Expanding salivary biomarker detection by creating a synthetic neuraminic acid sensor via chimeragenesis

to detect target molecules of interest. Detection of these analytes via minimally invasive sampling of biological fluids such as saliva will improve early detection and prognosis for a multitude of conditions. Our target compound is N-acetylneuraminic acid (Neu5Ac), associated with dysbiosis, inflammation, and oral squamous cell carcinoma. In this work, we present biosensor strain MG-Sphnx expressing the custom-made chimeric transcription factor Sphnx (LacI-LNK2-SiaP), which, upon binding Neu5Ac at physiopathological concentrations, enables the expression of a fluorescent reporter. We describe the construction and testing of a collection of chimeric transcriptional repressors, assay their functionality, characterize Sphnx among them, and test it with contrived samples. We also compared models of the 3D structure of functional and non-functional chimeras to widen our knowledge on the behavior of modular proteins. We expect these results to become the foundation of a platform for the screening of a panel of biomarkers with an array of WCBs, bringing attention to their potential for point-of-care and at-home diagnostics. IMPORTANCE: Whole-cell biosensors based on transcription factors are powerful tools with an enormous potential for point-of-care diagnostics, especially when designed to detect biomarkers from easy-to-extract biological fluids such as saliva. Detection of dysbiosis and cancer salivary biomarker N-acetylneuraminic acid (Neu5Ac) will improve patient prognosis. A common problem when designing biosensors for compounds such as Neu5Ac is the lack in the literature of a transcription factor capable of detecting the analyte of interest. Here, we describe a straightforward approach for the creation of a whole-cell biosensor based on a synthetic allosteric transcription factor capable of detecting Neu5Ac. We provide a scaled-down, affordable pipeline for the construction of a candidate library; we also characterize a candidate transcription factor and validate it with contrived salivary samples. Finally, we compare a functional and a non-functional synthetic transcription factor from our collection, hypothesizing on what are the structural differences influencing their functionality.