An alr-based antibiotic-marker-free platform reveals distinct fermentation and metabolic responses to T6P- and Leloir-associated lactose utilization modules in Lacticaseibacillus paracasei Zhang

Lactose utilization is an important determinant of the growth and fermentation performance of lactic acid bacteria in dairy environments. Whether different lactose-entry routes that restore this capability produce equivalent fermentation outcomes remains unclear. Here, the tagatose-6-phosphate (T6P)-associated lacTEGF module and the Leloir-associated lacS-lacLM module were compared in the same Lacticaseibacillus paracasei Zhang background using an alr -based antibiotic-marker-free complementation system. Both modules restored lactose-supported growth, lactose consumption, and acidification, and the corresponding enzyme-activity profiles provided functional evidence of module activity. However, the engineered strains exhibited distinct fermentation phenotypes. Zhang-T6P showed slightly higher acidification and lactose-consumption rates in MRS-lactose medium and greater titratable acidity in reconstituted skim milk, with a tendency toward shorter coagulation time. Transcriptomic and metabolomic analyses revealed shared responses together with module-associated differences. Zhang-Leloir showed pronounced induction of endogenous galactose PTS components and broader changes in nucleotide-, cofactor-, organic-acid-, and amino-sugar-related metabolism. These findings indicate that restoration of lactose utilization can be accompanied by distinct downstream physiological and metabolic responses associated with the introduced module. This highlights the value of phenotype-oriented evaluation when selecting lactose-utilization strategies for dairy-relevant lactic acid bacteria.