{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T15:18:20Z","timestamp":1780327100637,"version":"3.54.1"},"reference-count":39,"publisher":"Oxford University Press (OUP)","license":[{"start":{"date-parts":[[2026,5,22]],"date-time":"2026-05-22T00:00:00Z","timestamp":1779408000000},"content-version":"vor","delay-in-days":141,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100011747","name":"NovoNordisk Foundation Center for Basic Metabolic Research","doi-asserted-by":"publisher","award":["NNF19OC0054329"],"award-info":[{"award-number":["NNF19OC0054329"]}],"id":[{"id":"10.13039\/501100011747","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100014438","name":"Business Finland","doi-asserted-by":"publisher","award":["2616\/31\/2023"],"award-info":[{"award-number":["2616\/31\/2023"]}],"id":[{"id":"10.13039\/501100014438","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,1,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Methanosarcina acetivorans is a model methanogen because of its metabolic versatility and genetic tractability. This microbe is not known to natively utilize hydrogen as a catabolic electron donor, despite its genome encoding hydrogenases, and the fact that this microbe can be used to heterologously express functional hydrogenases. Its native hydrogenases are expressed at a very low level and are suggested to have a role in recycling hydrogen that is produced as a byproduct of nitrogen fixation. To explore whether hydrogen can act as a catabolic electron donor, we utilized a previously constructed strain (str. JB-MF) that has the operon encoding for the methyl-H4MPT:CoM methyltransferase (Mtr) disrupted which makes growth dependent on oxidation of electron donors other than methanol. We showed that this M. acetivorans strain can grow by methanol reduction to methane with hydrogen as the sole electron donor. The strain was then used to test the putative electron donors for methyl-reducing methanogenesis: hydrogen, serine, and ethanol. Hydrogen and serine were identified to act as catabolic electron donors for methyl-reducing methanogenesis, which demonstrates the expanded metabolic versatility of M. acetivorans JB-MF. The methanophenazine-reducing hydrogenase (Vht) and the F420-reducing hydrogenase (Frh) were determined to be involved in hydrogen metabolism, because deletion of either made M. acetivorans JB-MF incapable of growing using H2-dependent methyl-reduction. We demonstrate that strain JB-MF is a suitable chassis to screen alternative electron donors for methanogenesis and that this strain may be used for targeted directed evolution of oxidoreductases.<\/jats:p>","DOI":"10.1093\/femsle\/fnag061","type":"journal-article","created":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T11:42:18Z","timestamp":1779363738000},"source":"Crossref","is-referenced-by-count":0,"title":["Disrupting the\n                    <i>mtr<\/i>\n                    -operon in\n                    <i>Methanosarcina\u00a0acetivorans<\/i>\n                    enables methyl-reducing methanogenesis with hydrogen and serine as the alternative electron donors"],"prefix":"10.1093","volume":"373","author":[{"given":"Jichen","family":"Bao","sequence":"first","affiliation":[{"name":"School of Chemical Engineering, Aalto University Department of Bioproducts and Biosystems, , Kemistintie 1, 02150 Espoo ,","place":["Finland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-5117-1625","authenticated-orcid":false,"given":"Tejas","family":"Somvanshi","sequence":"additional","affiliation":[{"name":"School of Chemical Engineering, Aalto University Department of Bioproducts and Biosystems, , Kemistintie 1, 02150 Espoo ,","place":["Finland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yufang","family":"Tian","sequence":"additional","affiliation":[{"name":"School of Chemical Engineering, Aalto University Department of Bioproducts and Biosystems, , Kemistintie 1, 02150 Espoo ,","place":["Finland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0667-9224","authenticated-orcid":false,"given":"Silvan","family":"Scheller","sequence":"additional","affiliation":[{"name":"School of Chemical Engineering, Aalto University Department of Bioproducts and Biosystems, , Kemistintie 1, 02150 Espoo ,","place":["Finland"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,5,22]]},"reference":[{"key":"2026060110470231600_bib1","doi-asserted-by":"publisher","first-page":"225171","DOI":"10.1111\/febs.17409","article-title":"Nature AND nurture: enabling formate-dependent growth in Methanosarcina acetivorans","volume":"292","author":"Bao","year":"2025","journal-title":"FEBS J"},{"key":"2026060110470231600_bib2","doi-asserted-by":"publisher","first-page":"113","DOI":"10.1007\/BF00419199","article-title":"Ethane production by Methanosarcina barkeri during growth in ethanol supplemented medium","volume":"54","author":"Belay","year":"1988","journal-title":"Antonie Van Leeuwenhoek"},{"key":"2026060110470231600_bib3","doi-asserted-by":"publisher","first-page":"31325","DOI":"10.1007\/s00203-004-0714-0","article-title":"Tetrahydrofolate-specific enzymes in Methanosarcina barkeri and growth dependence of this methanogenic archaeon on folic acid or p-aminobenzoic acid","volume":"182","author":"Buchenau","year":"2004","journal-title":"Arch Microbiol"},{"key":"2026060110470231600_bib4","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1111\/mmi.15377","article-title":"ModE regulates alternative nitrogenase expression in the methanogen Methanosarcina acetivorans","volume":"124","author":"Chanderban","year":"2025","journal-title":"Mol Microbiol"},{"key":"2026060110470231600_bib5","doi-asserted-by":"publisher","first-page":"88996","DOI":"10.1111\/j.1432-1033.1997.00889.x","article-title":"Purification and characterization of an alcohol: N,N -dimethyl-4-nitrosoaniline oxidoreductase from the methanogen Methanosarcina Barkeri DSM 804 strain fusaro","volume":"248","author":"Daussmann","year":"1997","journal-title":"Eur J Biochem"},{"key":"2026060110470231600_bib6","doi-asserted-by":"publisher","first-page":"e3647","DOI":"10.1371\/journal.pone.0003647","article-title":"A one pot, one step, precision cloning method with high throughput capability","volume":"3","author":"Engler","year":"2008","journal-title":"PLoS One"},{"key":"2026060110470231600_bib7","doi-asserted-by":"publisher","first-page":"fnaa137","DOI":"10.1093\/femsle\/fnaa137","article-title":"The hydrogen threshold of obligately methyl-reducing methanogens","volume":"367","author":"Feldewert","year":"2020","journal-title":"FEMS Microbiol Lett"},{"key":"2026060110470231600_bib8","doi-asserted-by":"publisher","first-page":"53242","DOI":"10.1101\/gr.223902","article-title":"The genome of M. acetivorans reveals extensive metabolic and physiological diversity","volume":"12","author":"Galagan","year":"2002","journal-title":"Genome Res"},{"key":"2026060110470231600_bib9","doi-asserted-by":"publisher","first-page":"72755","DOI":"10.1146\/annurev-micro-041020-024935","article-title":"Diversity and evolution of methane-related pathways in archaea","volume":"76","author":"Garcia","year":"2022","journal-title":"Annu Rev Microbiol"},{"key":"2026060110470231600_bib10","doi-asserted-by":"publisher","first-page":"282633","DOI":"10.1128\/JB.00563-08","article-title":"Differences in hydrogenase gene expression between Methanosarcina acetivorans and Methanosarcina barkeri","volume":"191","author":"Guss","year":"2009","journal-title":"J Bacteriol"},{"key":"2026060110470231600_bib11","doi-asserted-by":"publisher","first-page":"167180","DOI":"10.1111\/j.1365-2958.2005.04514.x","article-title":"Genetic analysis of mch mutants in two Methanosarcina species demonstrates multiple roles for the methanopterin-dependent C-1 oxidation\/reduction pathway and differences in H2 metabolism between closely related species","volume":"55","author":"Guss","year":"2005","journal-title":"Mol Microbiol"},{"key":"2026060110470231600_bib12","doi-asserted-by":"publisher","first-page":"237688","DOI":"10.1038\/ismej.2016.41","article-title":"Differences down-under: alcohol-fueled methanogenesis by archaea present in Australian macropodids","volume":"10","author":"Hoedt","year":"2016","journal-title":"ISME J"},{"key":"2026060110470231600_bib13","doi-asserted-by":"publisher","DOI":"10.1101\/2021.10.12.464174","article-title":"Vht hydrogenase is required for hydrogen cycling during nitrogen fixation by the non-hydrogenotrophic methanogen Methanosarcina acetivorans","author":"Hoerr","year":"2021","journal-title":"Biorxiv"},{"key":"2026060110470231600_bib14","doi-asserted-by":"publisher","first-page":"11228","DOI":"10.1021\/pr049832c","article-title":"Proteome of Methanosarcina acetivorans part I: an expanded view of the biology of the cell","volume":"4","author":"Li","year":"2005","journal-title":"J Proteome Res"},{"key":"2026060110470231600_bib15","doi-asserted-by":"publisher","first-page":"12935","DOI":"10.1021\/pr049831k","article-title":"Proteome of Methanosarcina acetivorans part II:\u2009 comparison of protein levels in acetate- and methanol-grown cells","volume":"4","author":"Li","year":"2005","journal-title":"J Proteome Res"},{"key":"2026060110470231600_bib16","doi-asserted-by":"publisher","first-page":"104853","DOI":"10.1139\/m95-146","article-title":"Oxidation\u2013reduction of methanol, formaldehyde, serine, and formate in Methanosphaera stadtmanae using 14C short- and long-term labelling","volume":"41","author":"Lin","year":"1995","journal-title":"Can J Microbiol"},{"key":"2026060110470231600_bib17","doi-asserted-by":"publisher","first-page":"6526","DOI":"10.1139\/w98-050","article-title":"Investigation of serine hydroxymethyltransferase in methanogens","volume":"44","author":"Lin","year":"1998","journal-title":"Can J Microbiol"},{"key":"2026060110470231600_bib18","doi-asserted-by":"publisher","first-page":"2479","DOI":"10.1128\/aem.49.1.247-249.1985","article-title":"Production and consumption of H2 during growth of Methanosarcina spp. on acetate","volume":"49","author":"Lovley","year":"1985","journal-title":"Appl Environ Microb"},{"key":"2026060110470231600_bib19","doi-asserted-by":"publisher","first-page":"e00342","DOI":"10.1128\/JB.00342-18","article-title":"Genetic, biochemical, and molecular characterization of Methanosarcina barkeri mutants lacking three distinct classes of hydrogenase","volume":"200","author":"Mand","year":"2018","journal-title":"J Bacteriol"},{"key":"2026060110470231600_bib20","doi-asserted-by":"publisher","first-page":"e00020","DOI":"10.1128\/MMBR.00020-19","article-title":"Energy conservation and hydrogenase function in methanogenic archaea, in particular the genus Methanosarcina","volume":"83","author":"Mand","year":"2019","journal-title":"Microbiol Mol Biol Rev"},{"key":"2026060110470231600_bib21","article-title":"Hydrogenase utilization and regulation in species of Methanosarcina","author":"Mand","year":"2018"},{"key":"2026060110470231600_bib22","doi-asserted-by":"publisher","first-page":"E3948","DOI":"10.1073\/pnas.1407927111","article-title":"Bacterial formate hydrogenlyase complex","volume":"111","author":"McDowall","year":"2014","journal-title":"Proc Natl Acad Sci USA"},{"key":"2026060110470231600_bib23","doi-asserted-by":"publisher","first-page":"262631","DOI":"10.1073\/pnas.94.6.2626","article-title":"A genetic system for Archaea of the genus Methanosarcina\u202f: liposome-mediated transformation and construction of shuttle vectors","volume":"94","author":"Metcalf","year":"1997","journal-title":"Proc Natl Acad Sci USA"},{"key":"2026060110470231600_bib24","doi-asserted-by":"publisher","first-page":"297681","DOI":"10.1073\/pnas.1618596114","article-title":"Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans","volume":"114","author":"Nayak","year":"2017","journal-title":"Proc Natl Acad Sci USA"},{"key":"2026060110470231600_bib25","doi-asserted-by":"publisher","first-page":"142533","DOI":"10.1128\/AEM.70.3.1425-1433.2004","article-title":"Development of a markerless genetic exchange method for Methanosarcina acetivorans C2A and its use in construction of new genetic tools for methanogenic archaea","volume":"70","author":"Pritchett","year":"2004","journal-title":"Appl Environ Microb"},{"key":"2026060110470231600_bib26","doi-asserted-by":"publisher","first-page":"62","DOI":"10.1186\/1471-2180-10-62","article-title":"Carbon-dependent control of electron transfer and central carbon pathway genes for methane biosynthesis in the Archaean, Methanosarcina acetivorans strain C2A","volume":"10","author":"Rohlin","year":"2010","journal-title":"BMC Microbiol"},{"key":"2026060110470231600_bib27","doi-asserted-by":"publisher","first-page":"1692934","DOI":"10.1073\/pnas.0407486101","article-title":"Anaerobic growth of Methanosarcina acetivorans C2A on carbon monoxide: an unusual way of life for a methanogenic archaeon","volume":"101","author":"Rother","year":"2004","journal-title":"Proc Natl Acad Sci USA"},{"key":"2026060110470231600_bib28","doi-asserted-by":"crossref","first-page":"e2113853119","DOI":"10.1073\/pnas.2113853119","article-title":"Deconstructing Methanosarcina acetivorans into an acetogenic archaeon","volume":"119","author":"Sch\u00f6ne","year":"2022","journal-title":"Proc Natl Acad Sci USA"},{"key":"2026060110470231600_bib29","doi-asserted-by":"publisher","first-page":"154","DOI":"10.1007\/s10482-025-02165-x","article-title":"Methanosarcina acetivorans requires methanol:coenzyme M methyltransferases for ethane formation from ethanol","volume":"118","author":"Somvanshi","year":"2025","journal-title":"Antonie van Leeuwenhoek"},{"key":"2026060110470231600_bib30","doi-asserted-by":"publisher","first-page":"9718","DOI":"10.1128\/aem.47.5.971-978.1984","article-title":"Methanosarcina acetivorans sp. nov., an acetotrophic methane-producing bacterium isolated from marine sediments","volume":"47","author":"Sowers","year":"1984","journal-title":"Appl Environ Microb"},{"key":"2026060110470231600_bib31","doi-asserted-by":"publisher","first-page":"38329","DOI":"10.1128\/aem.59.11.3832-3839.1993","article-title":"Disaggregation of Methanosarcina spp. and growth as single cells at elevated osmolarity","volume":"59","author":"Sowers","year":"1993","journal-title":"Appl Environ Microb"},{"key":"2026060110470231600_bib32","doi-asserted-by":"publisher","first-page":"54963","DOI":"10.1111\/febs.16165","article-title":"Process of energy conservation in the extremely haloalkaliphilic methyl-reducing methanogen Methanonatronarchaeum thermophilum","volume":"289","author":"Steiniger","year":"2022","journal-title":"FEBS J"},{"key":"2026060110470231600_bib33","doi-asserted-by":"publisher","first-page":"57991","DOI":"10.1038\/nrmicro1931","article-title":"Methanogenic archaea: ecologically relevant differences in energy conservation","volume":"6","author":"Thauer","year":"2008","journal-title":"Nat Rev Micro"},{"key":"2026060110470231600_bib34","doi-asserted-by":"publisher","first-page":"eabj1453","DOI":"10.1126\/sciadv.abj1453","article-title":"A methylotrophic origin of methanogenesis and early divergence of anaerobic multicarbon alkane metabolism","volume":"7","author":"Wang","year":"2021","journal-title":"Sci Adv"},{"key":"2026060110470231600_bib35","doi-asserted-by":"publisher","first-page":"106649","DOI":"10.1073\/pnas.0502623102","article-title":"Loss of the mtr operon in Methanosarcina blocks growth on methanol, but not methanogenesis, and reveals an unknown methanogenic pathway","volume":"102","author":"Welander","year":"2005","journal-title":"Proc Natl Acad Sci USA"},{"key":"2026060110470231600_bib36","doi-asserted-by":"publisher","first-page":"24013","DOI":"10.1038\/s41596-023-00850-7","article-title":"QurvE: user-friendly software for the analysis of biological growth and fluorescence data","volume":"18","author":"Wirth","year":"2023","journal-title":"Nat Protoc"},{"key":"2026060110470231600_bib37","doi-asserted-by":"publisher","first-page":"e00731","DOI":"10.1128\/AEM.00731-21","article-title":"Correlation of key physiological properties of Methanosarcina isolates with environment of origin","volume":"87","author":"Zhou","year":"2021","journal-title":"Appl Environ Microb"},{"key":"2026060110470231600_bib38","doi-asserted-by":"publisher","first-page":"1235616","DOI":"10.3389\/fmicb.2023.1235616","article-title":"CRISPR\/Cas12a toolbox for genome editing in Methanosarcina acetivorans","volume":"14","author":"Zhu","year":"2023","journal-title":"Front Microbiol"},{"key":"2026060110470231600_bib39","doi-asserted-by":"publisher","first-page":"99","DOI":"10.1007\/s12010-025-05387-x","article-title":"Unveiling the impact of indole derivatives on methanogenic archaea and microbial functions in anaerobic digestion of waste sewage sludge","volume":"198","author":"Zolkefli","year":"2025","journal-title":"Appl Biochem Biotechnol"}],"container-title":["FEMS Microbiology Letters"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/femsle\/advance-article-pdf\/doi\/10.1093\/femsle\/fnag061\/68368998\/fnag061.pdf","content-type":"application\/pdf","content-version":"am","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/femsle\/article-pdf\/doi\/10.1093\/femsle\/fnag061\/68368998\/fnag061.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/femsle\/article-pdf\/doi\/10.1093\/femsle\/fnag061\/68368998\/fnag061.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T14:47:10Z","timestamp":1780325230000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/femsle\/article\/doi\/10.1093\/femsle\/fnag061\/8690722"}},"subtitle":[],"editor":[{"given":"Dieter","family":"Jahn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026]]},"references-count":39,"URL":"https:\/\/doi.org\/10.1093\/femsle\/fnag061","relation":{},"ISSN":["1574-6968"],"issn-type":[{"value":"1574-6968","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2026]]},"published":{"date-parts":[[2026]]},"article-number":"fnag061"}}