Engineering of Escherichia coli for D-Tagatose Production from Lactose and Whey Permeate via the Tagatose-6-Phosphate Pathway

D-tagatose is a low-calorie natural rare sugar with significant potential in the food and pharmaceutical industries. Conventional production relies on the enzymatic isomerization of D-galactose, a process limited by an unfavorable thermodynamic equilibrium and high substrate costs. This study presents a novel whole-cell biocatalytic approach for direct production of D-tagatose from lactose, an inexpensive and abundant sugar in dairy waste streams, such as whey permeate. The main lactose permease (lacY) of Escherichia coli BL21(DE3) was deleted, creating a clean host chassis that, due to its native galactose auxotrophy, was incapable of utilizing galactose. Subsequently, genes from the tagatose-6-phosphate (T6P) pathway of Lactococcus lactis , comprising a lactose-specific phosphotransferase system (PTS), a 6-phospho-β-galactosidase, a galactose-6-phosphate isomerase and the general PTS proteins, were modularly introduced, which allowed for the functional validation of each component. The crucial final step involved the expression of a sugar phosphatase to convert the intracellular intermediate, tagatose-6-phosphate, into D-tagatose. The fully engineered strain yielded a 35% conversion ratio of the galactose moiety of lactose to tagatose. This demonstrates the repurposing of the tagatose-6-phosphate pathway in E. coli to enable direct lactose utilization and growth-coupled production of D-tagatose from both lactose and whey permeate, without prior hydrolysis or substrate enrichment. Fermentation and tagatose production using whey permeate as the sole carbon source was also demonstrated, highlighting the potential of this system for the valorization of dairy byproducts into a high-value low calorie sweetener. • Synthetic biology was used to engineer an E. coli auxotroph for the efficient conversion of lactose into the rare sugar D-tagatose. • The final SynTag pathway establishes a growth-coupled bioprocess • This approach successfully valorizes low-cost dairy side-streams by utilizing whey permeate as the sole carbon source • Modular assembly of the Tagatose-6-phosphate pathway and a specific sugar phosphatase was crucial for mitigating toxicity from phosphorylated intermediates and enabling tagatose synthesis.