Development of a UDP-Glucose Regeneration Cascade Using Thermophilic Enzymes

In vitro enzymatic glycosylation is an attractive approach for stoichiometrically producing high-value glycosides from corresponding aglycons. One technical challenge in in vitro glycosylation is ensuring a continuous supply of nucleotide sugars, represented by UDP-glucose, which are used as glycosyl donors. Several in vitro UDP-glucose regeneration systems have been developed; however, these systems often suffer from their inherent limitations, including consumption of a large concentration of sacrificial substrates and the need for additional energy to regenerate UDP-glucose. In this study, we developed a novel UDP-glucose regeneration cascade based on the non-oxidative glycolysis, where phosphorolysis of maltodextrin serves as a source of energy and glucose moieties for UDP-glucose regeneration. The entire cascade is composed of thermostable enzymes, enabling one-step preparation of the enzymes by simple heat treatment. The reaction equilibrium in this cascade is more strongly biased toward UDP-glucose formation than those in conventional systems, and therefore, the addition of an excess concentration of sacrificial substrates can be omitted. As a proof of concept, we demonstrated trehalose synthesis from glucose using this cascade, achieving an aglycon-based conversion yield of 98.1 mol %.