Abstract
d-Tagatose, a rare low-calorie sweetener, is ideal for beverages due to its high solubility and low viscosity. Current enzymatic production methods from d-galactose or d-galactitol are limited by reaction reversibility, affecting the yield and purity. This study demonstrates that Escherichia coli harbors a thermodynamically favorable pathway for producing d-tagatose from d-glucose via phosphorylation-epimerization-dephosphorylation steps. GatZ and KbaZ, annotated as aldolase chaperones, exhibit C4 epimerization activity, converting d-fructose-6-phosphate to d-tagatose-6-phosphate. Structural analysis reveals active site differences between these enzymes and class II aldolases, indicating functional divergence. By exploiting the strains' inability to metabolize d-tagatose, carbon starvation was applied to remove sugar byproducts. The engineered strains converted 45 g L(-1) d-glucose to d-tagatose, achieving a titer of 7.3 g L(-1) and a productivity of 0.1 g L(-1) h(-1) under test tube conditions. This approach highlights E. coli as a promising host for efficient d-tagatose production.