Engineering <i>Yarrowia lipolytica</i> to Enhance the Production of Malonic Acid via Malonyl‐CoA Pathway at High Titer

Malonic acid (MA) is a high-value-added chemical with significant applications in the polymers, pharmaceutical, and food industries. Microbial production of MA presents enzyme inefficiencies, competitive metabolic pathways, and dispersive carbon flux, which collectively limit its biosynthesis. Here, the non-conventional oleaginous yeast Yarrowia lipolytica is genetically engineered to enhance MA production. Initially, the malonyl-CoA pathway, comprising a malonyl-CoA hydrolase from Saccharomyces cerevisiae, is confirmed as the most efficient for MA production in Y. lipolytica. To further enhance MA production, two novel malonyl-CoA hydrolases exhibiting higher activity than the hydrolase from S. cerevisiae, are identified from Y. lipolytica and Fusarium oxysporum, respectively. The introduction of the malonyl-CoA hydrolase from F. oxysporum increases the MA titer to 6.3 g L^-1. Subsequently, advanced metabolic engineering strategies are performed to ensure a sufficient flux of the precursors acetyl-CoA and malonyl-CoA for MA production, resulting in a production of 13.8 g L^-1 MA in shaking-flasks. Finally, by employing the fermentation conditions and feeding strategies, a maximum concentration of 63.6 g L^-1 of MA is achieved at 156 h with a productivity of 0.41 g L^-1 h^-1 in fed-batch fermentation. This study provides a new way for engineering Y. lipolytica to enhance MA production at high titer.