biostudies-literatureGrafskaia ERussian Science Foundation1120https://www.ebi.ac.uk/biostudies/studies/S-EPMC12649535biostudies-literatureUnknown14(11)<b>Background/Objectives</b>: The global rise in antibiotic resistance necessitates the development of novel antimicrobial agents. Antimicrobial peptides (AMPs), key components of innate immunity, are promising candidates. This study aimed to develop novel therapeutic peptides with enhanced properties through the mutagenesis of natural AMPs and high-throughput screening. <b>Methods</b>: We constructed mutant libraries of three broad-spectrum AMPs-melittin, cecropin, and Hm-AMP2-using mutagenesis with partially degenerate oligonucleotides. Libraries were expressed in <i>Escherichia coli</i>, and antimicrobial activity was assessed through bacterial growth kinetics and droplet serial dilution assays. Candidate molecules were identified by DNA sequencing, and the most promising variants were Antibiotics (Basel, Switzerland)Development of New Antimicrobial Peptides by Directional Selection.PMC1264953523-15-00084Bobrovsky PManuvera VSerebrennikova MKharlampieva DGrafskaia EBrovina KAlieva SSelezneva OLazarev VBessonova EfalseDevelopment of New Antimicrobial Peptides by Directional Selection.<b>Background/Objectives</b>: The global rise in antibiotic resistance necessitates the development of novel antimicrobial agents. Antimicrobial peptides (AMPs), key components of innate immunity, are promising candidates. This study aimed to develop novel therapeutic peptides with enhanced properties through the mutagenesis of natural AMPs and high-throughput screening. <b>Methods</b>: We constructed mutant libraries of three broad-spectrum AMPs-melittin, cecropin, and Hm-AMP2-using mutagenesis with partially degenerate oligonucleotides. Libraries were expressed in <i>Escherichia coli</i>, and antimicrobial activity was assessed through bacterial growth kinetics and droplet serial dilution assays. Candidate molecules were identified by DNA sequencing, and the most promising variants were 2025-01-01T00:00:00Z2025 Nov2026-05-19T03:14:26.549Z2026-05-19T03:11:12.652ZS-EPMC126495354130161510.3390/antibiotics14111120