Unlocking the formate utilization of wild‐type Yarrowia lipolytica through adaptive laboratory evolution

Abstract Synthetic biology is contributing to the advancement of the global net‐negative carbon economy, with emphasis on formate as a member of the one‐carbon substrate garnering substantial attention. In this study, we employed base editing tools to facilitate adaptive evolution, achieving a formate tolerance of Yarrowia lipolytica to 1 M within 2 months. This effort resulted in two mutant strains, designated as M25‐70 and M25‐14, both exhibiting significantly enhanced formate utilization capabilities. Transcriptomic analysis revealed the upregulation of nine endogenous genes encoding formate dehydrogenases when cultivated utilizing formate as the sole carbon source. Furthermore, we uncovered the pivotal role of the glyoxylate and threonine‐based serine pathway in enhancing glycine supply to promote formate assimilation. The full potential of Y. lipolytica to tolerate and utilize formate establishing the foundation for pyruvate carboxylase‐based carbon sequestration pathways. Importantly, this study highlights the existence of a natural formate metabolic pathway in Y. lipolytica .