Peroxisomal Surface Display of Cat2 in <i>Yarrowia lipolytica</i> Enhances the Biosynthesis Acetyl-CoA-Derived Chemicals

The oleaginous yeast Yarrowia lipolytica is an outstanding chassis for producing high-value chemicals. Its highly active β-oxidation pathway generates a substantial pool of acetyl-CoA within peroxisomes; however, this abundant reservoir remains largely underutilized. Here, we developed a peroxisomal surface-display platform in Y. lipolytica, enabling enhanced biosynthesis of acetyl-CoA-derived chemicals. We first identified three peroxisomal membrane-targeting signals, derived from Pex3, Pex22, and Pex26 for protein display on the cytosolic face of the peroxisome. By anchoring the carnitine acetyl-CoA transferase Cat2 via these membrane-targeting signals, peroxisomal acetyl-CoA was redirected to the cytosol, increasing acetyl-CoA level and mevalonate production up to 2.4-fold and 2.9-fold, respectively. Further engineering improved squalene titers by 47% to 1.62 g/L. The same platform applied to 3-hydroxypropionic acid biosynthesis improved its production by 22% to 12.85 g/L. Our study establishes a peroxisomal surface display platform for harnessing subcellular acetyl-CoA and provides a versatile toolkit for subcellular engineering in Y. lipolytica .