A Copper-Inducible Regulatory System for Dynamic Enhancement of Gene Editing Efficiency in Yarrowia lipolytica

To circumvent the potential irreversible risks to cells posed by permanent genetic modifications, this study developed a copper-inducible system for dynamically regulating gene editing efficiency of Yarrowia lipolytica. By combining promoters pMT2 or enhanced pUAS16MT2 with genes scRAD52, scRAD59, and ylRAD52, homologous recombination efficiency was raised from 21.4% to 56.4%, while maintaining a transformation efficiency of 75 ± 5 CFU/OD₆₀₀. Employing a homology-mediated end joining strategy further improved DNA fragment integration efficiency from 15% to approximately 60%. Subsequently, the engineered chassis was subsequently applied to investigate stable conjugated linolenic acid (CLNA) production. Transcriptomic analysis and combinatorial gene knockouts identified POX2, POX3, and POX5 as key acyl-CoA oxidases involved in β-oxidation of long-chain fatty acids. A triple knockout strain increased extracellular lipid content by 64.19%, with POX5 playing a critical role in lipid-rich environments. Consequently, the CLNA degradation rate reduced from 92.9% to 19.2%, and final CLNA yield of muti-copy strains reached to 5.2 g/L. In summary, this study establishes a tunable gene editing system for Y. lipolytica and provides an effective engineering strategy for high-value lipid production.