Identification and functional characterization of DzS3GT, a cytoplasmic glycosyltransferase catalyzing biosynthesis of diosgenin 3-O-glucoside in Dioscorea zingiberensis

Ye, Ting; Song, Wei; Zhang, Jiajiao; An, Malaviya; Feng, Shan; Shan, Yan Shen; Li, Jiaru

doi:10.1007/s11240-017-1187-6

Cited by 14 publications

(14 citation statements)

References 53 publications

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“…MG488290), respectively, in this study. Dz3GT2 shows only 56% amino acid identity to Dz3GT1 but displays 99.8 % identity to the previously identified DzS3GT from D. zingiberensis, the enzyme catalyzing a C3-glucosylating activity on diosgenin [ 28 ]. To further characterize the biochemical properties of Dz3GT1 and Dz3GT2, their ORFs were transferred into E. coli cells, and the recombinant UGTs were purified by the use of their N-terminal fused tags.…”

Section: Resultsmentioning

confidence: 99%

Comparative Transcriptome Analysis Identifies Putative Genes Involved in Dioscin Biosynthesis in Dioscorea zingiberensis

Qin

et al. 2018

Molecules

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Dioscorea zingiberensis is a perennial herb native to China. The rhizome of D. zingiberensis has long been used as a traditional Chinese medicine to treat rheumatic arthritis. Dioscin is the major bioactive ingredient conferring the medicinal property described in Chinese pharmacopoeia. Several previous studies have suggested cholesterol as the intermediate to the biosynthesis of dioscin, however, the biosynthetic steps to dioscin after cholesterol remain unknown. In this study, a comprehensive D. zingiberensis transcriptome derived from its leaf and rhizome was constructed. Based on the annotation using various public databases, all possible enzymes in the biosynthetic steps to cholesterol were identified. In the late steps beyond cholesterol, cholesterol undergoes site-specific oxidation by cytochrome P450s (CYPs) and glycosylation by UDP-glycosyltransferases (UGTs) to yield dioscin. From the D. zingiberensis transcriptome, a total of 485 unigenes were annotated as CYPs and 195 unigenes with a sequence length above 1000 bp were annotated as UGTs. Transcriptomic comparison revealed 165 CYP annotated unigenes correlating to dioscin biosynthesis in the plant. Further phylogenetic analysis suggested that among those CYP candidates four of them would be the most likely candidates involved in the biosynthetic steps from cholesterol to dioscin. Additionally, from the UGT annotated unigenes, six of them were annotated as 3-O-UGTs and two of them were annotated as rhamnosyltransferases, which consisted of potential UGT candidates involved in dioscin biosynthesis. To further explore the function of the UGT candidates, two 3-O-UGT candidates, named Dz3GT1 and Dz3GT2, were cloned and functionally characterized. Both Dz3GT1 and Dz3GT2 were able to catalyze a C3-glucosylation activity on diosgenin. In conclusion, this study will facilitate our understanding of dioscin biosynthesis pathway and provides a basis for further mining the genes involved in dioscin biosynthesis.

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Section: Resultsmentioning

confidence: 99%

Comparative Transcriptome Analysis Identifies Putative Genes Involved in Dioscin Biosynthesis in Dioscorea zingiberensis

Qin

et al. 2018

Molecules

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“…Among the steroid substrates tested, diosgenin was the best aglycone substrate of TfS3GT2. To the best of our knowledge, in a pure form or as a crude extract, UGTs capable of catalyzing 3- O -glucosylation of diosgenin or yamogenin were only isolated from a few species, including Solanum melongena L ( Pazkowski et al, 2001 ; Potocka and Zimowski, 2008 ), W. somnifera ( Madina et al, 2007 ), and D. zingiberensis ( Ye et al, 2017 ). In comparison with these previously reported S3GTs, TfS3GT2 was judged to be a novel diosgenin 3- O -glucosyltransferase based on the following observations: (1) The previously reported diosgenin 3- O -glucosyltransferases all give low activity on diosgenin, however, which is the best substrate here for TfS3GT2; (2) TfS3GT2 shares relatively low amino acid sequence identity (50–67%) with the previously reported diosgenin 3- O -glucosyltransferases.…”

Section: Discussionmentioning

confidence: 99%

“…Very recently, cytochrome P450s capable of converting cholesterol to diosgenin have been characterized from T. foenum-graecum and Paris polyphylla ( Christ et al, 2019 ). Some researchers have proposed that dioscin is directly biosynthesized from diosgenin, simply by adding one glucose and two rhamnose groups at its C-3 OH position (see the route 1 of Figure 1 ; Ye et al, 2017 ; Li et al, 2018 ). However, this hypothesis is challenged by the natural occurrence of furostanol saponins (e.g., protodisocin, see its structure in Figure 1 ) in T. foenum-graecum ( Kang et al, 2013 ; Krol-Kogus et al, 2020 ) and dioscorea genus ( Li et al, 2010 ; Zhang et al, 2014 ).…”

Section: Introductionmentioning

confidence: 99%

Molecular Cloning and Functional Characterization of a Sterol 3-O-Glucosyltransferase Involved in Biosynthesis of Steroidal Saponins in Trigonella foenum-graecum

Gao

Hua

et al. 2021

Front. Plant Sci.

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Fenugreek (Trigonella foenum-graecum), a pharmacologically important herb, is widely known for its antidiabetic, hypolipidemic, and anticancer effects. The medicinal properties of this herb are accredited to the presence of bioactive steroidal saponins with one or more sugar moieties linked to the C-3 OH position of disogenin or its C25-epimer yamogenin. Despite intensive studies regarding pharmacology and phytochemical profiles of this plant, enzymes and/or genes involved in synthesizing the glycosidic part of fenugreek steroidal saponins are still missing so far. This study reports the molecular cloning and functional characterization of a key sterol-specific glucosyltransferase, designated as TfS3GT2 here, from fenugreek plant. The recombinant TfS3GT2 was purified via expression in Escherichia coli, and biochemical characterization of the recombinant enzyme suggested its role in transferring a glucose group onto the C-3 hydroxyl group of diosgenin or yamogenin. The functional role of TfS3GT2 in the steroidal saponin biosynthesis was also demonstrated by suppressing the gene in the transgenic fenugreek hairy roots via the RNA interference (RNAi) approach. Down-regulation of TfS3GT2 in fenugreek generally led to reduced levels of diosgenin or yamogenin-derived steroidal saponins. Thus, Tf3SGT2 was identified as a steroid-specific UDP-glucose 3-O-glucosyltransferase that appears to be involved in steroidal saponin biosynthesis in T. foenum-graecum.

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“…It also gives the basis for heterologous biosynthesis of dioscin and relevant steroidal saponins in other plants and microbial hosts. Diosgenin was successfully synthesized in the cholesterol-producing yeasts (Christ et al, 2019), and sterol 3-O-glucosyltransferases, converting diosgenin to trillin, have been isolated (Ye et al, 2017; et al, 2018). Integrating the sterol 3-O-glucosyltransferase gene and DzGT1 into the diosgenin-producing yeasts, PSA is most likely synthesized.…”

Section: Discussionmentioning

confidence: 99%

“…From diosgenin to dioscin, one glucose and two rhamnose groups need to be added at the C-3 position of diosgenin by UDP-glucosyltransferase and UDP-rhamnosyltransferase. Although genes encoding UDP-glucosyltransferase genes that convert diosgenin to its C-3 glycosylated product trillin were isolated from D. zingiberensis (Ye et al, 2017;Li et al, 2018), genes encoding UDP-rhamnosyltransferase involved in the biosynthesis of dioscin and other steroidal saponins have not been isolated and characterized yet. Only some UDPrhamnosyltransferases in the biosynthetic pathways of steroidal glycoalkaloids and triterpene saponins whose structure is similar to steroidal saponins have been studied.…”

Section: Introductionmentioning

confidence: 99%

Identification and Characterization of a Trillin Rhamnosyltransferase From Dioscorea zingiberensis

Mosongo

Han

et al. 2021

Front. Plant Sci.

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Dioscorea zingiberensis accumulates abundant steroidal saponins, such as dioscin, which is the principal bioactive ingredient displaying a wide range of pharmacological activities. Diosgenin is the aglycone of dioscin, and recently, genes encoding cytochrome P450 enzymes in the late steps of diosgenin biosynthesis have been isolated. Diosgenin was successfully synthesized in the cholesterol-producing yeasts. From diosgenin to dioscin, one glucose and two rhamnose groups need to be added. Although genes encoding UDP-glucosyltransferases converting diosgenin to trillin were isolated, genes encoding UDP-rhamnosyltransferases involved in dioscin biosynthesis remain unknown. In this study, we isolated the cDNA encoding the trillin rhamnosyltransferase (designated DzGT1) from D. zingiberensis. Heterologous expression of DzGT1 in Escherichia coli cells showed that the gene product exhibits an enzyme activity that glycosylates the trillin to form prosapogenin A of dioscin (PSA). The transcript level of DzGT1 is in accord with PSA accumulation in different organs of D. zingiberensis. Integration of the biochemical, metabolic, and transcriptional data supported the function of DzGT1 in dioscin biosynthesis. The identification and characterization of DzGT1 will help understand the metabolism of steroidal saponins in D. zingiberensis and provide candidate UDP-rhamnosyltransferase for efficient production of PSA, dioscin, and relevant steroidal saponins in microbial hosts.

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Identification and functional characterization of DzS3GT, a cytoplasmic glycosyltransferase catalyzing biosynthesis of diosgenin 3-O-glucoside in Dioscorea zingiberensis

Abstract: Abbreviations S3GT3-O-sterol glycosyltransferase GT Glycosyltransferase RACE Rapid-amplification of cDNA ends ORF Open reading frame HPLC High performance liquid chromatography TLC Thin-layer chromatography LC-MS Liquid chromatography-mass spectrometry

Cited by 14 publications

References 53 publications

Comparative Transcriptome Analysis Identifies Putative Genes Involved in Dioscin Biosynthesis in Dioscorea zingiberensis

Comparative Transcriptome Analysis Identifies Putative Genes Involved in Dioscin Biosynthesis in Dioscorea zingiberensis

Molecular Cloning and Functional Characterization of a Sterol 3-O-Glucosyltransferase Involved in Biosynthesis of Steroidal Saponins in Trigonella foenum-graecum

Identification and Characterization of a Trillin Rhamnosyltransferase From Dioscorea zingiberensis

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