ARCANE: ‘A Reprogrammable Circuit Architecture for Non-model Engineering’ enables sophisticated metabolic control in Yarrowia lipolytica

The rational engineering of non-model microorganisms is often constrained by the scarcity of high-performance genetic regulatory tools. To overcome this limitation, we developed ARCANE – a reprogrammable circuit architecture for non-model engineering – featuring a cascaded design in which an inducer controls orthogonal bacterial transcription factors (bTFs), which in turn drive both activation and repression of endogenous promoters. This architecture provides strong insulation from host regulatory networks, minimizes leakage, and enables a broad dynamic range. As a proof-of-concept, we implemented ARCANE in the industrially significant non-model yeast, Yarrowia lipolytica . The resulting genetic switch exhibited exceptionally precise and tunable control over both single reporter genes and a multi-gene carotenoid biosynthesis pathway. Beyond providing a powerful regulatory tool for Y. lipolytica , ARCANE offers a versatile and transferable modular foundation for programmable gene control in emerging microbial platforms, paving the way for scalable synthetic biology in non-model systems.