Engineering Saccharomyces cerevisiae for growth on formate, a C1 feedstock derived from electrochemical activation of CO 2 , is a key solution to boost the development of formate‐mediated biomanufacturing and sustainable bioeconomy. Here, we developed a tripartite coupling strategy centered on serine that integrates formate assimilation and methanol dissimilation, enabling yeast growth on dual one‐carbon substrates. We first established a formate‐dependent yeast through constructing a serine‐autotrophic yeast and redesigning the reductive glycine (rGly) pathway, rendering the strain strictly dependent on formate for growth. Meanwhile, we identified mitochondrial and cytosolic serine deaminases (CHA1 mito and SDL1 cyto ) as the essential genes directing formate flux into central metabolism. Subsequently, we enhanced the supply of energy and reducing power by introducing methanol dissimilation pathways, which resulted in an optimal growth rate and improved formate utilization. Random mutagenesis screening yielded a strain with obligate growth on dual‐carbon substrates (formate as primary, methanol as auxiliary). Overall, our results demonstrate that establishing synthetic formatotrophy via rGly pathway requires efficient energy and reducing power, which facilitates the application of one‐carbon feedstocks in biorefineries.
Guo et al. (Tue,) studied this question.