Functional characterization of engineered bacterial biosensors for kynurenine detection

The kynurenine (KYN) pathway is the major catabolic pathway for tryptophan in humans, producing several metabolites that influence health. In clinical settings, KYN levels serve as a valuable biomarker for the diagnosis and prognosis of inflammatory and neurological diseases. Nevertheless, KYN detection relies on mass spectrometry analysis, which requires specialized knowledge and expertise with high operational costs. The bacterial biosensor presents as a promising tool for rapid and cost-effective targeted substance detection due to its ease of genetic modification. Therefore, this study aimed to develop an engineered bacterial biosensor by integrating a genetic module in a plasmid designed for KYN detection harboured in an <i>Escherichia coli</i> chassis. The KYN biosensing component in the genetic module encodes a KYN pathway regulator (KynR) from <i>Pseudomonas aeruginosa</i>, driven by the <i>P_BAD</i> arabinose-inducible promoter. Upon expression, KynR would bind to the exogenous KYN and the bacterial responding <i>kyn</i> promoter to express the downstream green fluorescent protein gene to emit a fluorescence signal. However, despite successful induction by arabinose and the presence of KYN, biosensors with different gene orientations and genetic components failed to produce a significant fluorescence signal. These findings suggest that the sensitivity of <i>P. aeruginosa</i> KynR is insufficient to detect physiological levels of KYN. Further exploration of alternative biological sensing components is warranted.