Dissecting native plasmid functions in Lactococcus lactis: Synergistic regulation of metabolism and industrially relevant traits

In Lactococcus lactis, the presence of native plasmids holds significant biological and practical value. They commonly harbor functional genes related to metabolism, antibiotic resistance, bacteriocin synthesis, and stress responses, thereby enabling the strain to adapt to diverse environmental conditions. A comprehensive investigation into the 3 native plasmids in L. lactis C20 was conducted to elucidate their functions, evolutionary origins, and effects on growth, metabolic activity, enantiomeric purity of L/D-lactate, and nisin biosynthesis. Comprehensive genomic analyses revealed that these plasmids, ranging from 2 kb to 258 kb and carrying genes implicated in mobile element activity, metabolic adaptation, and defense mechanisms, contribute significantly to the strain's genomic plasticity and environmental adaptability. Functional characterization of native plasmids pLL1, pLL2, and pLL3 demonstrated their collaborative regulation of key metabolic processes, with pLL2 in particular proving critical for maintaining robust growth and product formation. Combinatorial elimination further underscored the synergistic interplay among these plasmids, as evidenced by notable shifts in lactic acid yield, nisin titer, and L/D-lactate enantiomer distribution. These findings underscore the functional importance of native plasmids in L. lactis C20 and provide a biotechnological foundation for rational L. lactis engineering. Consequently, leveraging native plasmid diversity through synthetic biology and gene editing strategies holds significant promise for developing next-generation L. lactis in the food, pharmaceutical, and broader biotechnology industries.