Shelf-life extension of lettuce using cell-penetrating peptide-peptide nucleic acid conjugates targeting Sphingomonas paucimobilis

Sphingomonas is a dominant bacterial genus within the indigenous microbiota of plant-derived foods. S. paucimobilis, a food spoilage species and potential human pathogen, is frequently found on plant surfaces, in soils, and in freshwater environments, and raises food safety concerns. Conventional preservation strategies are often insufficient for the selective control of specific spoilage-associated microorganisms without disrupting the surrounding microbiota, underscoring the need for precision-targeted spoilage control approaches. Here, we present a peptide nucleic acid (PNA)-based antimicrobial strategy that employs antisense inhibition for selective suppression of S. paucimobilis. To enhance their cellular uptake, PNAs were conjugated with cell-penetrating peptides (CPPs), and several candidates were compared to identify the most effective delivery system. Among the tested CPPs, (KFF)₃K-PNA conjugates exhibited the strongest antimicrobial activity. PNAs targeting the essential genes gyrA and rpoA showed potent sequence-specific activity, with (KFF)₃K-gyrA PNAs demonstrating greater efficacy (≥0.5 μM) than (KFF)₃K-rpoA PNAs (≥2 μM), whereas mismatch conjugates showed no detectable effect. Quantitative transcriptional analysis confirmed significant downregulation of target genes, particularly gyrA, after treatment. Application of (KFF)₃K-gyrA PNA (2 μM) to lettuce significantly reduced discoloration and suppressed S. paucimobilis growth. These results highlight the high target specificity and antimicrobial efficacy of (KFF)₃K-PNA conjugates and suggest their potential use as precision biocontrol tools for enhancing the microbial stability and shelf life of plant-derived foods.