Replacing the Calvin cycle with the reductive glycine pathway in Cupriavidus necator

Abstract

Formate can be directly produced from CO2 and renewable electricity, making it a promising microbial feedstock for sustainable bioproduction. Cupriavidus necator is one of the few biotechnologically-relevant hosts that can grow on formate, but it uses the inefficient Calvin cycle. Here, we redesign C. necator metabolism for formate assimilation via the highly efficient synthetic reductive glycine pathway. First, we demonstrate that the upper pathway segment supports glycine biosynthesis from formate. Next, we explore the endogenous route for glycine assimilation and discover a wasteful oxidation-dependent pathway. By integrating glycine biosynthesis and assimilation we are able to replace C. necators Calvin cycle with the synthetic pathway and achieve formatotrophic growth. We then engineer more efficient glycine metabolism and use short-term evolution to optimize pathway activity, doubling the growth yield on formate and quadrupling the growth rate. This study thus paves the way towards an ideal microbial platform for realizing the formate bioeconomy.