Tedesco, P.; Durand, J.; Tarquis, L.; Potocki-Veronese, G.; Letisse, F.

Enzymatic synthesis of rare disaccharides by reverse-phosphorolysis is a potentially sustainable route to produce high-value glycosides for human health and nutrition. We report the metabolic engineering of Escherichia coli for in vivo production of {beta}-mannobiose with different osidic linkages from hexose sugars. We demonstrate production of {beta}-1,2-mannobiose with this approach as proof of concept. Phosphotransferase system (PTS) inactivation enables import of non-phosphorylated mannose via heterologous permease GalP, restoring growth on mannose in a PTS- background and allowing mannose into the reverse biosynthetic pathway. Deletion of pfkA, which promotes intracellular accumulation of key sugar phosphates (G1P, M1P), establishes a favorable metabolic chassis for oligosaccharide production using glycoside-phosphorylases. Using this chassis, we expressed two beta-mannoside phosphorylases to enable the direct production of {beta}-1,2- and {beta}-1,4-mannobiose from mannose. The same chassis was also employed for laminaribiose production through the expression of a laminaribiose-phosphorylase. pfkA deletion significantly increased product titer (> 0.6 g.L-1) and yield (up to 9% g/g mannose), highlighting a favorable redistribution of carbon fluxes toward disaccharide formation. Moreover, a combination of mixed-substrate cultures using glycerol as carbon and energy source and further metabolic engineering enabled partial growth-production decoupling, redirecting mannose utilization primarily toward product synthesis, with a yield of 60%. These results demonstrate the modularity and efficiency of the proposed platform for fermentative production of non-conventional oligosaccharides and expand the scope of metabolic engineering strategies for glycoside biosynthesis.