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

ABSTRACT 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/OD 600 . 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.