Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious threat to public health and can form biofilms to enhance its drug resistance. Caffeic acid phenethyl ester (CAPE), which is primarily extracted from propolis, possesses diverse biological activities. However, its effect on anti-MRSA biofilms and the relevant mechanisms have not been fully clarified. Therefore, this study explored the ability of CAPE to combat MRSA biofilms. The results showed that CAPE has significant antibiofilm activities against MRSA. The minimum inhibitory concentration (MIC) values of CAPE were 256 µg/mL for the MRSA strains ATCC 33591, CI2, and CI3. Crystal violet (CV) assay and XTT assays demonstrated that CAPE could inhibit the formation and consolidation of MRSA CI2 biofilms. Experiments on scanning electron microscopy (SEM), bacterial adhesion assays, and the levels of extracellular polysaccharides confirmed that CAPE can inhibit bacterial adhesion, as well as the synthesis of extracellular polysaccharides in MRSA CI2. Real-time quantitative PCR (RT-qPCR) experiments confirmed that CAPE can affect the expression of MRSA icaADBC, sarA, fnbAB, and clfAB genes. Therefore, the proposed antibiofilm mechanism of CAPE involves the downregulation of aforementioned genes, leading to reduced production of extracellular polysaccharides and adhesion-related proteins, thereby weakening MRSA adhesion and ultimately exerting an antibiofilm effect. In conclusion, these findings suggest CAPE is a promising candidate drug as an antimicrobial agent for managing and preventing biofilm-associated infections caused by MRSA.

Carcinine is an imidazole dipeptide with potent antioxidant and antiglycation properties, although its chemical synthesis currently relies on severely environmentally harmful processes. In this work, a multi-enzyme cascade biotransformation system comprising 4′-phosphopantetheinyl transferase and non-ribosomal peptide synthetase was constructed. To overcome the limitations arising from stochastic spatial distribution and suboptimal mass transfer associated with independent enzymes, a fusion protein strategy was adopted. The two enzymes were fused via a flexible genetic linker within plasmid pET28a-SFP-L-Ebony, which enabled robust soluble expression in Escherichia coli. Concurrently, the endogenous peptidase genes (pepA, pepB, pepD, and pepN) were systematically knocked out using CRISPR/Cas9-mediated gene editing. This quadruple protease-deficient strain (designated SFP-L-Ebony-ΔpepABDN) effectively suppressed product degradation. Subsequent optimization revealed that optimal catalytic performance occurred at 25 °C and pH 7.0. The highest biotransformation efficiency was achieved using 15 g/L crude enzymes, in the presence of 2 mM ATP and 10 mM MgCl₂. Through a fed-batch substrate feeding strategy in a 50 mL reaction system, the final carcinine titer reached 7.0 g/L after 48 h. This study, therefore, provides an efficient and sustainable technological pathway for the green biomanufacturing of carcinine as well as other high-value dipeptides.