Exploring alternative pathways for the in vitro establishment of the HOPAC cycle for synthetic CO₂ fixation

Nature has evolved eight different pathways for the capture and conversion of CO₂, including the Calvin-Benson-Bassham cycle of photosynthesis. Yet, these pathways underlie constrains and only represent a fraction of the thousands of theoretically possible solutions. To overcome the limitations of natural evolution, we introduce the HydrOxyPropionyl-CoA/Acrylyl-CoA (HOPAC) cycle, a new-to-nature CO₂-fixation pathway that was designed through metabolic retrosynthesis around the reductive carboxylation of acrylyl-CoA, a highly efficient principle of CO₂ fixation. We realized the HOPAC cycle in a step-wise fashion and used rational engineering approaches and machine learning-guided workflows to further optimize its output by more than one order of magnitude. Version 4.0 of the HOPAC cycle encompasses 11 enzymes from six different organisms, converting ~3.0 mM CO₂ into glycolate within 2 hours. Our work moves the hypothetical HOPAC cycle from a theoretical design into an established in vitro system that forms the basis for different potential applications.