Engineering Lactose Utilization in Lacticaseibacillus rhamnosus GG for Lactic Acid Production from Milk Permeate Waste

Dairy processing generates large volumes of lactose-rich permeate whose disposal can be costly. Lacticaseibacillus rhamnosus GG (LGG), a widely used probiotic strain, grows poorly on lactose, limiting the potential to valorize this stream. An endogenous Cas9-based genome-editing system successfully enabled adhE deletion and was subsequently used for strain engineering. Partial deletion of the lactose operon t1 terminator was followed by selection in MRS–lactose medium. Growth curves of 75 mutants were assessed; the ten with the highest maximum growth rates (MaxV) were sequenced and further adapted to lactose. Two were tested on glucose, lactose, and treated milk permeate. Subsequent pta deletion reduced acetate but delayed growth, while plasmid-borne ldhL1 overexpression provided no final lactate advantage. In 2 mL tube fermentations, LGGm produced 40.71 ± 2.09 g/L lactic acid within 24 h, versus 2.14 ± 0.18 g/L for wild-type LGG. The apparent yield was 1.02 g/g initial lactose, with approximately 98% L-lactate. These findings demonstrate the potential of engineered LGG for lactic acid production in MRS-supplemented dairy permeate at laboratory scale, providing a basis for further evaluation with reduced supplementation and larger fermentation volumes.