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

• DOI: 10.1021/acssynbio.1c00642
• Publication Date: 2022-03-10T13:32:00Z
• Authors: Laichuang Han; Xinyue Liu; Zhongyi Cheng; Wenjing Cui; Junling Guo; Jian Yin; Zhemin Zhou
• Keywords: High-Throughput Screening; Amidase; Niacin

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