Engineering Yeasts to Grow Solely on Methanol or Formic acid coupled with CO2 fixation

Abstract

Abstract Synthetic microorganisms capable of using one-carbon compounds, such as methanol, formic acid or carbon dioxide, are of increasing interest. In this study, we converted the yeasts of Pichia pastoris and Saccharomyces cerevisiae to both synthetic methylotroph and formatotroph, allowing them to grow on methanol and formic acid alone coupled with CO2 fixation through a synthetic C1-compound assimilation pathway (MFORG pathway). This pathway consists of a methanol-formic acid oxidation module and the reductive glycine pathway. We first assembled the MFORG pathway in P. pastoris using only native enzymes, followed by overexpression of genes in the reductive glycine pathway, blocking the native methanol assimilation pathway, and compartmentalizing the methanol oxidation module. These modifications successfully redesigned the native methylotrophic yeast P. pastoris to grow on both methanol and formic acid, where higher growth rate and yield on methanol was obtained compared to the wild-type strain. We then introduced the MFORG pathway from P. pastoris into the model yeast S. cerevisiae, establishing full synthetic methylotrophy and formatotrophy in this organism. The resulting strain was able to successfully grow on methanol or formic acid alone with consumption rates of 24 mg/L*h and 15.2 mg/L*h, respectively. The CO2 fixation ability of synthetic P. pastoris and S. cerevisiae through the MFORG pathway was confirmed by 13C-tracer analysis. Finally, production of 5-aminolevulinic acid and lactic acid with methanol as the sole carbon source was demonstrated using synthetic P. pastoris and S. cerevisiae, indicating the potential of yeasts as promising hosts for biochemical production from various one-carbon compounds.