Construction and Application of a High-Throughput In Vivo Screening Platform for the Evolution of Nitrile Metabolism-Related Enzymes Based on a Desensitized Repressive Biosensor.

Transcription factor (TF)-based biosensors are expected to serve as powerful tools for the high-throughput screening of biocatalytic systems; however, most of them respond to ligands in a narrow concentration range, which limits their application. In this study, we constructed a heterogenous niacin biosensor using the repressive TF BsNadR and its target promoters from Bacillus subtilis . The fine-tunable output of the niacin biosensor was expanded to a wide range of niacin concentrations (0-50 mM) through desensitization engineering, which was suitable for the accurate identification of differences in enzyme activity. Structural mechanism analysis indicated that weakening the affinity of BsNadR with the ligand niacin and with DNA alters its regulatory properties. Based on the desensitized niacin biosensor, a high-throughput in vivo screening platform was developed for evolving nitrile metabolism-related enzymes. The evolved nitrilase, amidase, and nitrile hydratase with 6.6-, 2.1-, and 21.3-fold improvements in activity were achieved, respectively. In addition, these mutants also exhibited elevated activity toward other cognate substrates, indicating the broad applicability of the screening platform. This study not only provided a universal high-throughput screening platform for different nitrile metabolism-related enzymes but also demonstrated the advantages of repressive biosensors and the vital role of desensitization engineering of the TF in the development of high-throughput screening platforms for enzymes.