Abstract
Hydroxysalidroside is an important natural phenylethanoid glycoside with broad application prospects in the food and pharmaceutical fields. However, its low concentration in plants and complex extraction hinder its production. Despite being a promising way to synthesize hydroxysalidroside in Escherichia coli, glycosylation remains the limiting factor for its production. A de novo biosynthetic pathway for hydroxysalidroside was successfully constructed in E. coli via the screening of glycosyltransferase, overexpressing phosphoglucomutase (pgm) and UDP-glucose pyrophosphorylase (galU) to ensure a sufficient supply of UDP-glucose (UDPG). Additionally, a semi-rational design of UGT85A1 was conducted to expand the acceptor-binding pocket to eliminate steric hindrance interfering with the binding of hydroxytyrosol. The endogenous genes ushA and otsA were knocked out to further reduce the consumption of UDPG. Finally, a titer of 5837.2 mg/L was achieved in a 5 L fermenter by optimizing the feeding times of carbon sources. This laid the foundation for the subsequent biosynthesis of phenylethanoid glycosides.