Conferring methylotrophy on industrial microorganisms would enable the production of diverse products from one-carbon feedstocks and contribute to establishing a low-carbon society. Rebuilding methylotrophs, however, requires a thorough metabolic refactoring and is highly challenging. Only recently was synthetic methylotrophy achieved in model microorganismsEscherichia coli and baker’s yeast Saccharomyces cerevisiae. Here, we have engineered industrially important yeast Yarrowia lipolytica to assimilate methanol. Through rationally constructing a chimeric assimilation pathway, rewiring the native metabolism for improved precursor supply, and laboratory evolution, we improved the methanol assimilation from undetectable to a level of 1.1 g/L per 72 h and enabled methanol-supported cellular maintenance. By transcriptomic analysis, we further found that fine-tuning of methanol assimilation and ribulose monophosphate/xylulose monophosphate (RuMP/XuMP) regeneration and strengthening formate dehydrogenation and the serine pathway were beneficial for methanol assimilation. This work paves the way for creating synthetic methylotrophic yeast cell factories for low-carbon economy.
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