Cell-free expression for enzyme discovery: Screening putative polyester polyurethane degrading enzymes with semi-automated cell free expression and nitrophenyl probes

In recent years, Cell-free expression (CFE) has emerged as a valuable tool for the rapid prototyping of enzymes in discovery efforts. This study focuses on demonstrating the effectiveness of CFE in screening potential polyurethane degrading enzyme sequences obtained from biofilm samples collected from aircraft, followed by genomic analysis. To streamline the process and ensure consistent results, an automated fluid handling system using a low-cost Opentrons instrument was employed. This automated system allowed for the assembly of multiple 30 µL reactions in the screening process, while maintaining precise temperature control. A benchmarking comparison was conducted between the automated process and manual operation, revealing greater consistency and reliability with the automated protocol, particularly when incorporating pipette tip changes. In this study, a total of 13 putative hydrolase enzymes sourced from aircraft biofilm organisms, along with the previously validated polyester-degrading cutinase, were expressed using in-house E. coli extract and minimal linear templates. The expressed enzymes were then tested for activity directly within the extract, with the hydrolysis of nitrophenyl substrates measured through absorbance readings. The highest absorbance changes were observed for 4-Nitrophenyl Hexanoate and 4-Nitrophenyl Valerate. Through this screening process, two of the putative enzymes exhibited statistically significant activity above the baseline. This demonstrates the potential of CFE in conjunction with automated fluid handling to rapidly prototype and screen for polymer-degrading enzymes derived from environmental consortia. These findings signify the promising application of CFE and automated systems in the efficient identification and characterization of enzymes with the ability to degrade polymers.