Functional production of recombinant lysostaphin in Nicotiana benthamiana: A sustainable antimicrobial strategy against drug-resistant Staphylococcus aureus.

Plant-based expression platforms are emerging as a cost-effective and scalable alternative to traditional microbial and mammalian expression systems for producing protein-based biotherapeutics. Plant-based systems offer several advantages, including lower production costs, a reduced risk of contamination with human pathogens, and simplified scale-up processes. In this study, we focused on the transient expression and purification of lysostaphin, a class III bacteriocin with potent antimicrobial activity against Staphylococcus aureus. Lysostaphin targets the pentaglycine cross-bridges in the peptidoglycan layer of staphylococcal cell walls via glycyl-glycine endopeptidase activity, making it a valuable candidate for combating antimicrobial-resistant infections. Codon optimized lysostaphin was expressed in Nicotiana benthamiana, fused with a deca-histidine tag and a TEV protease site to facilitate purification. The peak expression was observed at six days post-infiltration, yielding approximately 907 mg/kg of fresh leaf tissue, which surpasses conventional bacterial yield (442.26 mg/L). Moreover, size exclusion chromatography (SEC) analysis revealed that the plant-derived lysostaphin existed in a monomeric state like its commercial counterpart, and it exhibited functional activity comparable to commercial lysostaphin, as demonstrated by well diffusion, inhibition in microbroth dilution and biofilm disruption assays. These results support the potential of plant-based systems to produce functional antimicrobials for use in both medical and food sectors.