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In this pathway, ethanol and acetate are converted into short- and medium-chain carboxylates. However, to date, no genetic system has been published in a peer-reviewed publication. Here, we report the development of versatile genetic tools for\n                      <jats:italic toggle=\"yes\">C. kluyveri<\/jats:italic>\n                      , utilizing the pMTL\n                      <jats:italic toggle=\"yes\">Clostridia<\/jats:italic>\n                      shuttle vector system and thiamphenicol as a selective marker. We identified the native restriction-modification system of\n                      <jats:italic toggle=\"yes\">C. kluyveri<\/jats:italic>\n                      as a critical barrier to DNA transfer and overcame it by identifying and characterizing the crucial methyltransferase. To mimic the native DNA methylation pattern of\n                      <jats:italic toggle=\"yes\">C. kluyveri<\/jats:italic>\n                      , we performed\n                      <jats:italic toggle=\"yes\">in-vivo<\/jats:italic>\n                      methylation of the shuttle vector plasmid by expressing the methyltransferase in\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      , followed by DNA transfer via conjugation. After validating the genetic system, we demonstrated heterologous expression of different combinations of both NADH- and NADPH-dependent alcohol dehydrogenases from\n                      <jats:italic toggle=\"yes\">Clostridium acetobutylicum<\/jats:italic>\n                      . The expression of these genes was controlled by the P\n                      <jats:italic toggle=\"yes\">\n                        <jats:sub>thl<\/jats:sub>\n                      <\/jats:italic>\n                      promoter, which is commonly used in\n                      <jats:italic toggle=\"yes\">Clostridia<\/jats:italic>\n                      , and the P\n                      <jats:italic toggle=\"yes\">\n                        <jats:sub>adhE2<\/jats:sub>\n                      <\/jats:italic>\n                      promoter, leading to\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      -butanol and\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      -hexanol production of the mutant strains. This genetic system for\n                      <jats:italic toggle=\"yes\">C. kluyveri<\/jats:italic>\n                      will not only enable further research on the metabolism of this microbe but also enable more profound insights into ethanol-based chain elongation in general.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Medium-chain carboxylates are required in various everyday products, including cosmetics, pharmaceuticals, and fragrances, and show a natural antimicrobial property. Furthermore, they represent food additives and serve as chemical building blocks for several other compounds. Traditionally, these carboxylates are produced from fossil resources, contributing to increased greenhouse gas emissions. Alternatively, they are derived from animal- or plant-based fat (e.g., coconut oil), which competes with agricultural land that is needed for food production. However, microbial chain elongation, which is a biotechnological approach relying on microbes, such as\n                      <jats:italic toggle=\"yes\">Clostridium kluyveri<\/jats:italic>\n                      , is sustainable and a promising alternative to the conventional production of medium-chain carboxylates. Notably, it enables the use of industrial waste streams (e.g., off-gases and carbohydrate-rich industrial waste) as substrates, making the process more environmentally friendly. By applying our genetic system for\n                      <jats:italic toggle=\"yes\">C. kluyveri<\/jats:italic>\n                      , a better understanding of microbial chain elongation can be achieved and potentially even enable an extension of its product portfolio.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aem.00058-26","type":"journal-article","created":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T13:10:36Z","timestamp":1773407436000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Genetic modification of\n                    <i>Clostridium kluyveri<\/i>\n                    for heterologous\n                    <i>n<\/i>\n                    -butanol and\n                    <i>n<\/i>\n                    -hexanol production"],"prefix":"10.1128","volume":"92","author":[{"given":"Caroline","family":"Schlai\u00df","sequence":"first","affiliation":[{"name":"AG Angenent, Max Planck Institute for Biology","place":["T\u00fcbingen, Germany"]},{"id":[{"id":"https:\/\/ror.org\/03a1kwz48","id-type":"ROR","asserted-by":"publisher"}],"name":"Environmental Biotechnology Group, Department of Geosciences, University of T\u00fcbingen","place":["T\u00fcbingen, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7186-9163","authenticated-orcid":true,"given":"Saskia T.","family":"Baur","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03a1kwz48","id-type":"ROR","asserted-by":"publisher"}],"name":"Environmental Biotechnology Group, Department of Geosciences, University of T\u00fcbingen","place":["T\u00fcbingen, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James W.","family":"Marsh","sequence":"additional","affiliation":[{"name":"Department of Microbiome Science, Max Planck Institute for Biology","place":["T\u00fcbingen, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kurt","family":"Gemeinhardt","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03a1kwz48","id-type":"ROR","asserted-by":"publisher"}],"name":"Environmental Biotechnology Group, Department of Geosciences, University of T\u00fcbingen","place":["T\u00fcbingen, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0180-1865","authenticated-orcid":true,"given":"Largus T.","family":"Angenent","sequence":"additional","affiliation":[{"name":"AG Angenent, Max Planck Institute for Biology","place":["T\u00fcbingen, Germany"]},{"id":[{"id":"https:\/\/ror.org\/03a1kwz48","id-type":"ROR","asserted-by":"publisher"}],"name":"Environmental Biotechnology Group, Department of Geosciences, University of T\u00fcbingen","place":["T\u00fcbingen, Germany"]},{"id":[{"id":"https:\/\/ror.org\/01aj84f44","id-type":"ROR","asserted-by":"publisher"}],"name":"The Novo Nordisk Foundation CO2 Research Center (CORC), Aarhus University","place":["Aarhus, Denmark"]},{"id":[{"id":"https:\/\/ror.org\/01aj84f44","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological and Chemical Engineering, Aarhus University","place":["Aarhus, Denmark"]},{"id":[{"id":"https:\/\/ror.org\/03a1kwz48","id-type":"ROR","asserted-by":"publisher"}],"name":"Cluster of Excellence \u2013 Controlling Microbes to Fight Infections, University of T\u00fcbingen","place":["T\u00fcbingen, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","volume-title":"Climate change 2021: the physical science basis, contribution of working group I to the sixth assessment report of the interfovernmetal panel on climate change","author":"Masson-Delmotte V","year":"2022","unstructured":"Masson-DelmotteV, ZhaiP, PiraniA, ConnorsSL. 2022. 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