Glycosyltransferases and non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease(NAFLD) is the most common form of chronic liver disease and its incidence is increasing worldwide. However, the underlying mechanisms leading to the development of NAFLD are still not fully understood. Glycosyltransferases(GTs) are a diverse class of enzymes involved in catalyzing the transfer of one or multiple sugar residues to a wide range of acceptor molecules. GTs mediate a wide range of functions from structure and storage to signaling, and play a key role in many fundamental biological processes. Therefore, it is anticipated that GTs have a role in the pathogenesis of NAFLD. In this article, we present an overview of the basic information on NAFLD, particularly GTs and glycosylation modification of certain molecules and their association with NAFLD pathogenesis. In addition, the effects and mechanisms of some GTs in the development of NAFLD are summarized.

The biosynthesis of asiaticoside and madecassoside reveals tandem duplication-directed evolution of glycoside glycosyltransferases in the Apiales

Certain plant species within the Apiales order accumulate triterpenoid saponins that feature a distinctive glucose-glucose-rhamnose(G-G-R)sugar chain attached at the C-28 position of the pentacyclic triterpene skeleton.Until recently,the genomic basis underlying the biosynthesis and evolution of this sugar chain has remained elusive.In this study,we identified two novel glycoside glycosyltransferases(GGTs)that can sequentially install the sugar chain’s second D-glucose and third L-rhamnose during the biosynthesis of asiaticoside and madecassoside,two representative G-G-R sugar chain-containing triterpenoid saponins produced by Centella asiatica.Enzymatic assays revealed the remarkable substrate promiscuity of the two GGTs and the key residues crucial for sugar-donor selectivity of the glucosyltransferase and rhamnosyltransferase.We further identified syntenic tandem gene duplicates of the two GGTs in the Apiaceae and Araliaceae families,suggesting a well-conserved genomic basis underlying sugar chain assembly that likely has evolved in the early ancestors of the Apiales order.Moreover,expression patterns of the two GGTs in pierced leaves of C.asiatica were found to be correlated with the production of asiaticoside and madecassoside,implying their involvement in host defense against herbivores and pathogens.Our work sheds light on the biosynthesis and evolution of complex saponin sugars,paving the way for future engineering of diverse bioactive triterpenoids with unique glycoforms.

Attractive but Toxic： Emerging Roles of Glycosidically Bound Volatiles and Glycosyltransferases Involved in Their Formation

Plants emit an overabundance of volatile compounds, which act in their producers either as appreciated attractants to lure beneficial animals or as repellent toxins to deter pests in a species-specific and concentration-dependent manner. Plants have evolved solutions to provide sufficient volatiles without poisoning themselves. Uridine-diphosphate sugar-dependent glycosyltransferases （UGTs） acting on vola-tiles is one important part of this sophisticated system, which balances the levels of bioactive metabolites and prepares them for cellular and long-distance transport and storage but enables the remobilization of disarmed toxins for the benefit of plant protection. This review provides an overview of the research history of glycosidically bound volatiles （GBVs）, a relatively new group of plant secondary metabolites, and discusses the role of UGTs in the production of GBVs for plant protection.

Construction of a Rice Glycosyltransferase Phylogenomic Database and Identification of Rice-Diverged Glycosyltransferases

Glycosyltransferases （GTs; EC 2.4.x.y） constitute a large group of enzymes that form glycosidic bonds through transfer of sugars from activated donor molecules to acceptor molecules. GTs are critical to the biosynthesis of plant cell walls, among other diverse functions. Based on the Carbohydrate-Active enZymes （CAZy） database and sequence similarity.searches, we have identified 609 potential GT genes （loci） corresponding to 769 transcripts （gene models） in rice （Oryza sativa）, the reference monocotyledonous species. Using domain composition and sequence similarity, these rice GTs were classified into 40 CAZy families plus an additional unknown class. We found that two Pfam domains of unknown function, PF04577 and PF04646, are associated with GT families GT61 and GT31, respectively. To facilitate functional analysis of this important and large gene family, we created a phylogenomic Rice GT Database （http：//ricephylogenomics. ucdavis.edu/cellwalls/gtJ）. Through the database, several classes of functional genomic data, including mutant lines and gene expression data, can be displayed for each rice GT in the context of a phylogenetic tree, allowing for comparative analysis both within and between GT families. Comprehensive digital expression analysis of public gene expression data revealed that most （-80%） rice GTs are expressed. Based on analysis with Inparanoid, we identified 282 ‘rice-diverged＇ GTs that lack orthologs in sequenced dicots （Arabidopsis thaliana, Populus tricocarpa, Medicago truncatula, and Ricinus communis）. Combining these analyses, we identified 33 rice-diverged GT genes （45 gene models） that are highly expressed in above-ground, vegetative tissues. From the literature and this analysis, 21 of these loci are excellent targets for functional examination toward understanding and manipulating grass cell wall qualities. Study of the remainder may reveal aspects of hormone and protein metabolism that are critical for rice biology. This list of 33 genes and the Rice GT Database will facilitate the study of GTs and cell wall synthesis in rice and other plants.

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[^(13)C_(6)]-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.

Glycosyltransferases:Mining,engineering and applications in biosynthesis of glycosylated plant natural products

UDP-Glycosyltransferases(UGTs)catalyze the transfer of nucleotide-activated sugars to specific acceptors,among which the GT1 family enzymes are well-known for their function in biosynthesis of natural product glycosides.Elucidating GT function represents necessary step in metabolic engineering of aglycone glycosylation to produce drug leads,cosmetics,nutrients and sweeteners.In this review,we systematically summarize the phylogenetic distribution and catalytic diversity of plant GTs.We also discuss recent progress in the identifi-cation of novel GT candidates for synthesis of plant natural products(PNPs)using multi-omics technology and deep learning predicted models.We also highlight recent advances in rational design and directed evolution engineering strategies for new or improved GT functions.Finally,we cover recent breakthroughs in the appli-cation of GTs for microbial biosynthesis of some representative glycosylated PNPs,including flavonoid glycosides(fisetin 3-O-glycosides,astragalin,scutellarein 7-O-glucoside),terpenoid glycosides(rebaudioside A,ginseno-sides)and polyketide glycosides(salidroside,polydatin).

Advance in glycosyltransferases, the important bioparts for production of diversified ginsenosides

Ginsenosides are a series of glycosylated triterpenoids predominantly originated from Panax species with multiple pharmacological activities such as anti-aging, mediatory effect on the immune system and the nervous system. During the biosynthesis of ginsenosides, glycosyltransferases play essential roles by transferring various sugar moieties to the sapogenins in contributing to form structure and bioactivity diversified ginsenosides, which makes them important bioparts for synthetic biology-based production of these valuable ginsenosides. In this review, we summarized the functional elucidated glycosyltransferases responsible for ginsenoside biosynthesis, the advance in the protein engineering of UDP-glycosyltransferases(UGTs) and their application with the aim to provide in-depth understanding on ginsenoside-related UGTs for the production of rare ginsenosides applying synthetic biology-based microbial cell factories in the future.

Enzymatic biosynthesis of benzylisoquinoline alkaloid glycosides via promiscuous glycosyltransferases from Carthamus tinctorius

Enzymatic glycosylation catalyzed by glycosyltransferases (GTs) has great potential in creating diverse novel and bioactive glycosides. Herein, three new GTs (UGT84 A33, UGT71 AE1 and UGT90 A14) from Carthamus tinctorius exhibited robust catalytic promiscuity to benzylisoquinoline alkaloids, and were used as enzymatic tools in glycosylation of bioactive benzylisoquinoline alkaloids. Seven novel benzylisoquinoline alkaloids O-glycosides were synthesized with high efficiency. These studies indicate the significant potential of promiscuous GTs in synthesis of benzylisoquinoline alkaloids glycosides for drug discovery.

Functional characterization of a cycloartenol synthase and four glycosyltransferases in the biosynthesis of cycloastragenol-type astragalosides from Astragalus membranaceus

Astragalosides are the main active constituents of traditional Chinese medicine Huang-Qi,of which cycloastragenol-type glycosides are the most typical and major bioactive compounds.This kind of compounds exhibit various biological functions including cardiovascular protective,neuroprotective,etc.Owing to the limitations of natural sources and the difficulties encountered in chemical synthesis,re-engineering of biosynthetic machinery will offer an alternative and promising approach to producing astragalosides.However,the biosynthetic pathway for astragalosides remains elusive due to their complex structures and numerous reaction types and steps.Herein,guided by transcriptome and phylogenetic analyses,a cycloartenol synthase and four glycosyltransferases catalyzing the committed steps in the biosynthesis of such bioactive astragalosides were functionally characterized from Astragalus membranaceus.AmCAS1,the first reported cycloartenol synthase from Astragalus genus,is capable of catalyzing the formation of cycloartenol;AmUGT15,AmUGT14,AmUGT13,and AmUGT7 are four glycosyltransferases biochemically characterized to catalyze 3-O-xylosylation,3-O-glucosylation,25-O-glucosylation/O-xylosylation and 2’-O-glucosylation of cycloastragenol glycosides,respectively.These findings not only clarified the crucial enzymes for the biosynthesis and the molecular basis for the structural diversity of astragalosides in Astragalus plants,also paved the way for further completely deciphering the biosynthetic pathway and constructing an artificial pathway for their efficient production.

The ZmHSF08-ZmUGT92A1 module regulates heat tolerance by altering reactive oxygen species levels in maize

GTs(Glycosyltransferases)are important in plant growth and abiotic stresses.However,its role in maize heat response is far from clear.Here,we describe the constitutively expressed UDP-glycosyltransferase ZmUGT92A1,which has a highly conserved PSPG box and is localized in chloroplasts,is induced under heat stress.Functional disruption of ZmUGT92A1 leads to heat sensitivity and reactive oxygen species accumulation in maize.Metabolomics analysis revealed that ZmUGT92A1 affected multiple metabolic pathways and altered the metabolic homeostasis of flavonoids under heat stress.In vitro assay showed ZmUGT92A1 exhibits glycosyltransferase activity on flavonoids and hormones.Additionally,we identified a rapidly heat-induced transcription factor,ZmHSF08,which can directly bind and repress the promoter region of ZmUGT92A1.The ZmHSF08 overexpression line exhibits heat sensitivity and reactive oxygen species accumulation.These findings reveal that the ZmHSF08-ZmUGT92A1 module plays a role in heat tolerance in maize and provide candidate strategies for the development of heat-tolerant varieties.

Effects of Glycosyltransferase Gene sm-Ngt1 on the Plant Height of Rice

[Objective] The aim was to study the effects of over-expressed sm-Ngt1,a glycosyltransferase gene induced by both methyl jasmonate and salicylic acid from tobacco,on the plant height of rice.[Method] The binary expression vector of the sm-Ngt1 gene was constructed and transferred to matured embryo of indica rice YTB with the method of Agrobacterium infection.The height of the positive transgenic plants was measured.[Result] 117 positive transgenic plants with sm-Ngt1 were obtained.The results showed that the rice plants dwarfed with different degrees after transferring the sm-Ngt1 gene,the height of 37% of transgenic plants is 71.4±9.8 cm,27% is 65.1±4.6 cm,and the average height of YTB（CK） is 130.0±4.3 cm.[Conclusion] These results aid a foundafion for further study on function of sm-Ngt1.

Genome-Wide Study Identifies the Regulatory Glycosyltransferase Genes Networks and Signaling Pathways from Keshan Disease

KD （Keshan disease） is an endemic cardiomyopathy occurring only in China. Its pathogenesis is unclear till now. In the study, gene expression profiles of the PBMC （peripheral blood mononuclear cell） derived respectively from KD patients and healthy in KD areas were compared. Total RNA was isolated, amplified, labeled and hybridized to Agilent 4 ~ 44 K Whole Human Genome Oligonucleotide Microarray. Significant canonical pathways were analyzed by IPA （ingenuity pathway analysis） to identify differently expressed genes and pathways involved in the cardiovascular system development and function. Quantitative RT-PCR was applied to further validate our microarray results. Eighty-three up-regulated （ratios 〉 2.0） and nine down-regulated glycosyltransferase genes （ratios 〈 0.5） in PBMC in KD patients were detected by significance analysis of microarrays. Two significant canonical pathways from glycosyltransferase gene expression profiles were screened by IPA. The results of qRT-PCR show that four up-regulated （BMP 1/7/10 and FGF 18） and one down-regulated （BMP2） genes are consistent with those in microarray experiment, confirming the validity of the microarray data. Based on the results of the study, it is suggested that bone morphogenetic proteins and fibroblast growth factors might play an important role in the pathogenesis of KD. This further helps us to understand the pathogenesis of KD, as well as dilated cardiomyopathy.

Functional characterization,structural basis,and protein engineering of a rare flavonoid 2′-O-glycosyltransferase from Scutellaria baicalensis

Glycosylation is an important post-modification reaction in plant secondary metabolism,and contributes to structural diversity of bioactive natural products.In plants,glycosylation is usually catalyzed by UDP-glycosyltransferases.Flavonoid 2′-O-glycosides are rare glycosides.However,no UGTs have been reported,thus far,to specifically catalyze 2′-O-glycosylation of flavonoids.In this work,UGT71AP2 was identified from the medicinal plant Scutellaria baicalensis as the first flavonoid 2′-O-glycosyltransferase.It could preferentially transfer a glycosyl moiety to 2′-hydroxy of at least nine flavonoids to yield six new compounds.Some of the 2′-O-glycosides showed noticeable inhibitory activities against cyclooxygenase 2.The crystal structure of UGT71AP2(2.15Å)was solved,and mechanisms of its regio-selectivity was interpreted by pKa calculations,molecular docking,MD simulation,MM/GBSA binding free energy,QM/MM,and hydrogen‒deuterium exchange mass spectrometry analysis.Through structure-guided rational design,we obtained the L138T/V179D/M180T mutant with remarkably enhanced regio-selectivity(the ratio of 7-O-glycosylation byproducts decreased from 48%to 4%)and catalytic efficiency of 2′-O-glycosylation(kcat/Km,0.23 L/(s·μmol),12-fold higher than the native).Moreover,UGT71AP2 also possesses moderate UDP-dependent de-glycosylation activity,and is a dual function glycosyltransferase.This work provides an efficient biocatalyst and sets a good example for protein engineering to optimize enzyme catalytic features through rational design.

Filling the Gaps to Solve the Extensin Puzzle

Extensins （EXTs） are highly repetitive plant O-glycoproteins that require several post-translational modifi- cations （PTMs） to become functional in plant cell walls. First, they are hydroxylated on contiguous proline residues; then they are O-glycosylated on hydroxyproline and serine. After secretion into the apoplast, O-glycosylated EXTs form a tridimensional network organized by inter- and intra-Tyr linkages. Recent studies have made significant progress in the identification of the enzymatic machinery required to process EXTs, which includes prolyl 4-hydroxylases, glycosyltransferases, papain-type cysteine endopeptidases, and peroxidases. EXTs are abundant in plant tissues and are particularly important in rapidly expanding root hairs and pollen tubes, which grow in a polar manner. Small changes in EXT PTMs affect fastgrowing cells, although the molecular mechanisms underlying this regulation are unknown. In this review, we highlight recent advances in our understanding of EXT modifications throughout the secretory pathway, EXT assembly in cell walls, and possible sensing mechanisms involving the Catharanthus roseus cell surface sensor receptor-like kinases located at the interface between the apoplast and the cytoplasmic side of the plasma membrane.

The Cooperative Activities of CSLD2, CSLD3, and CSLD5 Are Required for Normal Arabidopsis Development

Glycosyltransferases of the Cellulose Synthase Like D （CS/D） subfamily have been reported to be involved in tip growth and stem development in Arabidopsis. The csld2 and csld3 mutants are root hair defective and the csld5 mutant has reduced stem growth. In this study, we produced double and triple knockout mutants of CSLD2, CSLD3, and CSLD5. Unlike the single mutants and the csld2/csld3 double mutant, the csld2/csld5, csld3/csld5, and csld2/csld3/csld5 mutants were dwarfed and showed severely reduced viability. This demonstrates that the cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development, and that they are involved in important processes besides the specialized role in tip growth. The mutant phenotypes indicate that CSLD2 and CSLD3 have overlapping functions with CSLD5 in early plant development, whereas the CSLD2 and CSLD3 proteins are non-redundant. To determine the biochemical function of CSLD proteins, we used transient expression in tobacco leaves. Microsomes containing heterologously expressed CSLD5 transferred mannose from GDP-mannose onto endogenous acceptors. The same activity was detected when CSLD2 and CSLD3 were coexpressed but not when they were expressed separately. With monosaccharides as exogenous acceptors, microsomal preparations from CSLD5-expressing plants mediated the transfer of mannose from GDP-mannose onto mannose. These results were supported by immunodetection studies that showed reduced levels of a mannan epitope in the cell walls of stem interfascicular fibers and xylem vessels of the csld2/csld3/csld5 mutant.

Dietary fiber in plant cell walls-the healthy carbohydrates

Dietary fiber(DF)is one of the major classes of nutrients for humans.It is widely distributed in the edible parts of natural plants,with the cell wall being the main DF-containing structure.DF content varies significantly in different plant species and organs,and the processing procedure can have a dramatic effect on the DF composition of plant-based foods.Given the considerable nutritional value of DF,a deeper understanding of DF in food plants,including its composition and biosynthesis,is fundamental to the establishment of a daily intake reference of DF and is also critical to molecular breeding programs for modifying DF content.In the past decades,plant cell wall biology has seen dramatic progress,and such knowledge is of great potential to be translated into DF-related food science research and may provide future research directions for improving the health benefits of food crops.In this review,to spark interdisciplinary discussions between food science researchers and plant cell wall biologists,we focus on a specific category of DF--cell wall carbohydrates.We first summarize the content and composition of carbohydrate DF in various plant-based foods,and then discuss the structure and biosynthesis mechanism of each carbohydrate DF category,in particular the respective biosynthetic enzymes.Health impacts of DF are highlighted,and finally,future directions of DF research are also briefly outlined.

Unraveling the serial glycosylation in the biosynthesis of steroidal saponins in the medicinal plant Paris polyphylla and their antifungal action

Sugar-sugar glycosyltransferases play important roles in constructing complex and bioactive saponins.Here,we characterized a series of UDP-glycosyltransferases responsible for biosynthesizing the branched sugar chain of bioactive steroidal saponins from a widely known medicinal plant Paris polyphylla var.yunnanensis.Among them,a 2'-O-rhamnosyltransferase and three 6'-O-glucosyltrasferases catalyzed a cascade of glycosylation to produce steroidal diglycosides and triglycosides,respectively.These UDP-glycosyltransferases showed astonishing substrate promiscuity,resulting in the generation of a panel of 24 terpenoid glycosides including 15 previously undescribed compounds.A mutant library containing 44 variants was constructed based on the identification of critical residues by molecular docking simulations and protein model alignments,and a mutant UGT91AH1^(Y187A)with increased catalytic efficiency was obtained.The steroidal saponins exhibited remarkable antifungal activity against four widespread strains of human pathogenic fungi attributed to ergosterol-dependent damage of fungal cell membranes,and 2'-O-rhamnosylation appeared to correlate with strong antifungal effects.The findings elucidated the biosynthetic machinery for their production of steroidal saponins and revealed their potential as new antifungal agents.

Molecular Dissection of Xylan Biosynthesis during Wood Formation in Poplar

Xylan, being the second most abundant polysaccharide in dicot wood, is considered to be one of the factors contributing to wood biomass recalcitrance for biofuel production. To better utilize wood as biofuel feedstock, it is crucial to functionally characterize all the genes involved in xylan biosynthesis during wood formation. In this report, we investigated roles of poplar families GT43 and GT8 glycosyltransferases in xylan biosynthesis during wood formation. There exist seven GT43 genes in the genome of poplar （Populus trichocarpa）, five of which, namely PtrGT43A, PtrGT43B, PtrGT43C, PtrGT43D, and PtrGT43E, were shown to be highly expressed in the developing wood and their encoded proteins were localized in the Golgi. Comprehensive genetic complementation coupled with chemical analyses demonstrated that overexpression of PtrGT43A/B/E but not PtrGT43C/D was able to rescue the xylan defects conferred by the Arabidopsis irx9 mutant, whereas overexpression of PtrGT43C/D but not PtrGT43A/B/E led to a complementation of the xylan defects in the Arabidopsis irx14 mutant. The essential roles of poplar GT43 members in xylan biosynthesis was further substantiated by RNAi down-regulation of GT43B in the hybrid poplar （Populus alba x tremula） leading to reductions in wall thickness and xylan content in wood, and an elevation in the abundance of the xylan reducing end sequence. Wood digestibility analysis revealed that cellulase digestion released more glucose from the wood of poplar GT43B RNAi lines than the control wood, indicating a decrease in wood biomass recalcitrance. Furthermore, RNAi down-regulation of another poplar wood-associated glycosyltransferase, PoGTSD, was shown to cause decreases in wall thickness and xylan content as well as in the abundance of the xylan reducing end sequence. Together, these findings demonstrate that the poplar GT43 members form two functionally non-redundant groups, namely PtrGT43A/B/E as functional orthologs of Arabidopsis IRX9 and PtrGT43C/D as functional orthologs ofArabidopsis IRX14, all of which are involved in the biosynthesis of xylan backbones, and that the poplar GT8D is essential for the biosynthesis of the xylan reducing end sequence.

A UV-B-responsive glycosyltransferase,OsUGT706C2,modulates flavonoid metabolism in rice

Although natural variations in rice flavonoids exist, and biochemical characterization of a few flavonoid glycosyltransferases has been reported, few studies focused on natural variations in tricin-lignan-glycosides and their underlying genetic basis. In this study, we carried out metabolic profiling of tricin-lignan-glycosides and identified a major quantitative gene annotated as a UDPdependent glycosyltransferase OsUGT706C2 by metabolite-based genome-wide association analysis. The putative flavonoid glycosyltransferase OsUGT706C2 was characterized as a flavonoid 7-O-glycosyltransferas in vitro and in vivo. Although the in vitro enzyme activity of OsUGT706C2 was similar to that of OsUGT706D1, the expression pattern and induced expression profile of OsUGT706C2 were very different from those of OsUGT706D1. Besides, OsUGT706C2 was specifically induced by UV-B. Constitutive expression of OsUGT706C2 in rice may modulate phenylpropanoid metabolism at both the transcript and metabolite levels. Furthermore, overexpressing OsUGT706C2 can enhance UV-B tolerance by promoting ROS scavenging in rice. Our findings might make it possible to use the glycosyltransferase OsUGT706C2 for crop improvement with respect to UVB adaptation and/or flavonoid accumulation, which may contribute to stable yield.

The enzymatic biosynthesis of acylated steroidal glycosides and their cytotoxic activity

Herein we describe the discovery and functional characterization of a steroidal glycosyltransferase(SGT) from Ornithogalum saundersiae and a steroidal glycoside acyltransferase(SGA) from Escherichia coli and their application in the biosynthesis of acylated steroidal glycosides(ASGs). Initially,an SGT gene, designated as OsSGT1, was isolated from O. saundersiae. OsSGT1-containing cell free extract was then used as the biocatalyst to react with 49 structurally diverse drug-like compounds. The recombinant OsSGT1 was shown to be active against both 3β-and 17β-hydroxyl steroids. Unexpectedly,in an effort to identify OsSGT1, we found the bacteria lacA gene in lac operon actually encoded an SGA,specifically catalyzing the acetylations of sugar moieties of steroid 17β-glucosides. Finally, a novel enzymatic two-step synthesis of two ASGs, acetylated testosterone-17-O-β-glucosides(AT-17β-Gs) and acetylated estradiol-17-O-β-glucosides(AE-17β-Gs), from the abundantly available free steroids using OsSGT1 and EcSGA1 as the biocatalysts was developed. The two-step process is characterized by EcSGA1-catalyzed regioselective acylations of all hydroxyl groups on the sugar unit of unprotected steroidal glycosides(SGs) in the late stage, thereby significantly streamlining the synthetic route towards ASGs and thus forming four monoacylates. The improved cytotoxic activities of 30-acetylated testosterone17-O-β-glucoside towards seven human tumor cell lines were thus observable.