Engineering a stable high-copy episomal plasmid in Yarrowia lipolytica.

Episomal plasmids are widely used in yeast metabolic engineering for their flexibility and high transformation efficiency. However, their application in Yarrowia lipolytica is often limited by low copy number and insufficient genetic stability. In this study, a robust episomal plasmid that balanced genetic stability and protein expression capacity was developed in Y. lipolytica. The autonomously replicating sequence (ARS) elements were selected, and ARS1 was identified as a favorable element that balanced genetic stability and gene expression capacity. To improve plasmid copy number, different ubiquitin degrons were used to tune selection marker abundance, and Ubi-R-Δk-mediated marker resulted in an approximately fourfold increase in plasmid copy number (from ∼1 to ∼5 copies per cell). The copy number of the engineered plasmid was one of the highest‑copy episomal systems in Y. lipolytica. To further evaluate the engineered plasmid, we applied it to β‑carotene biosynthesis. The results showed that the β‑carotene titer was 3.43‑fold higher than that of the non‑optimized plasmid. Collectively, this work expands the episomal plasmid toolbox for Y. lipolytica and establishes a generalizable framework for robust protein expression plasmid engineering in non-conventional yeasts.