Restoration of lactose metabolism in Lacticaseibacillus rhamnosus GG for dairy fermentation applications.

Lacticaseibacillus rhamnosus GG (LGG) is a widely used probiotic but has limited applications in dairy fermentation because of its poor lactose utilization. Here, we generated a lactose-positive LGG derivative using the LGG endogenous CRISPR-Cas system achieving a marker-free, scarless 20-bp deletion to disrupt the t1 transcriptional terminator upstream of the lactose operon. Using lactose-based selection, lactose-positive derivatives were then isolated and confirmed a restored functional lacG allele by sequencing. After curing the editing plasmid, the final strain, MJM570, was obtained. Whole-genome sequencing verified the intended terminator deletion and lacG reversion. MJM570 grew robustly on lactose, supported growth and acidification in milk, and showed improved proliferation and viability in yogurt fermentation and refrigerated storage compared with the wild-type LGG. This marker-free strategy leaves no foreign DNA in the final strain and provides a practical platform for developing dairy-compatible probiotic cultures with improved performance in fermented dairy applications. Overall, this study demonstrates the potential of endogenous CRISPR-Cas systems for food-relevant genome editing for developing dairy-compatible probiotic cultures.