In the course of this research, we have developed and implemented the most advanced genetic manipulation strategies to date in methanogenic archaea, organisms traditionally regarded as genetically intractable. Specifically, we successfully established a CRISPR–Cas12a-based genome editing system, coupled with a highly efficient transformation protocol, tailored for these absolute non-standard and slow-growing microorganisms. This breakthrough now enables precise, targeted genetic modifications in methanogens, unlocking experimental possibilities that were previously inaccessible. Parallel to these genetic advances, we significantly enhanced our cryo-electron tomography (cryo-ET) workflows, optimizing sample preparation, imaging conditions, and computational analysis pipelines. As a result, we achieved high-quality tomograms of multiple acetogenic and methanogenic species, providing structural insights into native cellular architectures under anaerobic conditions. These methodological advances set the stage for a series of ground-breaking discoveries in microbial cell biology and metabolism. Furthermore, we successfully implemented topological cross-linking mass spectrometry (XL-MS) as a robust integrative structural tool in our laboratory. This technique now complements our cryo-EM and tomography platforms, allowing us to resolve spatial protein organization and dynamic assemblies in vivo with higher confidence. Consequently, the ERC grant has allowed me to not only advance our acetogen research to a level that we can start to confidently say, we are on a good track to obtain a full mechanistic understanding. On top of that allowed it us to establish the work on the other major branch of the WLP, Methanogenesis. This definitely is the most important significant achievement as it positions the lab optimally for future challenges. As a noteworthy example of our work, our publication on the MCR complex received substantial media attention and was widely recognized as a thought-provoking contribution to understanding the interplay between the global carbon and nitrogen cycles. The findings have been highlighted for their potential impact in identifying novel inhibitory mechanisms targeting methanogenesis in ruminants, one of the major biological sources of atmospheric methane, a potent greenhouse gas.
