A Novel Fluorescence Resonance Energy Transfer-Based High-Throughput Screening Method for Generation of Lysozyme with Improved Antimicrobial Activity against Escherichia coli Strains

Lysozyme has emerged to be a promising alternative to traditional antibiotics to deal with the increasing antibiotic resistance of bacteria. However, its application is hampered by its inferior bactericidal activity against Gram-negative bacteria. To address this problem, a novel "enzyme-cascade fluorescent high-throughput screening (HTS) method" was designed and constructed based on detection of fluorescence resonance energy transfer (FRET) and enzyme-cascade reaction of lysozyme and protease. As a proof of concept, site-saturation mutagenesis libraries targeting at residues of the unstructured stretch at the N-terminus of Antheraea pernyi lysozyme were constructed and screened by the proposed HTS method. The isolated lysozyme variants proved to exhibit higher antibacterial activity against Escherichia coli K12, demonstrating the significance of this region for the bactericidal function of lysozyme. The presented cell-based fluorescent HTS method is a new tool for screening lysozyme variants with improved bactericidal efficacy against Gram-negative bacteria and for exploring the sequence-structure-function relationship of lysozyme.