Synergistic enhancement of nisin production through metabolic and regulatory engineering of Lactococcus lactis ATCC 11454: thermal and pH stability and application in food model system

Nisin is one of the most studied bacteriocins due to its broad antimicrobial spectrum and Generally Recognized as Safe status. In this study, nisRK was overexpressed in lactic acid-deficient Lactococcus lactis ATCC 11454, generating a recombinant Lactococcus lactis ATCC 11454 Δldh carrying pMG36e-P8-nisRK (ldhΔnisRKOE), to enhance nisin biosynthesis. Growth kinetics (OD600 monitoring) and antimicrobial activity agar (well diffusion assay) were compared among wildtype, ldh∆, nisRKOE, and double mutant strains. The ldhΔnisRKOE strain exhibited significantly higher antimicrobial activity than all other strains, with inhibition zones increased by up to 107% against Gram-positive and 78% against Gram-negative bacteria, compared with the wildtype. Thermal and pH stability tests showed that antimicrobial activity was well preserved at low temperatures (0-4 °C) and across a wide pH range (2-10), with optimal performance near pH 6. Activity declined by 26%-34% following heat treatment at 100-121 °C. Application in raw chicken demonstrated that CFS from the engineered strain slowed microbial growth and extended shelf life. After seven days at 4 °C, treated samples maintained lower bacterial counts (8.41 ± 0.02 log CFU/g) compared with controls (9.10 ± 0.07 log CFU/g). At 30 °C, untreated samples spoiled within 72 h, while treated meats remained stable until day 7. These findings highlight that combining metabolic redirection with regulatory overexpression can substantially improves nisin yield and antimicrobial activity. L. lactis ATCC 11454-ldhΔnisRKOE strain holds strong potential as a microbial cell factory for bacteriocin-based applications in food preservation and biotechnology.

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