UDP-dependent Glycosyltransferases Research Articles

Progress in Identification of UDP-Glycosyltransferases for Ginsenoside Biosynthesis.

Ginsenosides, the primary pharmacologically active constituents of the Panax genus, have demonstrated a variety of medicinal properties, including anticardiovascular disease, cytotoxic, antiaging, and antidiabetes effects. However, the low concentration of ginsenosides in plants and the challenges associated with their extraction impede the advancement and application of ginsenosides. Heterologous biosynthesis represents a promising strategy for the targeted production of these natural active compounds. As representative triterpenoids, the biosynthetic pathway of the aglycone skeletons of ginsenosides has been successfully decoded. While the sugar moiety is vital for the structural diversity and pharmacological activity of ginsenosides, the mining of uridine diphosphate-dependent glycosyltransferases (UGTs) involved in ginsenoside biosynthesis has attracted a lot of attention and made great progress in recent years. In this paper, we summarize the identification and functional study of UGTs responsible for ginsenoside synthesis in both plants, such as Panax ginseng and Gynostemma pentaphyllum, and microorganisms including Bacillus subtilis and Saccharomyces cerevisiae. The UGT-related microbial cell factories for large-scale ginsenoside production are also mentioned. Additionally, we delve into strategies for UGT mining, particularly potential rapid screening or identification methods, providing insights and prospects. This review provides insights into the study of other unknown glycosyltransferases as candidate genetic elements for the heterologous biosynthesis of rare ginsenosides.

Integrated metabolome and transcriptome analyses reveal the molecular mechanism underlying dynamic metabolic processes during taproot development of Panax notoginseng

BackgroundPanax notoginseng (Burk) F. H. Chen is one of the most famous Chinese traditional medicinal plants. The taproot is the main organ producing triterpenoid saponins, and its development is directly linked to the quality and yield of the harvested P. notoginseng. However, the mechanisms underlying the dynamic metabolic changes occurring during taproot development of P. notoginseng are unknown.ResultsWe carried out metabolomic and transcriptomic analyses to investigate metabolites and gene expression during the development of P. notoginseng taproots. The differentially accumulated metabolites included amino acids and derivatives, nucleotides and derivatives, and lipids in 1-year-old taproots, flavonoids and terpenoids in 2- and 3-year-old taproots, and phenolic acids in 3-year-old taproots. The differentially expressed genes (DEGs) are related to phenylpropanoid biosynthesis, metabolic pathway and biosynthesis of secondary metabolites at all three developmental stages. Integrative analysis revealed that the phenylpropanoid biosynthesis pathway was involved in not only the development of but also metabolic changes in P. notoginseng taproots. Moreover, significant accumulation of triterpenoid saponins in 2- and 3-year-old taproots was highly correlated with the up-regulated expression of cytochrome P450s and uridine diphosphate-dependent glycosyltransferases genes. Additionally, a gene encoding RNase-like major storage protein was identified to play a dual role in the development of P. notoginseng taproots and their triterpenoid saponins synthesis.ConclusionsThese results elucidate the molecular mechanism underlying the accumulation of and change relationship between primary and secondary metabolites in P. notoginseng taproots, and provide a basis for the quality control and genetic improvement of P. notoginseng.

Transcriptome-Wide Identification and Integrated Analysis of a UGT Gene Involved in Ginsenoside Ro Biosynthesis in Panax ginseng.

Panax ginseng as a traditional medicinal plant with a long history of medicinal use. Ginsenoside Ro is the only oleanane-type ginsenoside in ginseng, and has various pharmacological activities, including anti-inflammatory, detoxification, and antithrombotic activities. UDP-dependent glycosyltransferase (UGT) plays a key role in the synthesis of ginsenoside, and the excavation of UGT genes involved in the biosynthesis of ginsenoside Ro has great significance in enriching ginsenoside genetic resources and further revealing the synthesis mechanism of ginsenoside. In this work, ginsenoside-Ro-synthesis-related genes were mined using the P. ginseng reference-free transcriptome database. Fourteen hub transcripts were identified by differential expression analysis and weighted gene co-expression network analysis. Phylogenetic and synteny block analyses of PgUGAT252645, a UGT transcript among the hub transcripts, showed that PgUGAT252645 belonged to the UGT73 subfamily and was relatively conserved in ginseng plants. Functional analysis showed that PgUGAT252645 encodes a glucuronosyltransferase that catalyzes the glucuronide modification of the C3 position of oleanolic acid using uridine diphosphate glucuronide as the substrate. Furthermore, the mutation at 622 bp of its open reading frame resulted in amino acid substitutions that may significantly affect the catalytic activity of the enzyme, and, as a consequence, affect the biosynthesis of ginsenoside Ro. Results of the in vitro enzyme activity assay of the heterologous expression product in E. coli of PgUGAT252645 verified the above analyses. The function of PgUGAT252645 was further verified by the result that its overexpression in ginseng adventitious roots significantly increased the content of ginsenoside Ro. The present work identified a new UGT gene involved in the biosynthesis of ginsenoside Ro, which not only enriches the functional genes in the ginsenoside synthesis pathway, but also provides the technical basis and theoretical basis for the in-depth excavation of ginsenoside-synthesis-related genes.

Leveraging glycoside-targeted metabolomics to gain insight into biological function.

In plants, uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) catalyze glycosylation of secondary metabolites, but assigning physiological functions to UGTs is still a daunting task. The recent study of Wu et al. presents a useful method to resolve this problem by elegantly combining modification-specific metabolomics with isotope tracing.

Reconstruction of engineered yeast factory for high yield production of ginsenosides Rg3 and Rd.

Panax notoginseng is one of the most valuable traditional Chinese herbs. The main active ingredients, dammarane-type ginsenosides, show multiple pharmacological activities. Recently, the key UDP-dependent glycosyltransferases (UGTs) involved in the biosynthesis of common ginsenosides have been widely studied. However, only a few UGTs that catalyze ginsenoside formation have been reported. This study further investigated the new catalytic function of 10 characterized UGTs from the public database. PnUGT31(PnUGT94B2) and PnUGT53 (PnUGT71B8)exhibited promiscuous sugar-donor specificity of UDP-glucose and UDP-xylose, which could catalyze the glycosylation of C20-OH sites and elongation of the sugar chain at the C3 and/or C20 sites. We further analyzed the expression patterns in P. notoginseng and predicted the catalytic mechanisms of PnUGT31 and PnUGT53 using molecular docking simulations. Moreover, different gene modules were built to increase the yield of ginsenosides in engineered yeast. The metabolic flow of the proginsenediol (PPD) synthetic pathway was enhanced by LPPDS gene modules based on the engineered strain. The resulting yeast was constructed to produce 1.72 g/L PPD in a shaking flask, but cell growth was significantly inhibited. EGH and LKG gene modules were constructed to achieve high-level production of dammarane-type ginsenosides. The production of G-Rg3 controlled by LKG modules increased 3.84 times (254.07 mg/ L), whereas the G-Rd titer reached 56.68 mg/L after 96 h in shaking flask culture under the control of all modules, both of which yielded the highest values for known microbes.

Fusion glycosyltransferase design for enhanced conversion of rebaudioside A into rebaudioside M in cascade

Rebaudioside M (RM) is an attractive target for developing next-generation sweeteners due to its exceptional sweetness, up to 350 times sweeter than sucrose, and its non-caloric nature. However, its low natural abundance makes the traditional plant extraction method neither cost-effective nor environmentally sustainable. This study presents a fusion enzyme design of UGT76G1 and UGT91C1, both UDP-dependent glycosyltransferases (UTGs), to produce RM from naturally abundant rebaudioside A in a cascade reaction. The fusion enzyme was produced by Pichia pastoris with a yield of ∼25 mg purified protein per liter of culture, displaying optimal activity at ∼42°C and pH 7.0. It was found that a peptide linker consisting of three repeats of GGGGS enhanced substrate channeling and the synthesis rate of RM by 1.8-fold. Intracellular overexpression of the fusion enzyme in P. pastoris cells demonstrated higher catalytic efficiency, thermostability, and recyclability than mixed cells expressing UGT76G1 or UGT91C1 separately, or mixed purified enzymes. This study provides a promising approach for the industrial preparation of RM.

Production of the antidepressant orcinol glucoside in Yarrowia lipolytica with yields over 6,400-fold higher than plant extraction.

Orcinol glucoside (OG), mainly found in the rhizome of the traditional Chinese herb Curculigo orchioides Gaertn, is noted for its antidepressant effects. In this study, an efficient screening pipeline was established for identifying the highly active orcinol synthase (ORS) and UDP-dependent glycosyltransferase (UGT) involved in the biosynthesis of OG by combining transcriptome analysis, structure-based virtual screening, and in vitro enzyme activity assays. By enhancing the downstream pathway, metabolic engineering and fermentation optimization, the OG production in Yarrowia lipolytica was improved 100-fold, resulting in a final yield of 43.46 g/L (0.84 g/g DCW), which is almost 6,400-fold higher than the extraction yield from C. orchioides roots. This study provides a reference for rapid identification of functional genes and high-yield production of natural products.

Comparative structural analysis of plant uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) in plant specialized metabolism: structures of plant UGTs for biosynthesis of steviol glycosides

Comparative structural analysis of plant uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) in plant specialized metabolism: structures of plant UGTs for biosynthesis of steviol glycosides

IiUGT71B2 catalyzes lignan glycosylation in Isatis indigotica with substrates specificity

Lignans are a major class of secondary metabolites distributed widely in the plant kingdom that are derived from coupling of phenylpropanoid (C6-C3) units. Most lignans are 8–8'-linked, and are often found in their glycosylated forms, which are catalyzed by UDP-dependent glycosyltransferase (UGT). Up to now, all UGTs involved in lignans biosynthesis are determined with substrate promiscuity. Here, IiUGT71B2 is reported from Isatis indigotica that specific catalyzes 4-O and 4′-O glycosylation of furan (lariciresinol) and dibenzylbutyrolactone (matairesinol) type lignans. Transcriptional profile of IiUGT71B2 indicates its involvement in lignan glycosides biosynthesis, which is co-expressed with other lignan biosynthetic genes, and is consistent with metabolic distribution of lignans in organs and tissues as well. Both in vitro and in vivo experiments demonstrate IiUGT71B2 catalyzes the mono- and di- glycosylation of lariciresinol and mono-glycosylation of matairesinol. The ligand docking and site-directed mutagenesis suggest Asn261, Ser289, Ala356, and Tyr395 as key residues, which may determine its catalytic efficiency together with the narrow binding pocket of IiUGT71B2. This study provides insights into lignan metabolism and functional diversity of plant UGTs.

Highly efficient synthesis of mono-β-1,6-Glucosylated Rebaudioside A derivative catalyzed by glycosyltransferase YjiC

Steviol glycosides have attracted great interest because of their high levels of sweetness and safety, and absence of calories. Improvement of their sensory qualities via glycosylation modification by glycosyltransferase is a research hotspot. In this study, YjiC, a uridine diphosphate-dependent glycosyltransferase from Bacillus subtilis 168, was found with the ability to glycosylate rebaudioside A (Reb A) to produce a novel mono β-1, 6-glycosylated Reb A derivative rebaudioside L2 (Reb L2). It has an improved sweetness compared with Reb A. Next, a cascade reaction was established by combining YjiC with sucrose synthase AtSuSy from Arabidopsis thaliana for scale-up preparation of Reb L2. It shows that Reb L2 (30.94 mg/mL) could be efficiently synthesized with an excellent yield of 91.34% within 12 h. Therefore, this study provides a potential approach for the production and application of new steviol glycoside Reb L2, expanding the scope of steviol glycosides.

Subfunctionalization of a monolignol to a phytoalexin glucosyltransferase is accompanied by substrate inhibition

Uridine diphosphate-dependent glycosyltransferases (UGTs) mediate the glycosylation of plant metabolites, thereby altering their physicochemical properties and bioactivities. Plants possess numerous UGT genes, with the encoded enzymes often glycosylating multiple substrates and some exhibiting substrate inhibition kinetics, but the biological function and molecular basis of these phenomena are not fully understood. The promiscuous monolignol/phytoalexin glycosyltransferase NbUGT72AY1 exhibits substrate inhibition (Ki) at 4 μM scopoletin, whereas the highly homologous monolignol StUGT72AY2 is inhibited at 190 μM. We therefore used hydrogen/deuterium exchange mass spectrometry and structure-based mutational analyses of both proteins and introduced NbUGT72AY1 residues into StUGT72AY2 and vice versa to study promiscuity and substrate inhibition of UGTs. A single F87I and chimeric mutant of NbUGT72AY1 showed significantly reduced scopoletin substrate inhibition, whereas its monolignol glycosylation activity was almost unaffected. Reverse mutations in StUGT72AY2 resulted in increased scopoletin glycosylation, leading to enhanced promiscuity, which was accompanied by substrate inhibition. Studies of 3D structures identified open and closed UGT conformers, allowing visualization of the dynamics of conformational changes that occur during catalysis. Previously postulated substrate access tunnels likely serve as drainage channels. The results suggest a two-site model in which the second substrate molecule binds near the catalytic site and blocks product release. Mutational studies showed that minor changes in amino acid sequence can enhance the promiscuity of the enzyme and add new capabilities such as substrate inhibition without affecting existing functions. The proposed subfunctionalization mechanism of expanded promiscuity may play a role in enzyme evolution and highlights the importance of promiscuous enzymes in providing new functions.

Molecular identification and functional characterization of two glycosyltransferases genes from Fallopia multiflora.

The traditional Chinese medicine plant Fallopia multiflora (Thunb.) Harald. contains various pharmacodynamically active glycosides, such as stilbene glycosides, anthraquinone (AQ) glycosides, and flavonoid glycosides. Glycosylation is an important reaction in plant metabolism that is generally completed by glycosyltransferase in the last step of the secondary metabolite biosynthesis pathway, and it can improve the beneficial properties of many natural products. In this study, based on the transcriptome data of F. multiflora, we cloned two Uridine-diphosphate-dependent glycosyltransferases (UGTs) from the cDNA of F. multiflora (FmUGT1 and FmUGT2). Their full-length sequences were 1602 and 1449 bp, encoding 533 and 482 amino acids, respectively. In vitro enzymatic reaction results showed that FmUGT1 and FmUGT2 were promiscuous and could catalyze the glycosylation of 12 compounds, including stilbenes, anthraquinones, flavonoids, phloretin, and curcumin, and we also obtained and structurally identified 13 glycosylated products from both of them. Further experiments on the in vivo function of FmUGT1 and FmUGT2 showed that 2, 3, 5, 4'- tetrahydroxy stilbene-2-O-β-d-glucoside (THSG) content in hairy roots was elevated significantly when FmUGT1 and FmUGT2 were overexpressed and decreased accordingly in the RNA interference (RNAi) groups. These results indicate that FmUGT1 and FmUGT2 were able to glycosylate a total of 12 structurally diverse types of acceptors and to generate O-glycosides. In addition, FmUGT1 and FmUGT2 efficiently catalyzed the biosynthesis of THSG, and promoted the production of AQs in transgenic hairy roots.

Structure function relationships in plant UDP-glycosyltransferases

Uridine diphosphate-dependent glycosyltransferases (UGTs) in plants catalyze O -, C -, S - and N -glycosylation of diverse substrates to yield a wide variety of glycosides with broad bioactivities. Not all wild-type plant UGTs can exhibit excellent catalytic characteristics and achieve application requirements. Desirable enzymes with far greater catalytic properties than the wild type can be obtained through enzyme engineering. Here, we enumerate all currently resolved crystal structures of plant UGTs. The proposed mechanisms for the four types of glycosylation are then summarized. Structure-based protein engineering strategies for improving the catalytic properties of plant UGTs and their principles are further elaborated. Finally, aspects that need in-depth studies and trends for future application of plant UGTs are discussed. • This article itemizes all resolved crystal structures of plant UGTs and proposes mechanisms for four types of glycosylation. • Structure- function relationships are explored under the guidance of protein engineering. • The industrialization prospects and possible future research directions of plant UGTs are clarified.

Revisiting the transcriptome data of Centella asiatica identified an ester-forming triterpenoid: UDP-glucose 28-O-glucosyltransferase

Advances in sequencing technologies have produced a huge amount of transcriptome data, which facilitate the revelation of natural product biosynthetic pathways in plants. Asiaticoside and madecassoside are two famous triterpene saponins with unusual glucose-glucose-rhamnose chain at the C-28 position of the aglycone. To date, the complete glycosylation pathway underlying the assembly of the above sugar chain remains largely unknown. Here we successfully screened out 75 putative uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) by analyzing the latest transcriptome data of Centella asiatica. We further identified a UGT (CaUGT1) that could specifically transfer the glucose to the C-28 carboxyl group of asiatic acid and madecassic acid. Interestingly, the expression profile of CaUGT1 is highly correlated with UGT73AD1, a previously characterized UDP-glucose 28-O-glucosyltransferase, suggesting that the initial glycosylation step of asiatic acid and madecassic acid was synergistically catalyzed by these two UGTs. This study thus laid the foundation for a comprehensively understanding of the glycosylation network in the biosynthesis of asiaticoside and madecassoside.

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

Steviol glycosides have been widely applied as new sweeteners in food, beverages, health care, and daily chemical industry owing to the properties of high-intensity sweetness, low calorie, and good physiological characteristics. However, most of steviol glycosides have a bitter taste. Their organoleptic properties can be effectively improved by modifying the linked glycosyl units. In this study, UGT94D1, a uridine diphosphate-dependent glycosyltransferase from Sesamum indicum, was reported to selectively glycosylate rebaudioside A (Reb A) for the synthesis of rebaudioside D2 (Reb D2). Furthermore, a cascade reaction system was constructed to synthesize Reb D2 with 94.66% yield by coupling UGT94D1 with sucrose synthase AtSuSy from Arabidopsis thaliana. Thus, our study not only introduced a practical method for the synthesis of steviol glycosides but also provided the possibility for further exploration of Reb D2.

Glycoside-specific metabolomics combined with precursor isotopic labeling for characterizing plant glycosyltransferases

Glycosylation by uridine diphosphate-dependent glycosyltransferases (UGTs) in plants contributes to the complexity and diversity of secondary metabolites. UGTs are generally promiscuous in their use of acceptors, making it challenging to reveal the function of UGTs in vivo. Here, we described an approach that combined glycoside-specific metabolomics and precursor isotopic labeling analysis to characterize UGTs in Arabidopsis. We revisited the UGT72E cluster, which has been reported to catalyze the glycosylation of monolignols. Glycoside-specific metabolomics analysis reduced the number of differentially accumulated metabolites in the ugt72e1e2e3 mutant by at least 90% compared with that from traditional untargeted metabolomics analysis. In addition to the two previously reported monolignol glycosides, a total of 62 glycosides showed reduced accumulation in the ugt72e1e2e3 mutant, 22 of which were phenylalanine-derived glycosides, including 5-OH coniferyl alcohol-derived and lignan-derived glycosides, as confirmed by isotopic tracing of [13C6]-phenylalanine precursor. Our method revealed that UGT72Es could use coumarins as substrates, and genetic evidence showed that UGT72Es endowed plants with enhanced tolerance to low iron availability under alkaline conditions. Using the newly developed method, the function of UGT78D2 was also evaluated. These case studies suggest that this method can substantially contribute to the characterization of UGTs and efficiently investigate glycosylation processes, the complexity of which have been highly underestimated.

Synergistic effects of Azospirillum brasilense and Bacillus cereus on plant growth, biochemical attributes and molecular genetic regulation of steviol glycosides biosynthetic genes in Stevia rebaudiana

The current study aimed to scale up the favorable bio-stimulants for enhancing the growth and breeding strategies of Stevia rebaudiana to increase sugar productivity. Inoculation of 45-day-old S. rebaudiana plantlets with Bacillus cereus and Azospirillum brasilense alone or in combination for 30 days allowed comparisons among their effects on enhancement and improvement of plant growth, production of bioactive compounds and expression of steviol glycoside genes. B. cereus SrAM1 isolated from surface-sterilized Stevia rebaudiana leaves was molecularly identified using 16s rRNA and tested for its ability to promote plant growth. Beneficial endophytic B. cereus SrAM1 induced all plant growth-promoting traits, except solubilization of phosphate, therefore it showed high effectiveness in the promotion of growth and production of bioactive compounds. Treatment of plants with B. cereus SrAM1 alone revealed carbohydrates content of 278.99 mg/g, total soluble sugar of 114.17 mg/g, total phenolics content of 34.05 mg gallic acid equivalent (GAE)/g dry weight) and total antioxidants activity of 32.33 mg (A.A)/g dry weight). Thus, plantlets inoculated with B. cereus SrAM1 alone exhibited the greatest responses in physiological and morphological parameters, but plantlets inoculated with B. cereus SrAM1 + A. brasilense showed a maximal upregulation of genes responsible for the biosynthesis of steviol glycosides (Kaurene oxidase, ent-KO; UDP-dependent glycosyl transferases of UGT85C2, UGT74G1, UGT76G1). Taken together, the used bacterial strains, particularly B. cereus SrAM1 could significantly improve the growth of S. rebaudiana via dynamic interactions in plants.

Structure-guided engineering of key amino acids in UGT85B1 controlling substrate and stereo-specificity in aromatic cyanogenic glucoside biosynthesis.

SUMMARYCyanogenic glucosides are important defense molecules in plants with useful biological activities in animals. Their last biosynthetic step consists of a glycosylation reaction that confers stability and increases structural diversity and is catalyzed by the UDP‐dependent glycosyltransferases (UGTs) of glycosyltransferase family 1. These versatile enzymes have large and varied substrate scopes, and the structure–function relationships controlling scope and specificity remain poorly understood. Here, we report substrate‐bound crystal structures and rational engineering of substrate and stereo‐specificities of UGT85B1 from Sorghum bicolor involved in biosynthesis of the cyanogenic glucoside dhurrin. Substrate specificity was shifted from the natural substrate (S)‐p‐hydroxymandelonitrile to (S)‐mandelonitrile by combining a mutation to abolish hydrogen bonding to the p‐hydroxyl group with a mutation to provide steric hindrance at the p‐hydroxyl group binding site (V132A/Q225W). Further, stereo‐specificity was shifted from (S) to (R) by substituting four rationally chosen residues within 6 Å of the nitrile group (M312T/A313T/H408F/G409A). These activities were compared to two other UGTs involved in the biosynthesis of aromatic cyanogenic glucosides in Prunus dulcis (almond) and Eucalyptus cladocalyx. Together, these studies enabled us to pinpoint factors that drive substrate and stereo‐specificities in the cyanogenic glucoside biosynthetic UGTs. The structure‐guided engineering of the functional properties of UGT85B1 enhances our understanding of the evolution of UGTs involved in the biosynthesis of cyanogenic glucosides and will enable future engineering efforts towards new biotechnological applications.

CRISPR/Cas9 disruption of UGT71L1 in poplar connects salicinoid and salicylic acid metabolism and alters growth and morphology.

Salicinoids are salicyl alcohol-containing phenolic glycosides with strong antiherbivore effects found only in poplars and willows. Their biosynthesis is poorly understood, but recently a UDP-dependent glycosyltransferase, UGT71L1, was shown to be required for salicinoid biosynthesis in poplar tissue cultures. UGT71L1 specifically glycosylates salicyl benzoate, a proposed salicinoid intermediate. Here, we analyzed transgenic CRISPR/Cas9-generated UGT71L1 knockout plants. Metabolomic analyses revealed substantial reductions in the major salicinoids, confirming the central role of the enzyme in salicinoid biosynthesis. Correspondingly, UGT71L1 knockouts were preferred to wild-type by white-marked tussock moth (Orgyia leucostigma) larvae in bioassays. Greenhouse-grown knockout plants showed substantial growth alterations, with decreased internode length and smaller serrated leaves. Reinserting a functional UGT71L1 gene in a transgenic rescue experiment demonstrated that these effects were due only to the loss of UGT71L1. The knockouts contained elevated salicylate (SA) and jasmonate (JA) concentrations, and also had enhanced expression of SA- and JA-related genes. SA is predicted to be released by UGT71L1 disruption, if salicyl salicylate is a pathway intermediate and UGT71L1 substrate. This idea was supported by showing that salicyl salicylate can be glucosylated by recombinant UGT71L1, providing a potential link of salicinoid metabolism to SA and growth impacts. Connecting this pathway with growth could imply that salicinoids are under additional evolutionary constraints beyond selective pressure by herbivores.

Mutation of Stevia glycosyltransferase UGT76G1 for efficient biotransformation of rebaudioside E into rebaudioside M

Rebaudioside M (RebM) is a desired natural non-caloric and high potency sweetener with very low content in the leaves of Stevia rebaudiana. In this study, based on the crystal structure of UDP-dependent glycosyltransferase UGT76G1, a key enzyme responsible for RebM production in S. rebaudiana, a computational strategy combining protein structure, molecular docking, and single-point saturation mutation was adopted to engineer UGT76G1 to promote the transformation of rebaudioside E (RebE) into RebM via a two-step continuous glycosylation. An S195Q mutant was verified to have better performance, with a catalytic efficiency 1.2-fold higher towards RebE and 2-fold higher towards rebaudioside D (RebD) than wild-type UGT76G1. RebM (12.8 ± 0.6 g/L) and RebD (10.5 ± 0.1 g/L) were generated from RebE (20 g/L) catalyzed by the S195Q mutant coupling with a sucrose synthase from Micractinium conductrix at 40 °C for 32 h. This work would expand the application of structure-based enzyme design for glycosyltransferase engineering.