RÉSUMÉ
The development of synthetic microorganisms that could use one-carbon compounds, such as carbon dioxide, methanol, or formate, has received considerable interest. In this study, we engineered Pichia pastoris and Saccharomyces cerevisiae to both synthetic methylotrophy and formatotrophy, enabling them to co-utilize methanol or formate with CO2 fixation through a synthetic C1-compound assimilation pathway (MFORG pathway). This pathway consisted of a methanol-formate oxidation module and the reductive glycine pathway. We first assembled the MFORG pathway in P. pastoris using endogenous enzymes, followed by blocking the native methanol assimilation pathway, modularly engineering genes of MFORG pathway, and compartmentalizing the methanol oxidation module. These modifications successfully enabled the methylotrophic yeast P. pastoris to utilize both methanol and formate. We then introduced the MFORG pathway from P. pastoris into the model yeast S. cerevisiae, establishing the synthetic methylotrophy and formatotrophy in this organism. The resulting strain could also successfully utilize both methanol and formate with consumption rates of 20 mg/L/h and 36.5 mg/L/h, respectively. The ability of the engineered P. pastoris and S. cerevisiae to co-assimilate CO2 with methanol or formate through the MFORG pathway was also confirmed by 13C-tracer analysis. Finally, production of 5-aminolevulinic acid and lactic acid by co-assimilating methanol and CO2 was demonstrated in the engineered P. pastoris and S. cerevisiae. This work indicates the potential of the MFORG pathway in developing different hosts to use various one-carbon compounds for chemical production.