Engineering N-acyl-homoserine lactone-based quorum-sensing circuit for dynamic regulatory control in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is widely employed in industrial biotechnology for chemical and pharmaceutical production. However, engineering yeast for high product titre remains challenging due to metabolic imbalances and competition for cellular resources. To address this, we developed an orthogonal quorum-sensing (QS) system based on N-acyl-homoserine lactones (AHLs) for cell density-dependent regulation in yeast. Using metabolic engineering, we established AHL production in yeast. Next, we improved AHL-biosensors via directed evolution and a novel growth-based screening strategy with amdS as a counter-selectable marker. We identified three LuxR variants with enhanced sensitivity and confirmed N86 to play an important role in their sensitivity to ligands, corroborating literature on the native system in bacteria. These sensitive LuxR variants were engineered for QS-controlled expression of a reporter gene, demonstrated by delayed autonomous expression of yeGFP. Additionally, we engineered LuxR to function as a repressor, achieving QS-dependent repression. The QS system was applied to enhance aloesone production, a plant-derived metabolite with cosmetic and pharmaceutical applications. The established system showed 51% increased production through QS-controlled repression of FAS1. This work establishes a versatile QS-based regulatory platform to support dynamic pathway regulation for metabolic engineering in yeast.