybutanoic Acid","volume":"7","author":"Hernandez","year":"2017","journal-title":"ACS Catal."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/3\/1471\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:18:39Z","timestamp":1760159919000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/3\/1471"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,2,2]]},"references-count":22,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2021,2]]}},"alternative-id":["ijms22031471"],"URL":"https:\/\/doi.org\/10.3390\/ijms22031471","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,2,2]]}}}