Construction and modular implementation of the THETA cycle for synthetic CO2 fixation

Abstract Synthetic biology offers the opportunity to build solutions for improved capture and conversion of carbon dioxide (CO 2 ) that outcompete those evolved by nature. Here we demonstrate the design and construction of a new-to-nature CO 2 -fixation pathway, the reductive tricarboxylic acid branch/4-hydroxybutyryl-CoA/ethylmalonyl-CoA/acetyl-CoA (THETA) cycle. The THETA cycle encompasses 17 enzymes from 9 organisms and revolves around two of the most efficient CO 2 -fixing enzymes described in nature, crotonyl-CoA carboxylase/reductase and phosphoenolpyruvate carboxylase. Here using rational and machine learning-guided optimization approaches, we improved the yield of the cycle by two orders of magnitude and demonstrated the formation of different biochemical building blocks directly from CO 2 . Furthermore, we separated the THETA cycle into three modules that we successfully implemented in vivo by exploiting the natural plasticity of Escherichia coli metabolism. Growth-based selection and/or 13 C-labelling confirmed the activity of three different modules, demonstrating the first step towards realizing highly orthogonal and complex CO 2 -fixation pathways in the background of living cells.