Evolutionary engineering of Saccharomyces cerevisiae : Crafting a synthetic methylotroph via self-reprogramming

Methanol, as a non-edible feedstock, offers a promising sustainable alternative to sugar-based substrates in biochemical production. Despite progress in engineering methanol assimilation in nonmethylotrophs, the full transformation into methanol-dependent synthetic methylotrophs remains a formidable challenge. Here, moving beyond the conventional rational design principle, we engineered a synthetic methylotrophic Saccharomyces cerevisiae through genome rearrangement and adaptive laboratory evolution. This evolutionarily advanced strain unexpectedly shed the heterologous methanol assimilation pathway and demonstrated the robust growth on sole methanol. We discovered that the evolved strain likely realized methanol assimilation through a previously unidentified Adh2-Sfa1-rGly (ASrG) pathway, facilitating the concurrent assimilation of formate and CO 2 . Furthermore, the incorporation of electron transfer material C 3 N 4 quantum dots obviously enhanced methanol-dependent growth, emphasizing the role of energy availability in the ASrG pathway. This breakthrough introduces a previously unidentified C1 utilization pathway and highlights the exceptional adaptability and self-evolving capacity of the S. cerevisiae metabolic network.