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

A stable high-copy episomal plasmid in Yarrowia lipolytica was developed with approximately fourfold increase in copy number, achieving one of the highest levels reported in this species.

• Why it matters: Episomal plasmids are crucial for yeast metabolic engineering but are limited in Y. lipolytica by low copy number and poor stability, restricting their effectiveness for protein expression and pathway optimization.
• What they did: Researchers selected ARS1 as a favorable autonomously replicating sequence and used ubiquitin degrons to tune marker abundance, resulting in a fourfold increase in plasmid copy number from approximately 1 to 5 copies per cell.
• The result: The engineered plasmid enabled a 3.43-fold increase in β-carotene titer compared to non-optimized plasmids, expanding the genetic toolkit and providing a framework for robust protein expression in non-conventional yeasts.