One-pot biosynthesis of gastrodin using UDP-glycosyltransferase itUGT2 with an in situ UDP-glucose recycling system.

Gastrodin, the major effective ingredient in Tianma (Gastrodia elata), is a p-hydroxybenzoic acid derivative with various activities. Gastrodin has been widely investigated for food and medical applications. The last biosynthetic step for gastrodin is UDP-glycosyltransferase (UGT)-mediated glycosylation with UDP-glucose (UDPG) as glycosyl donor. In this study, we performed a one-pot reaction both in vitro and in vivo to synthesize gastrodin from p-hydroxybenzyl alcohol (pHBA) by coupling UDP-glucosyltransferase from Indigofera tinctoria (itUGT2) to sucrose synthase from Glycine max (GmSuSy) for regeneration of UDPG. The in vitro results showed that itUGT2 transferred a glucosyl group to pHBA to generate gastrodin. After 37 UDPG regeneration cycles with 2.5% (molar ratio) UDP, the pHBA conversion reached 93% at 8 h. Furthermore, a recombinant strain with itUGT2 and GmSuSy genes was constructed. Through optimizing the incubation conditions, a 95% pHBA conversion rate (220 mg/L gastrodin titer) was achieved in vivo without addition of UDPG, which was 2.6-fold higher than that without GmSuSy. This in situ system for gastrodin biosynthesis provides a highly efficient strategy for both in vitro gastrodin synthesis and in vivo biosynthesis of gastrodin in E. coli with UDPG regeneration.