Single site mutations of glycosyltransferase with improved activity and regioselectivity for directed biosynthesis of unnatural protopanaxatriol-type ginsenoside product

Protopanaxatriol (PPT)-type ginsenosides are functional components in Panax species and exhibit diverse significant bioactivities. Glycosyltransferase Bs-YjiC from Bacillus subtilis 168 has been demonstrated to generate ginsenoside Rh1 and several unnatural PPT-type ginsenosides. However, the glycodiversification of ginsenosides and poor regioselectivity of glycosyltransferase might hamper the low-cost synthesis process of the desired product. The semi-rational design of Bs-YjiC is newly performed by structure-based guidance, and the small change of Bs-YjiC could target both activity improvement and regioselectivity enhancement. The created mutant F168M has 5.24 times of specific activity compared with WT and the increased conversion of 95.56% for PPT, and shows significantly improved regioselectivity of 87.28% for unnatural ginsenoside of 3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxatriol, which is 8.83 times of the wild type (9.88%). The coupled catalytic strategy of mutant F168M and sucrose synthase AtSuSy (from Arabidopsis thaliana) is used for efficient regeneration of UDP-glucose, and unnatural PPT-type ginsenoside could reach a high titer of 8.66 g/L (regioselectivity 90.42%) in a fed-batch mode, which is 99 times of that by wild type (0.088 g/L). It represents the highest level of production reported to date, and achieves a maximum number of UDP-glucose regeneration cycles (RcMax = 39.92). This study exploits an effective biocatalyst for unnatural ginsenoside, thus facilitating study and medical application.