Periodic Reporting for period 2 - ProtoMet (Protometabolic pathways: exploring the chemical roots of systems biology)
Periodo di rendicontazione: 2020-11-01 al 2023-10-31
We assembled a team of early-stage researchers and principal investigators to gain insight into the origins of life through the lens of metabolism. Very little is known about how life began because we lack artifacts from that era that can be directly interrogated. Similarly, skillful bioinformaticians can take us back in time to understand likely scenarios of evolution from ancient cells that were nevertheless quite complex. However, such analyses cannot directly reveal the chemistry that led to the emergence of the first, primitive cells. Our ESRs approached this problem by exploiting the tools of chemistry and microfluidics with input from philosophy. In this way, the team sought to recreate plausible scenarios that could have contributed to the emergence of life. To do so, ESRs focused on uncovering prebiotic analogues of central metabolic pathways necessary for the exploitation of fuel and the synthesis of the building blocks of life. Emphasis was also placed on the compartments that house living cells, since compartments are key cellular constituents for metabolism and Darwinian evolution. In addition to informing our understanding of ourselves and our planet, this work also gave ESRs a solid grounding in the chemistry that underpins biology with clear applications to synthetic biology and medicinal chemistry in either industry or academia.
The scientific pursuits can be roughly categorized as efforts to elucidate the chemistry of potential protometabolic pathways and the synergies that arise between such chemistry and prebiotic compartments. Both tracks were highly successful revealing new facets of prebiotic chemistry. The investigated protometabolic pathways included glycolysis and the citric acid cycle, since both are believed to be ancient and the former feeds into the latter in extant biology. When run in the forward direction, the citric acid cycle extracts and captures energy from fuel sources. When run in reverse, this same cycle can be used to synthesize the building blocks of life, e.g. amino acids. For example, we found that amino acids could be produced from the ketoacids of the citric acid cycle by reductive amination with hydrogen as the electron donor and iron containing meteorites as the catalyst. Chains of amino acids, i.e. peptides, can function as effective scaffolds for the coordination of iron-sulfur clusters capable of binding substrates, features present in the contemporary citric acid cycle. Additionally, the amino acid proline was found to function as an aldol catalyst within a prebiotic triose glycolysis pathway by facilitating the reaction of glycoladlehyde-phosphate with formaldehyde to give enantioenriched glyceraldehyde-2-phosphate. NADH can be produced by the nonenzymatic reduction of NAD+ by alpha-ketoacids, and NADH can participate in the enantioselective synthesis of amino acids during nonenzymatic reductive amination. Interestingly, the production and consumption of NADH could be tied to the formation and dissipation of compartments, since NADH was found to form coacervates with positively charged polypeptides, such as poly-Arg. Lattice Model Simulations were used to assess the impact of affinity and aggregation and the impact of the micro environments within such aggregates on flux through protometabolic pathways. In addition to coacervates, the role of lipids was assessed by demonstrating how the partitioning of hemin to aggregates of single-chain amphiphiles could generate catalytic pockets with peroxidase activity. Throughout these studies, new microfluidic technologies with tunable temperature and pH were developed for the investigation of compartments and protometabolism. The completion of ProtoMet led to the awarding of 6 PhDs and one more planned shortly. One additional ESR that was brought in later and was not initially enrolled in a PhD program is now enrolled in a PhD program. All ESR went through training, had career plans, and all that have graduated have secured positions either in industry or academia. The final symposium held by the consortium was a tremendous success that brought together leading figures in the field and communicated their findings through public events.
ProtoMet has uncovered the potential role of prebiotic metal and amino acid catalysts in the origins of life by facilitating the emergence of protometabolism. More importantly, when the different subprojects are looked at together, it becomes clearer how seemingly disparate processes are in fact tied together through synergies, e.g. the synthesis of amino acids in the reverse citric acid cycle could potentially catalyze steps of glycolysis or the production of NADH from protometabolism can give rise to coacervate compartments that then increase metabolic flux and begin to provide identify to nascent cell-like structures. Importantly, these prebiotic studies have already begun to impact our understanding of extant biology. Catalysts identified in prebiotic triose glycolysis were found to be active in E. coli, and the techniques developed to study iron-sulfur peptides were used to decipher in vivo regulatory mechanisms of glutathione homeostasis. Additionally, new microfluidic technologies were developed that very well may find their way into the market. Perhaps most importantly, this consortium has trained young, enthusiastic scientists equipped with a deeper understanding of the chemistry behind biology. Some have already joined companies, while others have, thus far, remained in academia. We expect both to significantly contribute to our economy and our understanding of biology.