Download MendeleyCharacterization of Sr UGT76G4 reveals a key residue for regioselectivity and efficient Reb M synthesis.
Wang, Y., Li, T., Zheng, Y., Guo, C., Li, K., Jia, X., Zhu, L., Chen, K., Yin, H.(2025) Proc Natl Acad Sci U S A 122: e2504698122-e2504698122
- PubMed: 40961143
- DOI: https://doi.org/10.1073/pnas.2504698122
- Primary Citation of Related Structures:
8ZI6, 8ZI7 - PubMed Abstract:
Steviol glycosides (SGs) from Stevia rebaudiana are prized as noncaloric sweeteners, with rebaudioside M (Reb M)-a next-generation SG known for its sucrose-like sweetness and lack of off-tastes-standing out for its superior sensory profile. However, Reb M's limited natural abundance impedes its commercial production. Here, we report the identification of a glucosyltransferase, UGT76G4 that efficiently catalyzes the conversion of Reb D to Reb M with a strong preference for C19 glycosylation. Structural and functional analyses, including X-ray crystallography, molecular dynamics simulations, and mutagenesis, revealed key residues in UGT76G4 that dictate its regioselectivity, with residue 200 playing a pivotal role. Engineered UGT76G4 variants, including Q199I/G200Y and H155S/Q199I/G200Y, enhanced Reb E and Reb D conversion efficiency by 1.46-fold and 23-fold, respectively, compared to UGT76G1. The engineered variants offer a promising pathway for increasing Reb M production, advancing biotechnological strategies for steviol glycoside biosynthesis and optimizing plant metabolic engineering approaches. Our findings deepen the understanding of SG biosynthesis and provide a basis for sustainable production of high-value sweeteners.
- Dalian Engineering Research Center for Carbohydrate Agricultural Preparations, Dalian Technology Innovation Center for Green Agriculture, Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, People's Republic of China.
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