Highly Efficient Biosynthesis of Rebaudioside M9 through Enzyme Screening and Structure-Guided Engineering

Area of Science:

• Biochemistry
• Enzymology
• Food Science

Background:

• Steviol glycosides (SGs) are natural sweeteners with limitations in taste and solubility.
• Glycosylation modification is a key strategy to improve SG properties.
• Rebaudioside M2 (Reb M2) is a precursor for enhanced SG production.

Purpose of the Study:

• To identify and engineer enzymes for improved SG glycosylation.
• To produce a novel SG, rebaudioside M9 (Reb M9), with enhanced properties.
• To develop an efficient enzymatic system for Reb M9 synthesis.

Main Methods:

• Phylogenetic analysis and enzyme screening to identify UGT94E13.
• Structure-guided enzyme engineering to enhance UGT94E13 activity.
• Construction and optimization of an enzymatic cascade system.

Main Results:

• Identified UGT94E13 catalyzing the production of novel Reb M9 from Reb M2.
• Enhanced UGT94E13 activity 12.0-fold via site-directed mutagenesis.
• Achieved 98.3% yield and 42.8 g/L titer of Reb M9 using an optimized enzymatic cascade.

Conclusions:

• Developed an effective enzyme engineering strategy for SG glycosylation.
• Established an efficient enzymatic cascade for novel Reb M9 synthesis.
• Provided a reference for glycosylation modification of natural products.