Discovery of Antibacterial Piperazic Acid-containing Pseudopeptides from a <i>Saccharothrix</i> Strain Using Targeted Genetic Screening and Molecular Networking Strategies

Piperazic acid (Piz)-containing compounds are a distinctive class of microbial metabolites characterized by a unique N-N bond and diverse bioactivities, rendering them as promising scaffolds for drug discovery. Herein, we utilized an integrated discovery strategy combining PCR-based genetic screening, genome mining and MS/MS-based molecular networking to target Piz-containing metabolites from an actinomycetes library. This effort led to the isolation of nine new Piz-containing linear pseudopeptides, saccharothriotides A-I (<b>1</b>-<b>9</b>), along with the known compound Sch 382583 (<b>10</b>), from a desert-derived actinomycete <i>Saccharothrix</i> sp. 275. Their planar structures and absolute configurations were elucidated by spectroscopic analysis, advanced Marfey's method, phenylglycine methyl ester (PGME) derivatization, and biosynthetic pathway deduction. Bioactivity assays revealed compounds <b>1</b>-<b>3</b>, which feature a hydroxamic acid moiety, exhibited significant activity against Gram-positive pathogens (MIC = 0.5-16 μg/mL). Notably, antibacterial efficacy of <b>1</b>-<b>3</b> against vancomycin-resistant <i>Enterococcus faecium</i> (MIC = 2-4 μg/mL) markedly outperformed the antibiotic levofloxacin (MIC = 64 μg/mL). They also moderately inhibited Gram-negative bacteria such as <i>Escherichia coli</i> and <i>Acinetobacter baumannii</i> (MIC = 16-64 μg/mL). As the first Piz-containing metabolites reported from the genus <i>Saccharothrix</i>, this work underscores the effectiveness of combining genetic and metabolomic strategies for discovering bioactive natural products from underexplored microorganisms.