Engineering an olfactory biosensor for preferential monitoring medium-chain fatty acids in wastewater

Monitoring medium-chain fatty acids (MCFAs) in wastewater is essential for environmental protection and resource recovery, as these compounds contribute to malodor issues while holding significant value as precursors to biofuels and fine chemicals. However, conventional techniques such as GC–MS and HPLC are limited by operational complexity and high cost, restricting their applicability for real-time monitoring. To overcome these challenges, we developed a biomimetic biosensor based on engineered human olfactory receptor OR51L1, a member of the OR51 subfamily known for its affinity toward short- and medium-chain fatty acids. Through systematic functional screening, we found that the wild-type OR51L1 receptor responds primarily to pentanoic acid (C5) and hexanoic acid (C6). Using structure-guided mutagenesis, we generated the OR51L1(T161A) mutant, which exhibits enhanced sensitivity toward these MCFAs and, importantly, gains the ability to detect heptanoic acid (C7) — a ligand not recognized by the wild-type receptor. Molecular dynamics (MD) simulations indicated that the T161A substitution optimizes the ligand-binding pocket, promoting stronger interactions with MCFAs. The engineered receptor was incorporated into a cell-based sensing platform, enabling sensitive and selective detection of MCFAs at the micromolar level, and additionally demonstrated robustness through high reproducibility and strong anti-interference capability against common wastewater constituents. This work demonstrates the promise of protein-engineered olfactory receptors as versatile biosensing tools for monitoring environmentally relevant MCFAs, offering scalable strategies for wastewater management and resource recovery. • Elucidates fatty acid recognition mechanism in human olfactory receptor 51 subfamily. • Engineered olfactory receptor gains expanded detection range and enhanced sensitivity. • Molecular dynamics simulations reveal the structural basis for ligand binding. • Biosensor achieves micromolar detection of medium-chain fatty acids in wastewater. • Biosensor exhibits high interferent specificity and excellent reproducibility.