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Key Message: CRISPR-Cas9-mediated disruption of a licorice cellulose synthase-derived glycosyltransferase gene, GuCSyGT, demonstrated the in planta role of GuCSyGT as the enzyme catalyzing 3-O-glucuronosylation of triterpenoid aglycones in soyasaponin biosynthesis. Triterpenoid glycosides (saponins) are a large, structurally diverse group of specialized metabolites in plants, including the sweet saponin glycyrrhizin produced by licorice (Glycyrrhiza uralensis) and soyasaponins that occur widely in legumes, with various bioactivities. The triterpenoid saponin biosynthetic pathway involves the glycosylation of triterpenoid sapogenins (the non-sugar part of triterpenoid saponins) by glycosyltransferases (GTs), leading to diverse saponin structures. Previously, we identified a cellulose synthase-derived GT (CSyGT), as a newly discovered class of triterpenoid GT from G. uralensis. GuCSyGT expressed in yeast, which could transfer the sugar glucuronic acid to the C3 position of glycyrrhetinic acid and soyasapogenol B, which are the sapogenins of glycyrrhizin and soyasaponin I, respectively. This suggested that GuCSyGT is involved in the biosynthesis of glycyrrhizin and soyasaponin I. However, the in planta role of GuCSyGT in saponin biosynthesis remains unclear. In this study, we generated GuCSyGT-disrupted licorice hairy roots using CRISPR-Cas9-mediated genome editing and analyzed the saponin content. This revealed that soyasaponin I was completely absent in GuCSyGT-disrupted lines, demonstrating the in planta role of GuCSyGT in saponin biosynthesis., (© 2023. The Author(s).)

Licorice (Glycyrrhiza) produces glycyrrhizin, a valuable triterpenoid saponin, which exhibits persistent sweetness and broad pharmacological activities. In the genus Glycyrrhiza, three species, Glycyrrhiza uralensis, Glycyrrhiza glabra and Glycyrrhiza inflata, produce glycyrrhizin as their main triterpenoid saponin, which has a ketone group at C-11. Other Glycyrrhiza species produce mainly oleanane-type saponins, which harbor homoannular or heteroannular diene structures that lack the C-11 ketone. Although the glycyrrhizin biosynthetic pathway has been fully elucidated, the pathway involving saponins with diene structures remains unclear. CYP88D6 from G. uralensis is a key enzyme in glycyrrhizin biosynthesis, catalyzing the sequential two-step oxidation of β-amyrin at position C-11 to produce 11-oxo-β-amyrin. In this study, we evaluated the functions of CYP88D6 homologs from the glycyrrhizin-producing species G. glabra and G. inflata and from the non-glycyrrhizin-producing species Glycyrrhiza pallidiflora and Glycyrrhiza macedonica, using yeast engineered to supply β-amyrin as a substrate. Yeast expressing CYP88D6 homologs from glycyrrhizin-producing species produced 11-oxo-β-amyrin. However, yeast expressing CYP88D6 homologs (such as CYP88D15) from the non-glycyrrhizin-producing Glycyrrhiza species accumulated oleana-9(11),12-dien-3β-ol and oleana-11,13(18)-dien-3β-ol; these diene compounds are non-enzymatic or yeast endogenous enzymatic dehydration derivatives of 11α-hydroxy-β-amyrin, a direct reaction product of CYP88D15. These results suggest that the activities of CYP88D6 homologs, particularly their ability to catalyze the second oxidation, could influence glycyrrhizin productivity and diversify the chemical structures of saponins in Glycyrrhiza plants. A synthetic biological approach to engineer CYP88D15 could enable the production of pharmacologically active saponins with diene structures, such as saikosaponins, whose biosynthetic pathways have yet to be fully characterized., (� The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Triterpenoid saponins are specialised metabolites distributed widely in the plant kingdom that consist of one or more sugar moieties attached to triterpenoid aglycones. Despite the widely accepted view that glycosylation is catalysed by UDP-dependent glycosyltransferase (UGT), the UGT which catalyses the transfer of the conserved glucuronic acid moiety at the C-3 position of glycyrrhizin and various soyasaponins has not been determined. Here, we report that a cellulose synthase superfamily-derived glycosyltransferase (CSyGT) catalyses 3-O-glucuronosylation of triterpenoid aglycones. Gene co-expression analyses of three legume species (Glycyrrhiza uralensis, Glycine max, and Lotus japonicus) reveal the involvement of CSyGTs in saponin biosynthesis, and we characterise CSyGTs in vivo using Saccharomyces cerevisiae. CSyGT mutants of L. japonicus do not accumulate soyasaponin, but the ectopic expression of endoplasmic reticulum membrane-localised CSyGTs in a L. japonicus mutant background successfully complement soyasaponin biosynthesis. Finally, we produced glycyrrhizin de novo in yeast, paving the way for sustainable production of high-value saponins.

Triterpenoid saponins are specialized metabolites, which are abundant in soybean seeds. They have a wide variety of effects on human health and physiology. The composition of sugar chain attached to the aglycone moiety of saponins can be controlled by genetic loci, such as Sg-1, 3, and 4. Among these, the homozygous recessive sg-4 impairs the accumulation of saponins that have an arabinose moiety at the second position of the C-3 sugar chain (i.e., saponins Ad and βa) in the hypocotyls. In this study, we found that sg-4 cultivars are disabled in Glyma.01G046300 expression in hypocotyls. This gene encodes a putative glycosyltransferase (UGT73P10) and is a homolog of GmSGT2 (UGT73P2) whose recombinant protein has been previously shown, in vitro, to conjugate the second galactose moiety at the C-3 position of soyasapogenol B monoglucuronide (SBMG). The sg-4 phenotype (absence of saponins Ad and βa in hypocotyls) was restored by introducing the Glyma.01G046300 genomic DNA fragment that was obtained from the Sg-4 cultivar 'Ibarakimame 7'. Although Glyma.01G046300 is expressed in the cotyledons even in the sg-4 cultivars such as 'Enrei', the induced premature stop codon mutation (W244*) resulted in impaired accumulation of saponin βa in this tissue also in the 'Enrei' genetic background. Furthermore, the recombinant Glyma.01G046300 protein was shown to conjugate the second Ara moiety at the C-3 position of SBMG using UDP-Ara as a sugar donor. These results demonstrate that Sg-4 is responsible for conjugation of the second Ara moiety at the C-3 position of soybean saponins., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Soyasaponins are specialized metabolites present in soybean seeds that affect the taste and quality of soy-based foods. The composition of the sugar chains attached to the aglycone moiety of soyasaponins is regulated by genetic loci such as sg-1, sg-3 and sg-4. Here, we report the cloning and characterization of the Sg-3 gene, which is responsible for conjugating the terminal (third) glucose (Glc) at the C-3 sugar chain of soyasaponins. The gene Glyma.10G104700 is disabled in the sg-3 cultivar, 'Mikuriya-ao', due to the deletion of genomic DNA that results in the absence of a terminal Glc residue on the C-3 sugar chain. Sg-3 encodes a putative glycosyltransferase (UGT91H9), and its predicted protein sequence has a high homology with that of the product of GmSGT3 (Glyma.08G181000; UGT91H4), which conjugates rhamnose (Rha) to the third position of the C-3 sugar chain in vitro. A recombinant Glyma.10G104700 protein could utilize UDP-Glc as a substrate to conjugate the third Glc to the C-3 sugar chain, and introducing a functional Glyma.10G104700 transgene into the mutant complemented the sg-3 phenotype. Conversely, induction of a premature stop codon mutation in Glyma.10G104700 (W270*) resulted in the sg-3 phenotype, suggesting that Glyma.10G104700 was Sg-3. The gmsgt3 (R339H) mutant failed to accumulate soyasaponins with the third Rha at the C-3 sugar chain, and the third Glc and Rha conjugations were both disabled in the sg-3 gmsgt3 double mutant. These results demonstrated that Sg-3 and GmSGT3 are non-redundantly involved in conjugation of the third Glc and Rha at the C-3 sugar chain of soyasaponins, respectively.

Glycyrrhiza uralensis (licorice) is a widely used medicinal plant belonging to the Fabaceae. Its main active component, glycyrrhizin, is an oleanane-type triterpenoid saponin widely used as a medicine and as a natural sweetener. Licorice also produces other triterpenoids, including soyasaponins. Recent studies have revealed various oxidosqualene cyclases and cytochrome P450 monooxygenases (P450s) required for the biosynthesis of triterpenoids in licorice. Of these enzymes, β-amyrin synthase (bAS) and β-amyrin C-24 hydroxylase (CYP93E3) are involved in the biosynthesis of soyasapogenol B (an aglycone of soyasaponins) from 2,3-oxidosqualene. Although these biosynthetic enzyme genes are known to be temporally and spatially expressed in licorice, the regulatory mechanisms underlying their expression remain unknown. Here, we identified a basic helix-loop-helix (bHLH) transcription factor, GubHLH3, that positively regulates the expression of soyasaponin biosynthetic genes. GubHLH3 preferentially activates transcription from promoters of CYP93E3 and CYP72A566, the second P450 gene newly identified and shown to be responsible for C-22β hydroxylation in soyasapogenol B biosynthesis, in transient co-transfection assays of promoter-reporter constructs and transcription factors. Overexpression of GubHLH3 in transgenic hairy roots of G. uralensis enhanced the expression levels of bAS, CYP93E3 and CYP72A566. Moreover, soyasapogenol B and sophoradiol (22β-hydroxy-β-amyrin), an intermediate between β-amyrin and soyasapogenol B, were increased in transgenic hairy root lines overexpressing GubHLH3. We found that soyasaponin biosynthetic genes and GubHLH3 were co-ordinately up-regulated by methyl jasmonate (MeJA). These results suggest that GubHLH3 regulates MeJA-responsive expression of soyasaponin biosynthetic genes in G. uralensis. The regulatory mechanisms of triterpenoid biosynthesis in legumes are compared and discussed.

The roots of Platycodon grandiflorus are widely used as a crude drug. The active components include a variety of triterpenoid saponins. Recent studies have revealed that Cyt P450 monooxygenases (P450s) function as triterpene oxidases in triterpenoid saponin biosynthesis in many plant species. However, there have been no reports regarding triterpene oxidases in P. grandiflorus. In this study, we performed transcriptome analysis of three different P. grandiflorus tissues (roots, leaves and petals) using RNA sequencing (RNA-Seq) technology. We cloned six P450 genes that were highly expressed in roots, and classified them as belonging to the CYP716A, CYP716D and CYP72A subfamilies. We heterologously expressed these P450s in an engineered yeast strain that produces β-amyrin, one of the most common triterpenes in plants. Two of the CYP716A subfamily P450s catalyzed oxidation reactions of the β-amyrin skeleton. One of these P450s, CYP716A140v2, catalyzed a three-step oxidation reaction at C-28 on β-amyrin to produce oleanolic acid, a reaction performed by CYP716A subfamily P450s in a variety of plant species. The other P450, CYP716A141, catalyzed the hydroxylation of β-amyrin at C-16β. This reaction is unique among triterpene oxidases isolated to date. These results enhance our knowledge of functional variation among CYP716A subfamily enzymes involved in triterpenoid biosynthesis, and provide novel molecular tools for use in synthetic biology to produce triterpenoid saponins with pre-defined structures., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

We evaluated the hemolytic activity of 41 commercially available triterpenoid saponins and sapogenins derived from three types of structural skeletons. Structure-activity relationships were established by comparing the structural characteristics of both the aglycone and sugar moieties among the tested compounds. The majority of oleanane-type sapogenins had stronger hemolytic effects than those of the ursane and dammarane types. The presence of polar regions on sapogenins, such as a carboxyl (COOH) at position 28, an α-hydroxyl (α-OH) at position 16, and/or a β-hydroxyl (β-OH) at position 2, significantly enhanced hemolysis. Meanwhile, the introduction of an α-OH at position 2 or a methyl hydroxyl (CH 2 OH) at positions 23 or 24 was closely associated with reduced activity. Our findings suggest that not only the complexity of sugar moieties but also the types and stereochemical configurations of functional groups at different positions, as well as the skeleton types, are important structural features affecting hemolytic potential. Our results provide a baseline in terms of the toxicity of saponins and sapogenins to erythrocytes, which holds promise for drug development.

The ability to evolve novel metabolites has been instrumental for the defence of plants against antagonists. A few species in the Barbarea genus are the only crucifers known to produce saponins, some of which make plants resistant to specialist herbivores, like Plutella xylostella, the diamondback moth. Genetic mapping in Barbarea vulgaris revealed that genes for saponin biosynthesis are not clustered but are located in different linkage groups. Using co-location with quantitative trait loci (QTLs) for resistance, transcriptome and genome sequences, we identified two 2,3-oxidosqualene cyclases that form the major triterpenoid backbones. LUP2 mainly produces lupeol, and is preferentially expressed in insect-susceptible B. vulgaris plants, whereas LUP5 produces β-amyrin and α-amyrin, and is preferentially expressed in resistant plants; β-amyrin is the backbone for the resistance-conferring saponins in Barbarea. Two loci for cytochromes P450, predicted to add functional groups to the saponin backbone, were identified: CYP72As co-localized with insect resistance, whereas CYP716As did not. When B. vulgaris sapogenin biosynthesis genes were transiently expressed by CPMV-HT technology in Nicotiana benthamiana, high levels of hydroxylated and carboxylated triterpenoid structures accumulated, including oleanolic acid, which is a precursor of the major resistance-conferring saponins. When the B. vulgaris gene for sapogenin 3-O-glucosylation was co-expressed, the insect deterrent 3-O-oleanolic acid monoglucoside accumulated, as well as triterpene structures with up to six hexoses, demonstrating that N. benthamiana further decorates the monoglucosides. We argue that saponin biosynthesis in the Barbarea genus evolved by a neofunctionalized glucosyl transferase, whereas the difference between resistant and susceptible B. vulgaris chemotypes evolved by different expression of oxidosqualene cyclases (OSCs)., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

The recent spread of next-generation sequencing techniques has facilitated transcriptome analyses of non-model plants. As a result, many of the genes encoding enzymes related to the production of specialized metabolites have been identified. Compounds derived from 2,3-oxidosqualene (the common precursor of sterols, steroids and triterpenoids), a linear compound of 30 carbon atoms produced through the mevalonate pathway, are called triterpenes. These include essential sterols, which are structural components of biomembranes; steroids such as the plant hormones, brassinolides and the toxin in potatoes, solanine; as well as the structurally diverse triterpenoids. Triterpenoids containing one or more sugar moieties attached to triterpenoid aglycones are called triterpenoid saponins. Triterpenoid saponins have been shown to have various medicinal properties, such as anti-inflammatory, anticancerogenic and antiviral effects. This review summarizes the recent progress in gene discovery and elucidates the biochemical functions of biosynthetic enzymes in triterpenoid saponin biosynthesis. Special focus is placed on key players in generating the structural diversity of triterpenoid saponins, cytochrome P450 monooxygenases (P450s) and the UDP-dependent glycosyltransferases (UGTs). Perspectives on further gene discovery and the use of biosynthetic genes for the microbial production of plant-derived triterpenoid saponins are also discussed., (© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

CYP51 has been recognized as a unique CYP family that consists of one isolated molecular species, a sterol 14-demethylase essential for sterol biosynthesis. However, another CYP51 gene classified as the CYP51H subfamily has been identified in higher plants, in addition to a sterol 14-demethylase gene, CYP51G1. To shed light on the function of this "second CYP51", oat CYP51H10 was introduced into the β-amyrin-producing yeast cells, and the effect of the expressed CYP51H10 on β-amyrin metabolism in the host cells was examined. In the CYP51H10-introduced cells, β-amyrin was converted to a metabolite with 12,13-epoxy and one additional hydroxyl group. Since the 12,13-epoxy group introduced into β-amyrin ring is an essential structure of avenacin A-1, a triterpene glycoside produced in oat from β-amyrin, the present findings indicate the contribution of CYP51H10 to avenacin A-1 biosynthesis from β-amyrin. This is the first study showing a second function of the CYP51 family.

Solanaceous plants contain steroid saponins that have diverse biological and pharmacological activities. The structures of their sugar chains play an important role in their activities. A functional glucosyltransferase SaGT4A from Solanum aculeatissimum glucosylates both steroidal sapogenins and steroidal alkaloids. A potato (S. tuberosum) glycosyltransferase StSGT, which has a high degree of sequence homology with SaGT4A, exhibits the same substrate specificity toward steroidal compounds as SaGT4A. To identify the residues or domain structures responsible for these enzymatic activities, we determined the residues that are essential for SaGT4A activity, compared the specific activities of SaGT4A and StSGT, and constructed several SaGT4A/StSGT chimeric proteins, focusing on the donor-sugar recognition domain. These proteins were heterogeneously expressed in E. coli and purified, and their glycosyltransferase activities were evaluated using a coupled assay. His369 and Glu377, located in the consensus motif for plant glycosyltransferases, and Cys121, Cys247, and Cys370 were shown to be important for SaGT4A activity. StSGT exhibited more activity with UDP-galactose as a sugar donor than with UDP-glucose, whereas SaGT4A exhibited glucosyltransferase activity exclusively. The sugar selectivities of SaGT4A and StSGT were not altered by exchanging their domains, and some of the chimeric proteins showed no activity. These results suggest that the differences in the SaGT4A and StSGT amino acid sequences do not simply reflect their distinct sugar-donor specificities. We also successfully converted the non-functional SaGT4A homolog, SaGT4R, into an active glucosyltransferase.

An enzyme-linked immunosorbent assay (ELISA) was developed for determination of aculeatisides. Aculeatiside A was conjugated with bovine serum albumin (BSA) for immunization. The ratio of hapten in an antigen conjugate was determined by matrix-assisted laser desorption/ionization TOF mass spectrometry. Polyclonal antibody was developed in rabbits against an aculeatiside A-BSA conjugate. The antibody was specific for aculeatiside A and aculeatiside B. The range of the immunoassay extended from 100 ng ml(-1) to 5 pg ml(-1) of aculeatisides. Good correlation between ELISA and HPLC methods was obtained when crude extracts of plant samples were analyzed. The optimized ELISA was found to be applicable to the determination of total aculeatisides in various plant samples.