A Modular Biosensor Platform for the Detection of Plastic Monomers and the Engineering of Promiscuous Amidases Toward Challenging Substrates

Stable chemical bonds dictate the properties of industrial chemicals and materials. Particularly the persistence of synthetic polymers like polyurethanes (PUs) contributes to the global issues of waste accumulation and environmental pollution. To accelerate the discovery and engineering of plastic-degrading biocatalysts, a genetically encoded biosensor platform is established that enzymatically converts polyfunctionalized monomers into (aliphatic) aldehydes and allows their robust detection by a bacterial luciferase. In vivo, in vitro, and hybrid applications of the investigated biosensor system facilitate the bioluminescence-based assessment of the promiscuous esterase, amidase, and urethanase activity of amidase signature family enzymes, circumventing chromatographic analysis. Furthermore, the biosensor platform guides the selection of improved variants in a site-saturated enzyme library, exhibiting up to 5.5-fold enhanced activity toward difficult to hydrolyze screening molecules, including N-substituted decanamides, a representative polyether dicarbamate, and a commercial polyester-PU. The latter contain polyols like diethylene glycol, for which biosensor applications are scarce. Hence, this biosensor platform is not only the first to enable the monitoring of amidase and urethanase activity independent of chromogenic/fluorogenic molecules in real-time. It expands the detection scope of bacterial luciferases toward plastic monomers like polyols, which will aid advancing current recycling strategies for PU waste and beyond.