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

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
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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