Reversal of the Leloir pathway to promote galactose and tagatose synthesis from glucose

D-Tagatose is a low-calorie rare sugar with health benefits as a low-glycemic sweetener. Current production methods are limited, often relying on galactose isomerization, and remain inefficient and costly. Here, we report a whole-cell process in Escherichia coli that converts glucose directly to tagatose by reversing the Leloir pathway. Central to this approach is a galactose-1-phosphate-specific phosphatase that drives equilibrium toward galactose. Computational analyses reveal hydrogen-bond networks that underlie stringent substrate selectivity. By co-expressing this phosphatase with an L-arabinose isomerase in a metabolically engineered strain, we demonstrate direct glucose-to-tagatose conversion. Cultures produced ∼10.5 g/L galactose from 30 g/L glucose (35% yield) and >1 g/L tagatose. While this is a proof-of-principle demonstration and further optimization is required to improve tagatose production, this strategy eliminates dependence on lactose-derived galactose and provides a framework for scalable, glucose-based biosynthesis of tagatose and other galactose-derived molecules, supporting sustainable rare-sugar production.