A Fungal Bioluminescent Pathway (FBP)-Based Yeast Biosensor for Caffeic Acid Quantification in Food and Beverages

Caffeic acid is a natural hydroxycinnamic acid widely distributed in plant tissues and abundant in the human diet through fruits, vegetables, and a variety of plant-based beverages. This compound exhibits strong antioxidant, metal-chelating, and biological activities, being one of the most studied phenylpropanoids for therapeutic and biotechnological applications. The discovery of the fungal bioluminescent pathway (FBP), which converts caffeic acid into visible light through enzymatic luciferin biosynthesis followed by luciferase-catalyzed oxidation, has opened new opportunities for bioanalytical applications. In this work, we developed a simple, fast, and low-cost methodology to detect and quantify caffeic acid using Komagataella phaffii cells expressing the FBP enzymes. The system is based on bioluminescent light emission and, under optimized assay conditions with the working buffer (50 mM phosphate buffer containing 1% YPD, pH 6.0, and a yeast cell density of OD600 = 5), achieved LODs ranging from 1.4 to 19.1 μM and LOQs ranging from 4.3 to 57.9 μM in six commercial beverage matrices. Using only a few microliters of sample, we rapidly quantified caffeic acid in roasted coffee (0.05 mg/g) and yerba mate (Ilex paraguariensis, 11.1 mg/g) infusions; red and white wines (8.9-15.4 and 3.6-6.3 mg/L, respectively); wildflower honey (11.6 mg/kg); and grape juice (6.5-8.5 mg/L). The whole-cell bioluminescent approach for caffeic acid quantification provides a sustainable and accessible alternative to conventional chromatographic or electrochemical methods, reducing environmental impact while preserving analytical reliability.