Metabolic engineering of Escherichia coli strains for the in vivo phosphorylase-mediated synthesis of disaccharides.

Abstract

Background: Enzymatic synthesis of rare disaccharides by reverse-phosphorolysis is a potentially sustainable route to produce high-value glycosides for human health and nutrition. In vivo synthesis using microorganisms expressing recombinant glycoside phosphorylases is an attractive strategy, as it may overcome key limitations associated with conventional chemical and enzymatic approaches. However, successful implementation requires dedicated metabolic chassis enabling efficient precursor supply and carbon flux redirection. In Escherichia coli, inactivation of the phosphotransferase system (PTS) combined with deletion of pfkA constitutes a promising strategy to decouple sugar uptake from their concomitant phosphorylation, promote the accumulation of key sugar phosphates, and enable reverse biosynthetic pathways for oligosaccharide production.

Results: We first established β-1,2-mannobiose production as a proof of concept. The expression of heterologous permease GalP restored the growth on mannose in a PTS⁻ background and allowed mannose into the reverse biosynthetic pathway. Deletion of pfkA, which promotes intracellular accumulation of glucose 1-phosphate (G1P) and mannose 1-phosphate (M1P), combined with the expression of a β-1,2-mannoside-phosphorylase, enabled the direct synthesis of β-1,2-mannobiose from mannose. The pfkA deletion significantly increased product titers (> 0.6 g·L⁻¹) and yields (up to 9% g/g mannose), reflecting an efficient redistribution of carbon fluxes toward disaccharide formation. Furthermore, mixed-substrate cultures using glycerol as the carbon and energy source, together with additional metabolic engineering, enabled partial growth-production decoupling and redirected mannose utilization primarily toward product synthesis, achieving yields of up to 60%. The same metabolic chassis was further employed for the production of β-1,4-mannobiose and laminaribiose through the expression of a β-mannoside-phosphorylase and a laminaribiose-phosphorylase, using mannose and glucose as substrates, respectively.

Conclusion: Here, we report the metabolic engineering of Escherichia coli for in vivo production of oligosaccharides with different glycosidic linkages from hexose sugars. 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.