Directed Evolution Boosts UGT85A1 Glycosylation for Enhanced Phenylethanoid Glucoside Production

Area of Science:

• Biotechnology
• Metabolic Engineering
• Enzyme Engineering

Background:

• Glycosides are vital plant compounds with significant food and pharmaceutical potential.
• Low natural abundance of glycosides like salidroside hinders large-scale production.
• Microbial biosynthesis presents a sustainable alternative, contingent on efficient glycosyltransferases.

Purpose of the Study:

• To engineer a highly efficient glycosyltransferase for enhanced microbial production of phenylethanoid glycosides.
• To develop a robust platform for the biosynthesis of high-value plant metabolites.

Main Methods:

• Iterative saturation mutagenesis (ISM) was employed to engineer the UGT85A1 enzyme.
• The high-activity mutant DHG (G114D/F217H/C128G) was identified and characterized.
• Molecular dynamics simulations were used to analyze the structural basis of enhanced enzyme activity.
• DHG was expressed in yeast strains engineered for tyrosol and hydroxytyrosol production.

Main Results:

• The engineered mutant DHG exhibited a 23.6-fold increase in catalytic efficiency compared to the wild-type UGT85A1.
• Molecular dynamics revealed stabilized substrate binding and optimized catalytic geometry in DHG.
• DHG enabled the production of salidroside at 2.70 g/L and hydroxysalidroside at 1.63 g/L in engineered yeast.
• These titers represent the highest reported for these compounds in Saccharomyces cerevisiae under shake-flask conditions.

Conclusions:

• Engineering plant glycosyltransferases, such as UGT85A1, can significantly enhance microbial glycoside production.
• The mutant DHG provides a highly efficient biocatalyst for salidroside and hydroxysalidroside biosynthesis.
• This study establishes a scalable platform for the sustainable production of valuable glycosides through yeast metabolic engineering.