| Autor: |
Soudier P; Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78352 Jouy-en-Josas, France.; Université de Montpellier, INSERM, CNRS, Centre de Biologie Structurale, 34090 Montpellier, France., Zúñiga A; Université de Montpellier, INSERM, CNRS, Centre de Biologie Structurale, 34090 Montpellier, France., Duigou T; Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78352 Jouy-en-Josas, France., Voyvodic PL; Université de Montpellier, INSERM, CNRS, Centre de Biologie Structurale, 34090 Montpellier, France., Bazi-Kabbaj K; Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78352 Jouy-en-Josas, France., Kushwaha M; Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78352 Jouy-en-Josas, France., Vendrell JA; Laboratoire de Biologie des Tumeurs Solides, Département de Pathologie et Oncobiologie, CHU Montpellier, Université de Montpellier, 34295 Montpellier, France., Solassol J; Laboratoire de Biologie des Tumeurs Solides, Département de Pathologie et Oncobiologie, CHU Montpellier, Université de Montpellier, 34295 Montpellier, France.; IRCM, INSERM, Univ Montpellier, ICM, 34298 Montpellier, France., Bonnet J; Université de Montpellier, INSERM, CNRS, Centre de Biologie Structurale, 34090 Montpellier, France., Faulon JL; Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78352 Jouy-en-Josas, France. |
| Abstrakt: |
Cell-free systems have great potential for delivering robust, inexpensive, 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 cell-free detection of different metabolites. H 2 O 2 is a central metabolite and a byproduct 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 a cell-free system, highlighting the need for preincubation 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 retrosynthesis workflow publicly available on the SynBioCAD Galaxy portal. We find that expressing the transducer during the preincubation step is crucial for optimal sensor operation. We then show that different reporters can be connected to PeroxiHUB, providing high adaptability for various applications. Finally, we demonstrate that a peroxiHUB lactate biosensor can detect endogenous levels of this metabolite in clinical samples. 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. |
