Efficient synthesis of rebaudioside D2 through UGT94D1-catalyzed regio-selective glycosylation

Qian, Ping; Yang, Lifeng; Jiang, Jiejuan; Yuan, Jiachen; Ai, Si; Sun, Sheng; Ni, Zihan; Yang, Sai; Yuan, Zhenbo; Rao, Yijian; Zhang, Yan

doi:10.1016/j.carres.2022.108687

Cited by 6 publications

(8 citation statements)

References 36 publications

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“…Lastly, we aimed to establish a cascade reaction for scale-up preparation of Reb M8. To facilitate UDPG regeneration, 39,41,43,44 sucrose synthase AtSuSy from A. thaliana was introduced, enabling the utilization of cost-effective sucrose and eliminating the need for UDPG in preparing Reb M8. UGT94E13-F169G/I185G and AtSuSy were coexpressed in E. coli BL21 (DE3), as confirmed by SDS-PAGE (Figure S2) and Western blot analyses (Figure S3), showing their recombinant coexpression and presence in the soluble fraction.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Highly Efficient Biosynthesis of Rebaudioside M8 through Structure-Guided Engineering of Glycosyltransferase UGT94E13

Yang,

Deng

et al. 2024

J. Agric. Food Chem.

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Given the low-calorie, high-sweetness characteristics of steviol glycosides (SGs), developing SGs with improved taste profiles is a key focus. Rebaudioside M8 (Reb M8), a novel non-natural SG derivative obtained through glycosylation at the C-13 position of rebaudioside D (Reb D) using glycosyltransferase UGT94E13, holds promise for further development due to its enhanced sweetness. However, the low catalytic activity of UGT94E13 hampers further research and commercialization. This study aimed to improve the enzymatic activity of UGT94E13 through semirational design, and a variant UGT94E13-F169G/I185G was obtained with the catalytic activity improved by 13.90 times. A cascade reaction involving UGT94E13-F169G/I185G and sucrose synthase AtSuSy was established to recycle uridine diphosphate glucose, resulting in an efficient preparation of Reb M8 with a yield of 98%. Moreover, according to the analysis of the distances between the substrate Reb D and enzymes as well as between Reb D and the glucose donor through molecular dynamics simulations, it is found that the positive effect of shortening the distance on glycosylation reaction activity accounts for the improved catalytic activity of UGT94E13-F169G/I185G. Therefore, this study addresses the bottleneck in the efficient production of Reb M8 and provides a foundation for its widespread application in the food industry.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Highly Efficient Biosynthesis of Rebaudioside M8 through Structure-Guided Engineering of Glycosyltransferase UGT94E13

Yang,

Deng

et al. 2024

J. Agric. Food Chem.

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“…In the present study, Mc SuSy_S31D and UGT76G1_S195Q prepared from E. coli BL21 (pRSF-S31D-S195Q) were coupled to form a SuSy-GT system (S31D-S195Q). A control experiment ( At SuSy1-S195Q) was performed under the same conditions using UGT76G1_S195Q and At SuSy1, which was widely applied in various glycosylation reactions ( Chen T. Y. et al, 2021 ; Chu et al, 2021 ; Ping et al, 2022 ). The cascade reactions were carried out at pH 7.2 and 40°C, and the mass ratio of RebE to sucrose was set to 1:10 ( Yu et al, 2022 ).…”

Section: Resultsmentioning

confidence: 99%

Identification of sucrose synthase from Micractinium conductrix to favor biocatalytic glycosylation

Chen,

Lin,

et al. 2023

Front. Microbiol.

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Sucrose synthase (SuSy, EC 2.4.1.13) is a unique glycosyltransferase (GT) for developing cost-effective glycosylation processes. Up to now, some SuSys derived from plants and bacteria have been used to recycle uridine 5′-diphosphate glucose in the reactions catalyzed by Leloir GTs. In this study, after sequence mining and experimental verification, a SuSy from Micractinium conductrix (McSuSy), a single-cell green alga, was overexpressed in Escherichia coli, and its enzymatic properties were characterized. In the direction of sucrose cleavage, the specific activity of the recombinant McSuSy is 9.39 U/mg at 37°C and pH 7.0, and the optimum temperature and pH were 60°C and pH 7.0, respectively. Its nucleotide preference for uridine 5′-diphosphate (UDP) was similar to plant SuSys, and the enzyme activity remained relatively high when the DMSO concentration below 25%. The mutation of the predicted N-terminal phosphorylation site (S31D) significantly stimulated the activity of McSuSy. When the mutant S31D of McSuSy was applied by coupling the engineered Stevia glycosyltransferase UGT76G1 in a one-pot two-enzyme reaction at 10% DMSO, 50 g/L rebaudioside E was transformed into 51.06 g/L rebaudioside M in 57 h by means of batch feeding, with a yield of 76.48%. This work may reveal the lower eukaryotes as a promising resource for SuSys of industrial interest.

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“…Recombinant protein expression was performed essentially as described previously ( Ping et al, 2022 ). Escherichia coli BL21 (DE3) with the relevant plasmids were cultured in 2 × YT medium with 100 mg/L ampicillin, incubated at 37°C with shaking.…”

Section: Methodsmentioning

confidence: 99%

“…For structure confirmation, the sample was purified with semi prepared HPLC, and the instrument and operative parameters were consistent with our previous work (Ping et al, 2022). The purified sample was further analyzed with nuclear magnetic resonance (NMR) spectroscopy.…”

Section: Cell-free Cascade Reaction For the Preparation Of Reb M2mentioning

confidence: 99%

“…UGTs have demonstrated exceptional stereo- and regio-selectivity in glycosylation of various natural products ( Caputi et al, 2012 ; Wen et al, 2018 ; Li et al, 2020 ; Teze et al, 2021 ), showcasing their significant potential in the preparation of steviol glycoside derivatives. In our previous research ( Ping et al, 2022 ), the glycosylation of Reb A to synthesize Reb D2 through the construction of a β 1→6 glycosidic bond at the C-19 position of Reb A was facilitated by a UGT known as UGT94D1, which was originally identified from Sesamum indicum ( Noguchi et al, 2008 ; Ono et al, 2020 ; Brandt et al, 2021 ). Hence, it is plausible to achieve a highly selective and efficient synthesis of Reb M2 through the forming of a similar β 1→6 glycosidic bond using Reb D as the substrate.…”

Section: Introductionmentioning

confidence: 99%

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Selective synthesis of rebaudioside M2 through structure-guided engineering of glycosyltransferase UGT94D1

Yang,

Deng

et al. 2024

Front. Bioeng. Biotechnol.

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Rebaudioside M2 (Reb M2), a novel steviol glycoside derivative, has limited industrial applications due to its low synthetic yield and selectivity. Herein, we identify UGT94D1 as a selective glycosyltransferase for rebaudioside D (Reb D), leading to the production of a mono β-1,6-glycosylated derivative, Reb M2. A variant UGT94D1-F119I/D188P was developed through protein engineering. This mutant exhibited a 6.33-fold improvement in catalytic efficiency, and produced Reb M2 with 92% yield. Moreover, molecular dynamics simulations demonstrated that UGT94D1-F119I/D188P exhibited a shorter distance between the nucleophilic oxygen (OH6) of the substrate Reb D and uridine diphosphate glucose, along with an increased Ophosphate-C1-Oacceptor angle, thus improving the catalytic activity of the enzyme. Therefore, this study provides an efficient method for the selective synthesis of Reb M2 and paves the way for its applications in various fields.

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Efficient synthesis of rebaudioside D2 through UGT94D1-catalyzed regio-selective glycosylation

Cited by 6 publications

References 36 publications

Highly Efficient Biosynthesis of Rebaudioside M8 through Structure-Guided Engineering of Glycosyltransferase UGT94E13

Highly Efficient Biosynthesis of Rebaudioside M8 through Structure-Guided Engineering of Glycosyltransferase UGT94E13

Identification of sucrose synthase from Micractinium conductrix to favor biocatalytic glycosylation

Selective synthesis of rebaudioside M2 through structure-guided engineering of glycosyltransferase UGT94D1

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