Development of a Pre-Modification Strategy to Overcome Restriction–Modification Barriers and Enhance Genetic Engineering in Lactococcus lactis for Nisin Biosynthesis

Due to the barriers imposed by the restriction-modification (RM) system, Nisin-producing industrial strains of <i>Lactococcus lactis</i> often encounter low transformation efficiency, which seriously hinders the widespread application of genetic engineering in non-model <i>L. lactis</i>. Herein, we present a novel pre-modification strategy (PMS) coupled with optimized plasmid delivery systems designed to systematically evade RM barriers and substantially improve Nisin biosynthesis in <i>L. lactis</i>. Through the use of engineered <i>Escherichia coli</i> strains with methylation profiles specifically optimized for <i>L. lactis</i> C20, we have effectively evaded RM barriers, thereby facilitating the efficient introduction of large Nisin biosynthetic gene clusters into <i>L. lactis</i>. The PMS tools, which significantly improve the transformation efficiency (~10³ transformants per microgram of DNA), have been further improved in combination with a Rolling Circle Amplification, resulting in a higher enhancement in transformation efficiency (~10⁴ transformants per microgram of DNA). Using this strategy, large Nisin biosynthetic gene clusters and the expression regulation of all genes within the cluster were introduced and analyzed in <i>L. lactis</i>, leading to a highest Nisin titer of 11,052.9 IU/mL through a fed-batch fermentation in a 5 L bioreactor. This is the first systematic report on the expression regulation and application of a complete Nisin biosynthesis gene cluster in <i>L. lactis</i>. Taken together, our studies provide a versatile and efficient strategy for systematic evasion and enhancement of RM barriers and Nisin biosynthesis, thereby paving the way for genetic modification and metabolic engineering in <i>L. lactis</i>.