Improving Thermostability and Catalytic Activity of Glycosyltransferase From Panax ginseng by Semi-Rational Design for Rebaudioside D Synthesis

As a natural sweetener and sucrose substitute, the biosynthesis and application of steviol glycosides containing the component rebaudioside D have attracted worldwide attention. Here, a glycosyltransferase PgUGT from Panax ginseng was first reported for the biosynthesis of rebaudioside D. With the three-dimensional structures built by homology modeling and deep-learning-based modeling, PgUGT was semi-rationally designed by FireProt. After detecting 16 site-directed variants, eight of them were combined in a mutant Mut8 with both improved enzyme activity and thermostability. The enzyme activity of Mut8 was 3.2-fold higher than that of the wild type, with an increased optimum reaction temperature from 35 to 40 degrees C. The activity of this mutant remained over 93% when incubated at 35 degrees C for 2 h, which was 2.42 times higher than that of the wild type. Meanwhile, when the enzymes were incubated at 40 degrees C, where the wild type was completely inactivated after 1 h, the residual activity of Mut8 retained 59.0% after 2 h. This study would provide a novel glycosyltransferase with great potential for the industrial production of rebaudioside D and other steviol glycosides.

Automated summary

This study reports the application of a glycosyltransferase PgUGT from Panax ginseng for the biosynthesis of rebaudioside D. By using rational design and site-directed mutagenesis, a mutant Mut8 with improved enzyme activity and thermostability was obtained. Mut8 showed a 3.2-fold increase in enzyme activity compared to the wild type and an increased optimum reaction temperature of 40°C. This study provides a potential novel glycosyltransferase for the industrial production of rebaudioside D and other steviol glycosides.