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 CO 2 , 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 CO 2 -fixation pathway that was designed through metabolic retrosynthesis around the reductive carboxylation of acrylyl-CoA, a highly efficient principle of CO 2 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 CO 2 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.