Periodic Reporting for period 1 - Two-CO2-One (CO2 Fixation and Energy Conservation in the ancient Wood-Ljungdahl Pathway)
Période du rapport: 2023-05-01 au 2025-10-31
Carbon dioxide (CO2) receives a lot of attention as a greenhouse gas that promotes human-induced climate change. On the other hand, CO2 is also the starting point for the production of virtually all biomass on our planet. Therefore, nature has developed sophisticated methods to fix CO2 and make it available for biochemical reactions. Of all known biological CO2 fixation pathways, the Wood-Ljungdahl pathway (WLP) is the simplest way to fix two CO2 molecules to form acetyl-CoA, the key metabolic intermediate for biomass formation. It is the only pathway directly related to energy conservation and regarded to be the be the most ancient. The Two-CO2-One project aims to gain a comprehensive structural and mechanistic understanding of CO2 fixation and energy conservation in acetogenic bacteria and methanogenic archaea. These ecologically highly relevant organisms can live under conditions of extreme energy limitation in the absence of oxygen and feed exclusively on CO2 and hydrogen. In the project we will elucidate how these species fix CO2 and conserve energy through their WLP by using the innovative structural approach of redox-guided cryogenic electron microscopy (Cryo-EM) to study the oxygen-sensitive metalloprotein machinery of the WLP. The mechanistic insights gained will be challenged by microbiological and genetic approaches in these anaerobic, non-standard model organisms. This project will have an impact beyond basic science, as using autotrophic organisms that can sequester gaseous CO2 to produce biogas or ethanol from abundant waste gas resources is one way to reduce the human carbon footprint. Therefore, the Two-CO2-One project will not only lead to a deeper understanding of the unique mechanistic principles of WLP, but also provide new perspectives for developing biotechnological applications based on improved microbes that capture and sequester CO2 to produce industrially relevant chemicals and to combat human-induced climate change.
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.
All of our current findings significantly advance the field well beyond the existing state of the art, and importantly, many of these discoveries were highly unexpected. Our ongoing work continually reveals novel biological insights, underscoring the transformative nature of this project. Notably, we have established redox-controlled cryo-electron tomography (cryoET) workflows, setting new methodological standards within anaerobic structural biochemistry. And indeed, our work on the membrane attachment of HDCR is beyond state of the art, both in terms of the technical difficulty of the genetics of the model system, as well as the findings for what anchors the super barrels to the membranes. It is the first time ever that a direct connection between electron transfer and chemiosmotic gradient can be shown. Additionally, the discovery of L-clusters within the MCR activation machinery were an unanticipated breakthrough. These findings have unlocked profound evolutionary insights into the origin and diversification of bioenergetic systems. In particular, the presence of L-clusters outside the nitrogen fixation apparatus represents a radical departure from prevailing biochemical paradigms and challenges long-standing assumptions.