[P001] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P01-037] Computational Engineering of a Robust UGT with Enhanced Thermostability and Organic Solvent Tolerance

○Yong Zhang¹, Shuoyang Wang¹, Mohamed Yassin Ali¹, Jiashuai Zhu¹, Yuhan Guo¹, Yan Feng¹ (1. Shanghai Jiao Tong University (China))

Keywords:

Computation-aided enzyme design,UDP glycosyltransferase,Thermostability,Organic Solvent Tolerance

Glycosylation strongly affects the solubility, activity, and stability of natural products [1]. Due to the low aqueous solubility of most nature product substrates, UGT-catalyzed reactions often need organic solvents or higher temperatures to improve efficiency [2, 3], making enzyme stability a key concern.In this study, we first determined the complex structure of UDP-bound UGT109A3 from Bacillus subtilis. We then used a combined computational approach including EvolvePro, FireProt, and Residue Co-evolution analysis to engineer the UGT enzyme for better stability. After several rounds of screening, the mutant UGT109A3m (A45L/C127Y/T299I) showed greatly enhanced organic solvent tolerance compared with the wild type in 30% DMSO. Differential scanning fluorimetry confirmed that two designed mutants, UGT109A3m (A45L/C127Y/T299I) and UGT109A3m (S203C/N304W), had improved thermostability simultaneously, with Tm increases of 6.9 and 7.1 ℃, respectively. Structural and MD analyses indicated that the enhanced stability was caused by more intramolecular hydrogen bonds and rigidification of the Ala150-Leu170 loop region. This work provides useful information for understanding UGT family enzymes and designing more efficient and robust biocatalysts.

References:
[1] Ali MY, Chang Q, Guo X, Zhang Y, Feng Y. Highly Efficient Biosynthesis of Glycyrrhetinic Acid Glycoside Derivatives Using an Artificial Biocatalyst Cascade Coupling of Microbial Glycosyltransferase to Plant Sucrose Synthase. Front. Bioeng. Biotechnol. 2021. 06/fbioe.2021.645079
[2] Liu, Z., & Xie, L.. Advancement of uridine diphosphate-dependent glycosyltransferases (UGTs) in the glycosylation modification of natural products and their protein engineering. Food Qual. Saf., 2025, https://doi.org/10.1093/fqsafe/fyaf005
[3] Dai L, Liu C, Li J, et al. One-pot Synthesis of Ginsenoside Rh2 and Bioactive Unnatural Ginsenoside by Coupling Promiscuous Glycosyltransferase from Bacillus subtilis 168 to sucrose synthase. J. Agric. Food Chem., 2018, 66(11): 2830-2837.