Abstract
Cell-free systems have great potential for delivering robust, cheap, and field-deployable biosensors. Many cell-free biosensors rely on transcription factors responding to small molecules, but their discovery and implementation still remain challenging. Here we report the engineering of PeroxiHUB, an optimized H 2 O 2-centered sensing platform supporting cellfree detection of different metabolites. H 2 O 2 is a central metabolite and a by-product of numerous enzymatic reactions. PeroxiHUB uses enzymatic transducers to convert metabolites of interest into H 2 O 2 , enabling rapid reprogramming of sensor specificity using alternative transducers. We first screen several transcription factors and optimize OxyR for the transcriptional response to H 2 O 2 in cell-free, highlighting the need for pre-incubation steps to obtain suitable signal-to-noise ratios. We then demonstrate modular detection of metabolites of clinical interest-lactate, sarcosine, and choline-using different transducers mined via a custom retro-synthesis workflow publicly available on the SynBioCAD Galaxy portal. We find that expressing the transducer during the pre-incubation step is crucial for optimal sensor operation. Finally, we show that different reporters can be connected to PeroxiHUB, providing high adaptability for various applications. Given the wide range of enzymatic reactions producing H 2 O 2 , the PeroxiHUB platform will support cell-free detection of a large number of metabolites in a modular and scalable fashion.