Guo, H;
Li, W;
Zhu, C;
Chen, Y;
Dalby, PA;
Fan, D;
(2023)
Enhancement of thermal stability of Bacillus subtilis 168 glycosyltransferase YjiC based on PoPMuSiC algorithm and its catalytic conversion of rare ginsenoside PPD.
Process Biochemistry
, 132
pp. 1-12.
10.1016/j.procbio.2023.07.005.
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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.
| Type: | Article |
|---|---|
| Title: | Enhancement of thermal stability of Bacillus subtilis 168 glycosyltransferase YjiC based on PoPMuSiC algorithm and its catalytic conversion of rare ginsenoside PPD |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.procbio.2023.07.005 |
| Publisher version: | https://doi.org/10.1016/j.procbio.2023.07.005 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions. |
| Keywords: | Science & Technology, Life Sciences & Biomedicine, Technology, Biochemistry & Molecular Biology, Biotechnology & Applied Microbiology, Engineering, Chemical, Engineering, Glycosyltransferase Bs-YjiC, PoPMuSiC, Thermostability, Molecular dynamics simulations, Ginsenoside PPD, ALPHA-L-ARABINOFURANOSIDASE, RATIONAL DESIGN, THERMOSTABILITY, MECHANISM, SPECIFICITY, IMPROVEMENT, LANDSCAPE |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10178762 |
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