Enhancement of thermal stability of Bacillus subtilis 168 glycosyltransferase YjiC based on PoPMuSiC algorithm and its catalytic conversion of rare ginsenoside PPD

Abstract

YjiC, a glycosyltransferase from Bacillus subtilis 168, has great promise for natural product biosynthesis due to its aglycon promiscuity. In this study, the K125I/N178I variant with target residues located away from the substrate binding site was selected based on PoPMuSiC algorithm prediction and combined mutagenesis. The melting temperature (Tm) and t1/2 at 55 ℃ were increased by 7.2 ℃ and 18 min, respectively. Enzyme kinetic analysis revealed that the Km value of K125I/N178I was reduced by 11.4% but had a 1.21-fold increase in catalytic efficiency. Analysis of thermal stability mechanisms through fluorescence spectroscopy, LigPlot+ and molecular dynamics (MD) simulation showed that the increase in hydrophobic interactions and reduced structural flexibility are the main determinant factors for improved thermal stability. Finally, a ginsenoside PPD transformation system coupling Bs-YjiC and sucrose synthase (SuSy) was established at 45 ℃ using sucrose as the sugar donor. The space-time yield (STY) of ginsenoside F12 (415 mg L−1 h−1) was 1.3 times higher than in previous studies. This is the first report on engineered Bs-YjiC for thermal stability improvement by rational design in industrial production of rare ginsenosides.