{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T16:58:43Z","timestamp":1785430723946,"version":"3.56.0"},"reference-count":44,"publisher":"American Society for Microbiology","issue":"7","license":[{"start":{"date-parts":[[2026,7,22]],"date-time":"2026-07-22T00:00:00Z","timestamp":1784678400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,7,22]],"date-time":"2026-07-22T00:00:00Z","timestamp":1784678400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100008982","name":"National Science Foundation","doi-asserted-by":"publisher","award":["MCB 2225776"],"award-info":[{"award-number":["MCB 2225776"]}],"id":[{"id":"10.13039\/501100008982","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000015","name":"U.S. Department of Energy","doi-asserted-by":"publisher","award":["DE-AC05-00OR22725"],"award-info":[{"award-number":["DE-AC05-00OR22725"]}],"id":[{"id":"10.13039\/100000015","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2026,7,22]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Methylococcus capsulatus<\/jats:italic>\n                      Bath is a unique methanotrophic bacterium that uses methane (CH\n                      <jats:sub>4<\/jats:sub>\n                      ) as a carbon and energy source and encodes the RubisCO enzyme for inorganic carbon assimilation, positioning it as an excellent candidate for biotechnology to capture atmospheric CH\n                      <jats:sub>4<\/jats:sub>\n                      and carbon dioxide (CO\n                      <jats:sub>2<\/jats:sub>\n                      ) for chemical production. However, a limited genetic toolbox and minimal high-throughput methodologies for genetic engineering hinder the development of efficient\n                      <jats:italic toggle=\"yes\">M. capsulatus<\/jats:italic>\n                      biocatalysts. In this work, we developed and optimized an electroporation workflow to transfer circular and linear DNA fragments into\n                      <jats:italic toggle=\"yes\">M. capsulatus<\/jats:italic>\n                      with high efficiency by evading the host restriction-modification (RM) systems that recognize and degrade foreign DNA. To achieve this, we determined the\n                      <jats:italic toggle=\"yes\">M. capsulatus<\/jats:italic>\n                      methylome to identify RM system methyltransferases and express them in\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      , resulting in DNA methylation in\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      that mirrors that of\n                      <jats:italic toggle=\"yes\">M. capsulatus<\/jats:italic>\n                      , which improved methanotroph electroporation efficiency. Iterative site-directed mutagenesis of plasmid methylation sites identified a m\n                      <jats:sup>6<\/jats:sup>\n                      A motif necessary for the increase in electroporation efficiency. An\n                      <jats:italic toggle=\"yes\">M. capsulatus<\/jats:italic>\n                      knock-out strain with a disrupted Type I RM system linked to the m\n                      <jats:sup>6<\/jats:sup>\n                      A motif showed improved transformation efficiency compared to wild type, underscoring the importance of proper m\n                      <jats:sup>6<\/jats:sup>\n                      A methylation in successful DNA transfer to\n                      <jats:italic toggle=\"yes\">M. capsulatus<\/jats:italic>\n                      . Notably, plasmid methylation coupled to an optimized electroporation workflow enabled the facile generation of an\n                      <jats:italic toggle=\"yes\">M. capsulatus<\/jats:italic>\n                      genome-wide CRISPR interference library for functional genomic screening. Collectively, our results identify an RM system that has, up to now, limited\n                      <jats:italic toggle=\"yes\">M. capsulatus<\/jats:italic>\n                      transformation and establish a methodology to bypass the RM system and improve transformation efficiency, which will accelerate the development of engineered methanotrophs for CH\n                      <jats:sub>4<\/jats:sub>\n                      bioconversion technologies.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Aerobic CH\n                        <jats:sub>4<\/jats:sub>\n                        -oxidizing bacteria (methanotrophs) utilize CH\n                        <jats:sub>4<\/jats:sub>\n                        as a carbon and energy source in nature, also making them promising biocatalysts for converting CH\n                        <jats:sub>4<\/jats:sub>\n                        to valuable products. However, the development of these non-model microbes for use in biotechnology has been slowed by limitations in genetic tools and methodologies. Here, we established DNA electroporation in the methanotroph,\n                        <jats:italic toggle=\"yes\">Methylococcus capsulatus<\/jats:italic>\n                        Bath, which had poor baseline DNA transfer efficiency. By determining the\n                        <jats:italic toggle=\"yes\">M. capsulatus<\/jats:italic>\n                        methylome and identifying its restriction-modification systems that digest foreign DNA, thereby limiting DNA transformation, we developed an\n                        <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                        cloning strain expressing\n                        <jats:italic toggle=\"yes\">M. capsulatus<\/jats:italic>\n                        methyltransferases that modify plasmid DNA mirroring the host, significantly improving DNA transfer to\n                        <jats:italic toggle=\"yes\">M. capsulatus<\/jats:italic>\n                        . These improvements enabled the proof-of-concept construction of a genetic library via electroporation. The significant advancements herein bring us closer to realizing the use of methanotrophs for biomanufacturing and atmospheric CH\n                        <jats:sub>4<\/jats:sub>\n                        -mitigating biotechnologies.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/aem.00538-26","type":"journal-article","created":{"date-parts":[[2026,6,12]],"date-time":"2026-06-12T14:14:31Z","timestamp":1781273671000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Evading native restriction-modification systems improves electroporation efficiency of the methanotroph\n                    <i>Methylococcus capsulatus<\/i>\n                    Bath"],"prefix":"10.1128","volume":"92","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-4227-3180","authenticated-orcid":false,"given":"Yao-Chuan","family":"Yu","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00v97ad02","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, BioDiscovery Institute, and Advanced Environmental Research Institute, University of North Texas","place":["Denton, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melissa","family":"Tumen-Velasquez","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01qz5mb56","id-type":"ROR","asserted-by":"publisher"}],"name":"Biosciences Division, Oak Ridge National Laboratory","place":["Oak Ridge, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael Melesse","family":"Vergara","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01qz5mb56","id-type":"ROR","asserted-by":"publisher"}],"name":"Biosciences Division, Oak Ridge National Laboratory","place":["Oak Ridge, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William","family":"Alexander","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01qz5mb56","id-type":"ROR","asserted-by":"publisher"}],"name":"Biosciences Division, Oak Ridge National Laboratory","place":["Oak Ridge, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Grinffiel","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00v97ad02","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, BioDiscovery Institute, and Advanced Environmental Research Institute, University of North Texas","place":["Denton, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessica M.","family":"Henard","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00v97ad02","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, BioDiscovery Institute, and Advanced Environmental Research Institute, University of North Texas","place":["Denton, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5823-5329","authenticated-orcid":true,"given":"Adam M.","family":"Guss","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01qz5mb56","id-type":"ROR","asserted-by":"publisher"}],"name":"Biosciences Division, Oak Ridge National Laboratory","place":["Oak Ridge, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4574-587X","authenticated-orcid":true,"given":"Calvin A.","family":"Henard","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00v97ad02","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, BioDiscovery Institute, and Advanced Environmental Research Institute, University of North Texas","place":["Denton, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibtech.2020.07.007"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1039\/C9GC03722E"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2022.127141"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2021.125263"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jclepro.2017.03.123"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1038\/srep21585"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.91.5.1924-1931.1966"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tim.2005.03.003"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2015.03.010"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.00881-21"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.00881-21"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00203-001-0387-x"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1099\/00221287-147-8-2065"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.00340-19"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.synbio.2024.02.003"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-2273-5_21"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0702643105"},{"key":"e_1_3_4_19_2","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-12-386905-0.00008-5"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.1016\/bs.mie.2018.02.011"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.03724-15"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2021.717033"},{"key":"e_1_3_4_23_2","doi-asserted-by":"publisher","DOI":"10.3389\/fbioe.2024.1412410"},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","DOI":"10.1186\/s13068-020-01846-1"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00773-16"},{"key":"e_1_3_4_26_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.03795-14"},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","DOI":"10.1002\/047122197X.ch7"},{"key":"e_1_3_4_28_2","doi-asserted-by":"publisher","DOI":"10.1128\/MMBR.00044-12"},{"key":"e_1_3_4_29_2","doi-asserted-by":"publisher","DOI":"10.1186\/s13068-020-01872-z"},{"key":"e_1_3_4_30_2","doi-asserted-by":"publisher","DOI":"10.1093\/jimb\/kuaf022"},{"key":"e_1_3_4_31_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1820256116"},{"key":"e_1_3_4_32_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkn884"},{"key":"e_1_3_4_33_2","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.5c00370"},{"key":"e_1_3_4_34_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00271-17"},{"key":"e_1_3_4_35_2","doi-asserted-by":"publisher","DOI":"10.1007\/s10295-019-02218-x"},{"key":"e_1_3_4_36_2","doi-asserted-by":"publisher","DOI":"10.1134\/S0026261722602196"},{"key":"e_1_3_4_37_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/16.13.6127"},{"key":"e_1_3_4_38_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.170.6.2796-2801.1988"},{"key":"e_1_3_4_39_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.01875-18"},{"key":"e_1_3_4_40_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac975"},{"key":"e_1_3_4_41_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.00338-22"},{"key":"e_1_3_4_42_2","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.ade1285"},{"key":"e_1_3_4_43_2","doi-asserted-by":"publisher","DOI":"10.1016\/0378-1119(95)00584-1"},{"key":"e_1_3_4_44_2","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.aed4234"},{"key":"e_1_3_4_45_2","doi-asserted-by":"publisher","DOI":"10.1128\/msphere.00496-24"}],"container-title":["Applied and Environmental Microbiology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/aem.00538-26","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/aem.00538-26","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,22]],"date-time":"2026-07-22T13:05:26Z","timestamp":1784725526000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/aem.00538-26"}},"subtitle":[],"editor":[{"given":"Haruyuki","family":"Atomi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,7,22]]},"references-count":44,"journal-issue":{"issue":"7","published-print":{"date-parts":[[2026,7,22]]}},"alternative-id":["10.1128\/aem.00538-26"],"URL":"https:\/\/doi.org\/10.1128\/aem.00538-26","relation":{},"ISSN":["0099-2240","1098-5336"],"issn-type":[{"value":"0099-2240","type":"print"},{"value":"1098-5336","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,22]]},"assertion":[{"value":"2026-03-18","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-05-14","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-06-12","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e00538-26"}}