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Schlagwörter:
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Zusammenfassung:
The birth of the industrial revolution initiated a significant shift in the global carbon cycle. In the intervening centuries, the production of anthropogenic atmospheric carbon rose dramatically and has resulted in a pronounced climactic shift. The rate of this change is accelerating, largely irreversible in the short-term, and is expected to have a profound negative impact on nearly every aspect of human life from culture and economics to mental and physical health. It is now generally recognised that past practices are unsustainable and that we must take immediate action if we are to ameliorate this problem. Global efforts to reduce carbon emissions have begun, and many novel technologies are currently being developed both to make manufacturing more efficient and to actively remove carbon from the atmosphere. Despite these efforts, average atmospheric CO2 concentrations have continued to rise, and the climate has continued to change. While a multitude of different methods will be required if we are to be successful in reducing global atmospheric carbon, an intriguing approach is generating designer organisms capable of fixing CO2. Natural carbon fixation is the cornerstone of organic life, but there are potential improvements that could be made to generate more efficient carbon-fixing organisms. In addition to removing atmospheric CO2, the carbon can potentially be funnelled into any number of value-added products. In this work, we have pursued the artificial Hydroxypropionyl-CoA/Acrylyl-CoA Cycle in an in vitro system. We aim to demonstrate that the cycle is functional at ambient temperature and in the presence of oxygen making it an appealing candidate for future in vivo engineering efforts. In the first part, we investigate the oxidative portion of the cycle which involves the conversion of (2S)-methylmalonyl-CoA to malonyl-CoA. Originally this involved chemistry analogous to the TCA cycle, however due to difficulties with multiple steps of this pathway, we introduced a novel bypass that directly oxidises succinyl-CoA to the metabolically unusual fumaryl-CoA. This portion of the cycle also includes the first carbon-fixing reaction, the ATP-dependent carboxylation of acetyl-CoA. In the second part, we evaluate the reductive portion of the cycle which involves the conversion of malonyl-CoA back to (2S)-methylmalonyl-CoA. This portion involves three reduction reactions and we explored two potential pathways going through either 3-hydroxypropionyl-CoA or β-alanyl-CoA. While both versions are functional, the lack of β-alanine-specific enzymes, especially a β-alanyl-CoA synthetase made the 3-hydroxypropionyl-CoA pathway more practical. In either case, this portion terminates with the reductive carboxylation of acrylyl-CoA to (2S)-methylmalonyl-CoA. Finally, these pathways were combined to yield a continuous cycle. After flux through the cycle was achieved, we sought to resolve a variety of resultant issues including regeneration of ATP and NADPH, elimination of reactive oxygen species, protection from coenzyme B12 radical inactivation, regeneration of FAD-dependent enzymes, and the repair of chemically modified cofactors. The current HOPAC cycle was found to produce ~500 µM glycolate, or five CO2-equivalents per molecule of acetyl-CoA. Overall, in this work we established the HOPAC cycle, a new-to-nature synthetic CO2-fixation pathway, in vitro and further optimised its functioning. This works provides another proof-of-principle for synthetic CO2-fixation and opens the path for implementation of HOPAC in natural and synthetic cells in the future.
