Carbon dioxide (CO 2) is an important carbon feedstock for a future green economy. This requires the development of efficient strategies for its conversion into multi-carbon compounds. Here, we describe a synthetic cycle for the continuous fixation of CO 2 in vitro. The crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle is a reaction network of 17 enzymes that converts CO 2 into organic molecules at a rate of 5 nmol CO 2 min -1 mg -1 protein. The CETCH cycle was drafted by metabolic retrosynthesis, established with enzymes originating from nine different organisms of all three domains of life and optimized in several rounds by enzyme engineering and metabolic proofreading. The CETCH cycle expands the six naturally evolved CO 2-fixation pathways by a seventh, synthetic alternative, opening the way for in vitro and in vivo-applications.