Markerless mutagenesis enables isoleucine biosynthesis solely from threonine in Methanothermobacter marburgensis
- Autor(en)
- Maximilian Klein, Angus S Hilts, Ross T Fennessy, Nino Trattnig, Thomas Stehrer-Polášek, Simon K-M R Rittmann, Christian Fink
- Abstrakt
UNLABELLED: The archaeal model microorganism Methanothermobacter marburgensis has been studied for methane production for decades. However, genetic modifications are required to harness M. marburgensis 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 M. marburgensis. 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 M. marburgensis and deleted the CimA coding gene, resulting in auxotrophy for isoleucine. The complementation of cimA initiated through constitutive expression led to prototrophic growth similar to the wild type, demonstrating that cimA is essential for pyruvate-derived isoleucine biosynthesis in M. marburgensis. As it has been shown vice versa in Escherichia coli 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 Geobacillus stearothermophilus. 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.
IMPORTANCE: The autotrophic, hydrogenotrophic methanogen Methanothermobacter marburgensis is one of the best-studied model organisms in the field of thermophilic archaea. The fact that M. marburgensis 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 M. marburgensis. Scalable bioreactor cultivation, the ability to genetically engineer, and the recent discovery of natural amino acid secretion in M. marburgensis 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.
- Organisation(en)
- Zentraler Informatikdienst, Department für Funktionelle und Evolutionäre Ökologie
- Externe Organisation(en)
- Arkeon GmbH
- Journal
- Microbiology Spectrum
- Seiten
- e0306824
- ISSN
- 2165-0497
- DOI
- https://doi.org/10.1128/spectrum.03068-24
- Publikationsdatum
- 04-2025
- Peer-reviewed
- Ja
- ÖFOS 2012
- 209006 Industrielle Biotechnologie
- Link zum Portal
- https://ucrisportal.univie.ac.at/de/publications/4141ab95-f3a3-457a-b4a5-c438c3ca9985