{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T07:17:29Z","timestamp":1791875849534,"version":"4.3.4"},"reference-count":62,"publisher":"American Society for Microbiology","issue":"6","license":[{"start":{"date-parts":[[2025,6,3]],"date-time":"2025-06-03T00:00:00Z","timestamp":1748908800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,6,3]],"date-time":"2025-06-03T00:00:00Z","timestamp":1748908800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100006106","name":"Austrian Centre of Industrial Biotechnology","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100006106","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004955","name":"\u00d6sterreichische Forschungsf\u00f6rderungsgesellschaft","doi-asserted-by":"publisher","award":["FO999901140"],"award-info":[{"award-number":["FO999901140"]}],"id":[{"id":"10.13039\/501100004955","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2025,6,3]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      The archaeal model microorganism\n                      <jats:italic toggle=\"yes\">Methanothermobacter marburgensis<\/jats:italic>\n                      has been studied for methane production for decades. However, genetic modifications are required to harness\n                      <jats:italic toggle=\"yes\">M. marburgensis<\/jats:italic>\n                      for the generation of novel archaeal cell factories for industrial-scale production of commodity and high-value chemicals. Only the development of tools for genetic engineering opens up this possibility. Here, we present the establishment of the first markerless mutagenesis system for genetic modification of\n                      <jats:italic toggle=\"yes\">M. marburgensis<\/jats:italic>\n                      . This system allows the recycling of positive selection markers and enables multiple sequential gene deletions or integrations. As a demonstration, we clarified the postulated isoleucine biosynthesis pathway directly from pyruvate via citramalate synthase (CimA). In doing so, we identified a putative CimA in\n                      <jats:italic toggle=\"yes\">M. marburgensis<\/jats:italic>\n                      and deleted the CimA coding gene, resulting in auxotrophy for isoleucine. The complementation of\n                      <jats:italic toggle=\"yes\">cimA<\/jats:italic>\n                      initiated through constitutive expression led to prototrophic growth similar to the wild type, demonstrating that\n                      <jats:italic toggle=\"yes\">cimA<\/jats:italic>\n                      is essential for pyruvate-derived isoleucine biosynthesis in\n                      <jats:italic toggle=\"yes\">M. marburgensis<\/jats:italic>\n                      . As it has been shown\n                      <jats:italic toggle=\"yes\">vice versa<\/jats:italic>\n                      in\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      before, we were able to complement isoleucine biosynthesis with the integration of a synthetic isoleucine biosynthesis pathway from threonine for the first time in a methanogenic archaeon. This was achieved via genome integration of the characterized thermostable threonine deaminase from\n                      <jats:italic toggle=\"yes\">Geobacillus stearothermophilus<\/jats:italic>\n                      . The successful integration of an alternative pathway for isoleucine production paves the road for future application of multi-gene biosynthetic pathways to overproduce industrially relevant chemicals.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      The autotrophic, hydrogenotrophic methanogen\n                      <jats:italic toggle=\"yes\">Methanothermobacter marburgensis<\/jats:italic>\n                      is one of the best-studied model organisms in the field of thermophilic archaea. The fact that\n                      <jats:italic toggle=\"yes\">M. marburgensis<\/jats:italic>\n                      shows robust growth and scalability in bioreactor systems makes it a highly suitable candidate for industrial-scale bioprocesses. Additionally, the reported study provides the tools for genetic engineering that enable sequential genome modification in\n                      <jats:italic toggle=\"yes\">M. marburgensis<\/jats:italic>\n                      . Scalable bioreactor cultivation, the ability to genetically engineer, and the recent discovery of natural amino acid secretion in\n                      <jats:italic toggle=\"yes\">M. marburgensis<\/jats:italic>\n                      set the cornerstone for the generation of the first cell factories in archaeal biotechnology to economically produce carbon dioxide-derived commodity and high-value chemicals at industrial scale.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.03068-24","type":"journal-article","created":{"date-parts":[[2025,4,17]],"date-time":"2025-04-17T09:01:47Z","timestamp":1744880507000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":12,"title":["Markerless mutagenesis enables isoleucine biosynthesis solely from threonine in\n                    <i>Methanothermobacter marburgensis<\/i>"],"prefix":"10.1128","volume":"13","author":[{"given":"Maximilian","family":"Klein","sequence":"first","affiliation":[{"name":"Arkeon GmbH","place":["Tulln a.d. Donau, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angus S.","family":"Hilts","sequence":"additional","affiliation":[{"name":"Arkeon GmbH","place":["Tulln a.d. Donau, Austria"]},{"id":[{"id":"https:\/\/ror.org\/03prydq77","id-type":"ROR","asserted-by":"publisher"}],"name":"Archaea Physiology & Biotechnology Group, Department of Functional and Evolutionary Ecology, Universit\u00e4t Wien","place":["Vienna, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ross T.","family":"Fennessy","sequence":"additional","affiliation":[{"name":"Arkeon GmbH","place":["Tulln a.d. Donau, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nino","family":"Trattnig","sequence":"additional","affiliation":[{"name":"Arkeon GmbH","place":["Tulln a.d. Donau, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Stehrer-Pol\u00e1\u0161ek","sequence":"additional","affiliation":[{"name":"Arkeon GmbH","place":["Tulln a.d. Donau, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9746-3284","authenticated-orcid":false,"given":"Simon K.-M. R.","family":"Rittmann","sequence":"additional","affiliation":[{"name":"Arkeon GmbH","place":["Tulln a.d. Donau, Austria"]},{"id":[{"id":"https:\/\/ror.org\/03prydq77","id-type":"ROR","asserted-by":"publisher"}],"name":"Archaea Physiology & Biotechnology Group, Department of Functional and Evolutionary Ecology, Universit\u00e4t Wien","place":["Vienna, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4233-014X","authenticated-orcid":true,"given":"Christian","family":"Fink","sequence":"additional","affiliation":[{"name":"Arkeon GmbH","place":["Tulln a.d. 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