Construction of a novel UDP-rhamnose regeneration system by a two-enzyme reaction system and application in glycosylation of flavonoid.

Graphical abstract Highlights • A NADPH-independent fusion enzyme (VvRHM-NRS) was constructed. • VvRHM-NRS could convert UDP-glucose to UDP-rhamnose with NADH self-sufficient. • VvRHM-NRS was coupled with GmSUS to develop a UDP-rhamnose regeneration system. • A total of 0.57 mM UDP-rhamnose was generated from sucrose, NAD+, and UDP. Abstract UDP-rhamnose is synthesized by UDP-rhamnose synthase, but the reaction requires two kinds of cofactors, NAD+ and NADPH. In this work, a cofactor self-sufficient UDP-rhamnose regeneration system was described herein. The UDP-rhamnose synthase (VvRHM) gene from Vitis vinifera was cloned and expressed in Escherichia coli and characterizations of VvRHM were determined. The N-terminal region of VvRHM was fused with the bifunctional UDP-4-keto-6-deoxy- d -glucose 3,5-epimerase/UDP-4-keto-rhamnose 4-keto-reductase (NRS/ER) from Arabidopsis thaliana to obtain the fusion enzyme (VvRHM-NRS), which was NADPH-independent and could convert UDP-glucose to UDP-rhamnose with NADH self-sufficient. The optimal activity of VvRHM-NRS was at pH 7.5 and 30 °C. Apparent K m and V max of VvRHM-NRS for UDP-glucose were 88 ± 9 μM and 12.7 ± 0.6 nmol/min/mg, and for NAD+ were 69 ± 7 μM and 11.9 ± 0.5 nmol/min/mg. Then, a novel process for the synthesis of UDP-rhamnose from sucrose, NAD+, and UDP was developed by using VvRHM-NRS coupled with Glycine max sucrose synthase (GmSUS). By optimizing coupled reaction conditions, UDP-rhamnose production reached 0.57 mM. Finally, UDP-rhamnose regeneration system was tested to synthesize quercitrin with A. thaliana glycosyltransferase (AtUGT78D1). Therefore, VvRHM-NRS coupled with GmSUS presented a green chemistry approach for the UDP-rhamnose regeneration in glycosylation of flavonoids and other bioactive substances. [ABSTRACT FROM AUTHOR]