Efficient production of D-tagatose via DNA scaffold mediated oxidoreductases assembly in vivo from whey powder

Among the emerging sweeteners, D-tagatose occupies a significant niche due to its low calorific value, antidiabetic property and growth promoting effects on intestinal probiotics. Recently, the main approach for D-tagatose biosynthesis is L-arabinose isomerase-based isomerization reaction from galactose, which shows relatively low conversion rate because of unfavorable thermodynamic equilibria. Herein, oxidoreductases, D-xylose reductase and galactitol dehydrogenase, together with endogenous β-galactosidase were employed to catalyze the biosynthesis of D-tagatose from lactose with a yield of 0.282 g/g in Escherichia coli. Then, a deactivated CRISPR-associated (Cas) proteins-based DNA scaffold system was developed, which were proved to be efficient for assembling the oxidoreductases in vivo and got a 1.44-folds increase in D-tagatose titer and yield. Further, by employing D-xylose reductase with higher galactose affinity and activity, as well as overexpressing pntAB genes, the D-tagatose yield from lactose (0.484 g/g) increased to 92.0 % of the theoretical value, 1.72-times as that of original strain. Finally, whey powder, a lactose-rich food by-product, was bifunctionally utilized as an inducer and substrate. In the 5 L bioreactor, D-tagatose titer reached 32.3 g/L with little galactose detected, and the yield from lactose approached 0.402 g/g, which was the highest from waste biomass in the literature. The strategies used here might provide new insights into the biosynthesis of D-tagatose in future.