Ahsan_2024_Synth.Biol.(Oxf)_9_ysae005

Cell-free expression (CFE) has shown recent utility in prototyping enzymes for discovery efforts. In this work, CFE is demonstrated as an effective tool to screen putative polyester polyurethane degrading enzyme sequences sourced from metagenomic analysis of biofilms prospected on aircraft and vehicles. An automated fluid handler with a controlled temperature block is used to assemble the numerous 30 microL CFE reactions to provide more consistent results over human assembly. In sum, 13 putative hydrolase enzymes from the biofilm organisms as well as a previously verified, polyester-degrading cutinase were expressed using in-house E. coli extract and minimal linear templates. The enzymes were then tested for esterase activity directly in extract using nitrophenyl conjugated substrates, showing highest sensitivity to shorter substrates (4-nitrophenyl hexanoate and 4-nNitrophenyl valerate). This screen identified 10 enzymes with statistically significant activities against these substrates; however, all were lower in measured relative activity, on a CFE volume basis, to the established cutinase control. This approach portends the use of CFE and reporter probes to rapidly prototype, screen and design for synthetic polymer degrading enzymes from environmental consortia. Graphical Abstract.