Periodic Reporting for period 4 - CARBONFIX (Towards a Self-Amplifying Carbon-Fixing Anabolic Cycle) Reporting period: 2020-03-01 to 2020-08-31 Summary of the context and overall objectives of the project How can simple molecules self-organize into a growing synthetic reaction network? In other words, how could biological metabolism have gotten started? This project takes a novel synthesis-driven approach to the question by exploring simple chemistry that mimics biological CO2-fixing pathways found in ancient life such as the reverse Krebs cycle and the acetyl CoA pathway. Five of the intermediates of these pathways are the synthetic precursors to all major classes of biomolecules and are built only from CO2 and electrons from simple reducing agents. Based on the nature of the reactions in these biochemical pathways and the specific structural features of the intermediates that comprise them, we hypothesized that parts or all of these metabolic pathways might be enabled with a surprisingly small number of simple, mutually compatible catalysts and reagents. If such chemistry could be demonstrated, it would provide an astounding link between prebiotic chemistry and the metabolic pathways used by ancient life. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We demonstrated that more than half of the reverse Krebs cycle can run in water without enzymes driven by metallic Fe and catalyzed by the metal ions Zn2+ and Cr3+ (Muchowska et al. Nature Ecology and Evolution 2017). Next, we showed that two simple metabolites react in iron-rich warm water to recreate many of the reactions of the biological Krebs cycle and glyoxylate cycles (Muchowska et al. Nature 2019).The discoveries described above led us to develop a non-enzymatic version of another biological CO2-fixation pathway that is thought to be ancient, the Acetyl CoA pathway (also known as the Wood-Ljungdahl pathway). We found that metallic iron, and several other zero-valent metals, fix CO2 on their surface to give the intermediates and end-products of that pathway even at room temperature and ambient pressure (Varma et al. Nature Ecology and Evolution 2018). Later, we found that the biological pathway can be mimicked even more closely using hydrogen gas as the source of electrons and common iron-based minerals as catalysts (Preiner et al. Nature Ecology and Evolution 2020). These discoveries are key for understanding the origin of biological carbon fixation (and the origin of life) because it means that forming C-C bonds from CO2 is not as difficult as previously thought and could have happened before enzymes existed. It is also very important because it shows a direct connection between the most ancient known biological CO2 fixation pathway and prebiotic chemistry.Taken together, these discoveries have important implications for the origin of life, because it means that these central biological pathways might have started before enzymes existed. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We discovered that some biological CO2-fixation pathways could potential have initiated without enzymes using simple metals as reagents and catalysts. We reported C-C bond formation from only CO2 in water by simple metals at ambient temperature and pressure. As illustrated by a highlight of our work in F1000 Prime, many evolutionary biologists interested in early evolution and some chemists specializing in the origin of life field consider this to be a major breakthrough. Future work will involve the study of the mechanism of C-C bond formation during the discovered CO2-fixation reaction, as well as the study of complex reaction networks that can give rise to the “missing” intermediates of the reverse Krebs cycle. We expect this approach to give significant insight into how early metabolism may have arisen and evolved.