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SCFSkp2/Cks1is an E3 ubiquitin ligase, whose substrate specificity is determined by the oncogenic F-box protein Skp2 and the adaptor protein Cks1. A principal target of SCFSkp2/Cks1is the cyclin-dependent kinase inhibitor p27. Elevated levels of Skp2 and reduced levels of p27 are common in a variety of cancers, and there is consequently a need to develop effective inhibitors of the Skp2-p27 interaction. However, conventional small-molecule approaches are challenging due to the extended bi-molecular interface that spans both Skp2 and Cks1, the lack of suitable binding pockets on this surface, and the intrinsically disordered nature of p27. Here, we develop macrocyclic peptides capable of binding to SCFSkp2/Cks1with nanomolar affinities, an enhancement of almost two orders of magnitude over the natural p27 peptide. We show that these macrocyclic peptides inhibit p27 ubiquitination in vitro, restore p27 levels in a breast cancer cell line, and reduce cell proliferation.

A high-quality genome assembly is imperative to explore the evolutionary basis of characteristic attributes that define chemotype and provide essential resources for a molecular breeding strategy for enhanced production of medicinal metabolites. Here, using single-molecule high-fidelity (HiFi) sequencing reads, we report chromosome-scale genome assembly for Chinese licorice (Glycyrrhiza uralensis), a widely used herbal and natural medicine. The entire genome assembly was achieved in eight chromosomes, with contig and scaffold N50 as 36.02 and 60.2 Mb, respectively. With only 17 assembly gaps and half of the chromosomes having no or one assembly gap, the presented genome assembly is among the best plant genomes to date. Our results showed an advantage of using highly accurate long-read HiFi sequencing data for assembling a highly heterozygous genome including its complexed repeat content. Additionally, our analysis revealed that G. uralensis experienced a recent whole-genome duplication at approximately 59.02 million years ago post a gamma (γ) whole-genome triplication event, which contributed to its present chemotype features. The metabolic gene cluster analysis identified 355 gene clusters, which included the entire biosynthesis pathway of glycyrrhizin. The genome assembly and its annotations provide an essential resource for licorice improvement through molecular breeding and the discovery of valuable genes for engineering bioactive components and understanding the evolution of specialized metabolites biosynthesis.

Aerenchymatous phellem (AP) is important for internal aeration and adaptation to waterlogging in plants. Herein, the extensive accumulation of triterpenoids such as lupeol and betulinic acid was identified in AP. However, the biological and physiological roles of these triterpenoids in plants are largely unknown. Lupeol is converted from 2,3-oxidosqualene by lupeol synthase (LUS) and oxidized to betulinic acid. Functional analysis of LUS genes in soybean revealed that GmLUS1 is crucial for triterpenoid biosynthesis in AP. Lupeol and betulinic acid were found to be the major components of epicuticular wax on the surface of AP cells, and they contributed to tissue hydrophobicity and oxygen transport to roots. Additionally, the lus1 mutant produced a shallow root system due to less oxygen transport via AP under waterlogged conditions. In conclusion, triterpenoid accumulation in AP aids internal aeration and root development for adaptation to waterlogging.

Uridine 5'-diphosphate (UDP)-glucose dehydrogenase (UGD) produces UDP-glucuronic acid from UDP-glucose as a precursor of plant cell wall polysaccharides. UDP-glucuronic acid is also a sugar donor for the glycosylation of various plant specialized metabolites. Nevertheless, the roles of UGDs in plant specialized metabolism remain poorly understood. Glycyrrhiza species (licorice), which are medicinal legumes, biosynthesize triterpenoid saponins, soyasaponins and glycyrrhizin, commonly glucuronosylated at the C-3 position of the triterpenoid scaffold. Often, several different UGD isoforms are present in plants. To gain insight into potential functional differences among UGD isoforms in triterpenoid saponin biosynthesis in relation to cell wall component biosynthesis, we identified and characterized Glycyrrhiza uralensis UGDs (GuUGDs), which were discovered to comprise five isoforms, four of which (GuUGD1-4) showed UGD activity in vitro. GuUGD1-4 had different biochemical properties, including their affinity for UDP-glucose, catalytic constant, and sensitivity to feedback inhibitors. GuUGD2 had the highest catalytic constant and highest gene expression level among the GuUGDs, suggesting that it is the major isoform contributing to the transition from UDP-glucose to UDP-glucuronic acid in planta. To evaluate the contribution of GuUGD isoforms to saponin biosynthesis, we compared the expression patterns of GuUGDs with those of saponin biosynthetic genes in methyl jasmonate (MeJA)-treated cultured stolons. GuUGD1-4 showed delayed responses to MeJA compared to those of saponin biosynthetic genes, suggesting that MeJA-responsive expression of GuUGDs compensates for the decreased UDP-glucuronic acid pool due to consumption during saponin biosynthesis.

We herein report experimental applications of a novel, automated computational approach to chemical reaction network (CRN) identification. This report shows the first chemical applications of an autonomous tool to identify the kinetic model and parameters of a process, when considering both catalytic species and various integer and non-integer orders in the model's rate laws. This kinetic analysis methodology requires only the input of the species within the chemical system (starting materials, intermediates, products, etc.) and corresponding time-series concentration data to determine the kinetic information of the chemistry of interest. This is performed with minimal human interaction and several case studies were performed to show the wide scope and applicability of this process development tool. The approach described herein can be employed using experimental data from any source and the code for this methodology is also provided open-source., We herein report experimental applications of a novel, automated computational approach to chemical reaction network (CRN) identification.

Antibody-drug conjugates (ADCs) harness the highly specific targeting capabilities of an antibody to deliver a cytotoxic payload to specific cell types. They have garnered widespread interest in drug discovery, particularly in oncology, as discrimination between healthy and malignant tissues or cells can be achieved. Nine ADCs have received approval from the US Food and Drug Administration and more than 80 others are currently undergoing clinical investigations for a range of solid tumours and haematological malignancies. Extensive research over the past decade has highlighted the critical nature of the linkage strategy adopted to attach the payload to the antibody. Whilst early generation ADCs were primarily synthesised as heterogeneous mixtures, these were found to have sub-optimal pharmacokinetics, stability, tolerability and/or efficacy. Efforts have now shifted towards generating homogeneous constructs with precise drug loading and predetermined, controlled sites of attachment. Homogeneous ADCs have repeatedly demonstrated superior overall pharmacological profiles compared to their heterogeneous counterparts. A wide range of methods have been developed in the pursuit of homogeneity, comprising chemical or enzymatic methods or a combination thereof to afford precise modification of specific amino acid or sugar residues. In this review, we discuss advances in chemical and enzymatic methods for site-specific antibody modification that result in the generation of homogeneous ADCs.

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., Saponins such as glycyrrhizin, a natural sweetener found in licorice root, are a class of triterpenoids synthesized that are characterized by a glucoronic acid moiety at the C-3 position. Here the authors show that saponin glucuronosylation is catalyzed by cellulose-synthase like enzymes and reconstitute glycyrrhizin synthesisin yeast.

Site-selective modification of peptides and proteins has resulted in the development of a host of novel tools for the study of cellular systems or the synthesis of enhanced biotherapeutics. There is a need for useful methodologies that enable site-selective modification of native peptides or proteins, which is even more prevalent when modification of the biomolecule with multiple payloads is desired. Herein, we report the development of a novel dual functional divinylpyrimidine (dfDVP) platform that enables robust and modular modification of peptides, antibody fragments and antibodies. These biomacromolecules could be easily functionalised with a range of functional payloads (e.g. fluorescent dyes, cytotoxic warheads or cell-penetrating tags). Importantly, the dual functionalised peptides and antibodies demonstrated exquisite bioactivity in a range of in vitro cellular assays, showcasing the enhanced utility of these bioactive conjugates.

Summary Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side‐effects. Here, we report that UDP‐glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP‐glucuronic acid to glycyrrhetinic acid 3‐O‐monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin‐deficient (83‐555) strain of G. uralensis. The natural variant showed loss of specificity for UDP‐glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83‐555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP‐glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP‐glucuronic acid. The functional arginine residue and resultant specificity for UDP‐glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP‐sugar selectivity for the rational engineering of sweet triterpenoid saponins., Significance Statement Glycyrrhizin, a licorice‐derived sweet triterpenoid diglycoside, is one of the most important phytoconstituents in the pharmaceutical and food industries. Here, we identified UGT73P12 as the enzyme catalyzing the sugar–sugar linkage to produce glycyrrhizin from its less glycosylated precursor in the presence of UDP‐glucuronic acid, and found that Arg32 of UGT73P12 is essential for the high specificity for UDP‐glucuronic acid, which was probably acquired during evolution to enable the functional specialization of UGT73P12 for glycyrrhizin biosynthesis.

Triterpenoids are widely distributed among plants of the legume family. However, most studies have focused on triterpenoids and their biosynthetic enzymes in model legumes. We evaluated the triterpenoid aglycones profile of the medicinal legume tree Bauhinia forficata by gas chromatography-mass spectrometry. Through transcriptome analyses, homology-based cloning, and heterologous expression, we discovered four oxidosqualene cyclases (OSCs) which are responsible for the diversity of triterpenols in B. forficata. We also investigated the effects of the unique motif TLCYCR on α-amyrin synthase activity. B. forficata highly accumulated α-amyrin. We discovered an OSC with a preponderant α-amyrin-producing activity, which accounted for at least 95% of the total triterpenols. We also discovered three other functional OSCs (BfOSC1, BfOSC2, and BfOSC4) that produce β-amyrin, germanicol, and cycloartenol. Furthermore, by replacing the unique motif TLCYCR from BfOSC3 with the MWCYCR motif, we altered the function of BfOSC3 such that it no longer produced α-amyrin. Our results provide new insights into OSC cyclization, which is responsible for the diversity of triterpenoid metabolites in B. forficata, a non-model legume plant.

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.

Uridine 5′-diphosphate (UDP)-glucose dehydrogenase (UGD) produces UDP-glucuronic acid from UDP-glucose as a precursor of plant cell wall polysaccharides. UDP-glucuronic acid is also a sugar donor for the glycosylation of various plant specialized metabolites. Nevertheless, the roles of UGDs in plant specialized metabolism remain poorly understood. Glycyrrhiza species (licorice), which are medicinal legumes, biosynthesize triterpenoid saponins, soyasaponins and glycyrrhizin, commonly glucuronosylated at the C-3 position of the triterpenoid scaffold. Often, several different UGD isoforms are present in plants. To gain insight into potential functional differences among UGD isoforms in triterpenoid saponin biosynthesis in relation to cell wall component biosynthesis, we identified and characterized Glycyrrhiza uralensis UGDs (GuUGDs), which were discovered to comprise five isoforms, four of which (GuUGD1–4) showed UGD activity in vitro. GuUGD1–4 had different biochemical properties, including their affinity for UDP-glucose, catalytic constant, and sensitivity to feedback inhibitors. GuUGD2 had the highest catalytic constant and highest gene expression level among the GuUGDs, suggesting that it is the major isoform contributing to the transition from UDP-glucose to UDP-glucuronic acid in planta. To evaluate the contribution of GuUGD isoforms to saponin biosynthesis, we compared the expression patterns of GuUGDs with those of saponin biosynthetic genes in methyl jasmonate (MeJA)-treated cultured stolons. GuUGD1–4 showed delayed responses to MeJA compared to those of saponin biosynthetic genes, suggesting that MeJA-responsive expression of GuUGDs compensates for the decreased UDP-glucuronic acid pool due to consumption during saponin biosynthesis.

Artemisinin, a sesquiterpene lactone exhibiting effective antimalarial activity, is produced by only Artemisia annua plant. A key step in artemisinin biosynthesis is the cyclization of farnesyl pyrophosphate (FPP) to amorpha-4,11-diene catalyzed by amorpha-4,11-diene synthase (AaADS). Intriguingly, several non-artemisinin-producing Artemisia plants also express genes highly homologous to AaADS. Our previous functional analysis of these homologous enzymes revealed that they catalyzed the synthesis of rare natural sesquiterpenoids. In this study, we analyzed the function of another putative sesquiterpene synthase highly homologous to AaADS from A. maritima. Unlike AaADS, in vivo enzymatic assay showed that this enzyme cyclized FPP to 4-amorphen-11-ol, a precursor of several gastroprotective agents. The discovery of 4-amorphen-11-ol synthase (AmAOS) and the successful de novo production of 4-amorphen-11-ol in engineered yeast demonstrated herein provides insights into the methods used to enhance its production for future application.

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.

Artemisinin, a sesquiterpene lactone exhibiting effective antimalarial activity, is produced by only Artemisia annua plant. A key step in artemisinin biosynthesis is the cyclization of farnesyl pyrophosphate (FPP) to amorpha-4,11-diene catalyzed by amorpha-4,11-diene synthase (AaADS). Intriguingly, several non-artemisinin-producing Artemisia plants also express genes highly homologous to AaADS. Our previous functional analysis of these homologous enzymes revealed that they catalyzed the synthesis of rare natural sesquiterpenoids. In this study, we analyzed the function of another putative sesquiterpene synthase highly homologous to AaADS from A. maritima. Unlike AaADS, in vivo enzymatic assay showed that this enzyme cyclized FPP to 4-amorphen-11-ol, a precursor of several gastroprotective agents. The discovery of 4-amorphen-11-ol synthase (AmAOS) and the successful de novo production of 4-amorphen-11-ol in engineered yeast demonstrated herein provides insights into the methods used to enhance its production for future application.

Several enzymes of the CYP716A subfamily have been reported to be involved in triterpenoid biosynthesis. Members of this subfamily oxidize various positions along the triterpenoid backbone and the majority of them catalyze a three-step oxidation at the C-28 position. Interestingly, C-28 oxidation is a common feature in oleanolic acid, ursolic acid, and betulinic acid, which are widely distributed in plants and exhibit important biological activities. In this work, three additional CYP716A enzymes isolated from olive, sugar beet, and coffee, were characterized as multifunctional C-28 oxidases. Semi-quantitative comparisons of in vivo catalytic activity were made against the previously characterized enzymes CYP716A12, CYP716A15, and CYP716A52v2. When heterologously expressed in yeast, the isolated enzymes differed in both catalytic activity and substrate specificity. This study indicates that the screening of enzymes from different plants could be a useful means of identifying enzymes with enhanced catalytic activity and desired substrate specificity. Furthermore, we show that "naturally-evolved" enzymes can be useful in the heterologous production of pharmacologically and industrially important triterpenoids.

Morolic acid is a plant‐derived triterpenoid that possesses pharmacological properties such as cytotoxicity, as well as anti‐HIV, anti‐HSV, anti‐inflammatory, and antidiabetic effects. The significant therapeutic properties of morolic acid are desirable in the context of pharmacological and drug development research, but the low accessibility of morolic acid from natural resources limits its applications. In the present study, we developed a microbial system for the production of morolic acid. Using a combinatorial biosynthesis approach, a novel production pathway was constructed in Saccharomyces cerevisiae by co‐expressing BfOSC2 (germanicol synthase) from Bauhinia forficata and CYP716A49 (triterpene C‐28 oxidase) from Beta vulgaris. Moreover, we reconstructed the cellular galactose regulatory network by introducing a chimeric transcriptional activator (fusion of Gal4dbd.ER.VP16, GEV) to overdrive the genes under the control of the galactose promoter. We also overexpressed truncated HMG1, encoding feedback‐inhibition‐resistant form of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase 1 and sterol‐regulating transcription factor upc2‐1, to increase the isoprenoid precursors in the mevalonate pathway. Using this yeast system, we achieved morolic acid production up to 20.7 ± 1.8 mg/L in batch culture. To our knowledge, this is the highest morolic acid titer reported from a heterologous host, indicating a promising approach for obtaining rare natural triterpenoids. This article is protected by copyright. All rights reserved.

Plants produce various prenylated phenolic metabolites, including flavonoids, phloroglucinols, and coumarins, many of which have multiple prenyl moieties and display various biological activities. Prenylated phenylpropanes, such as artepillin C (3, 5-diprenyl-p-coumaric acid), exhibit a broad range of pharmaceutical effects. To date, however, no prenyltransferases (PTs) involved in the biosynthesis of phenylpropanes and no plant enzymes that introduce multiple prenyl residues to native substrates with different regio-specificities have been identified. This study describes the isolation from Artemisia capillaris of a phenylpropane-specific PT gene, AcPT1, belonging to UbiA superfamily. This gene encodes a membrane-bound enzyme, which accepts p-coumaric acid as its specific substrate and transfers two prenyl residues stepwise to yield artepillin C. These findings provide novel insights into the molecular evolution of this gene family, contributing to the chemical diversification of plant specialized metabolites. These results also enabled the design of a yeast platform for the synthetic biology of artepillin C., アルテピリンC合成酵素の発見とその生産 --雑草の遺伝子から生理活性物質の生産へ--. 京都大学プレスリリース. 2019-10-25.

Triquinane is a type of sesquiterpenoid with a unique structure that contains a fused tricyclopentane ring and exhibits a wide range of bioactivities. Like other sesquiterpenoids, the first committed step in triquinane-type sesquiterpenoid biosynthesis is the cyclization of farnesyl pyrophosphate (FPP), a common precursor of all sesquiterpenoids, catalyzed by sesquiterpene synthase. Artemisia abrotanum L. (Asteraceae), a common plant used in the culinary and cosmetics industries, has been reported to accumulate high levels of triquinane silphiperfol-5-en-3-one A. This compound is potentially biosynthesized from the cyclization of FPP into 7-epi-silphiperfol-5-ene followed by a multi-step oxidation to silphiperfol-5-en-3-one A. In this study, we aimed to identify the sesquiterpene synthase responsible for the synthesis of 7-epi-silphiperfol-5-ene. We performed RNA sequencing of A. abrotanum leaves and gene candidates were mined by homology searches using the triquinane α-isocomene synthase of chamomile (MrTPS2) as query. After gene cloning, we obtained five variants of putative sesquiterpene synthase showing greater than 85% amino acid identity to MrTPS2 and greater than 95% amino acid identity to each other. Heterologous expression of these variants in a FPP-high-producing yeast strain revealed the first four variants to be (+)-α-bisabolol synthases (AabrBOS1-4). However, the fifth candidate cyclized FPP into 7-epi-silphiperfol-5-ene and can therefore be defined as a 7-epi-silphiperfol-5-ene synthase (AabrSPS). These findings revealed the first committed enzyme involved in silphiperfol-5-en-3-one A and (+)-α-bisabolol biosyntheses in A. abrotanum. Furthermore, the results of this study will be useful for enhancing the production of these compounds for further applications.

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 biosynthetic pathway of cytosolic isoprenoids bifurcates after farnesyl diphosphate into sesquiterpene and triterpene pathways. “Metabolic switching” has been used to increase sesquiterpene content in plants by suppressing the competitive triterpene pathway using transgenic technology. To develop “metabolic switching” without using transgenic technology, we developed a model system of “chemical metabolic switching” using inhibitors of the competitive pathway. Arabidopsis plants that overexpress the amorpha-4,11-diene synthase gene were treated with squalestatin, a squalene synthase inhibitor, or terbinafine, a squalene epoxidase inhibitor. We then analyzed total sterol content as major triterpenes and amorpha-4,11-diene in the plant. Plants treated with squalestatin showed decreased total sterol content and increased amorpha-4,11-diene content. In contrast, plants treated with terbinafine showed decreased total sterol content, but amorpha-4,11-diene accumulation was quite low. These results suggest that inhibition of the enzyme just below the branch point is more effective than inhibition of enzymes far from the branch point for “chemical metabolic switching”. In addition, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme of the cytosolic isoprenoid biosynthetic pathway, was upregulated in plants treated with squalestatin, suggesting that feedback regulation of 3-hydroxy-3-methylglutaryl-CoA reductase may contribute to amorpha-4,11-diene production. Here we demonstrated the effectiveness of “chemical metabolic switching” in plants.

CYP716A179, a cytochrome P450 monooxygenase expressed predominantly in tissue-cultured stolons of licorice ( Glycyrrhiza uralensis ), functions as a triterpene C-28 oxidase in the biosynthesis of oleanolic acid and betulinic acid. Cytochrome P450 monooxygenases (P450s) play key roles in the structural diversification of plant triterpenoids. Among these, the CYP716A subfamily, which functions mainly as a triterpene C-28 oxidase, is common in plants. Licorice (Glycyrrhiza uralensis) produces bioactive triterpenoids, such as glycyrrhizin and soyasaponins, and relevant P450s (CYP88D6, CYP72A154, and CYP93E3) have been identified; however, no CYP716A subfamily P450 has been isolated. Here, we identify CYP716A179, which functions as a triterpene C-28 oxidase, by RNA sequencing analysis of tissue-cultured stolons of G. uralensis. Heterologous expression of CYP716A179 in engineered yeast strains confirmed the production of oleanolic acid, ursolic acid, and betulinic acid from β-amyrin, α-amyrin, and lupeol, respectively. The transcript level of CYP716A179 was about 500 times higher in tissue-cultured stolons than in intact roots. Oleanolic acid and betulinic acid were consistently detected only in tissue-cultured stolons. The discovery of CYP716A179 helps increase our understanding of the mechanisms of tissue-type-dependent triterpenoid metabolism in licorice and provides an additional target gene for pathway engineering to increase the production of glycyrrhizin in licorice tissue cultures by disrupting competing pathways.

Chinese liquorice/licorice (Glycyrrhiza uralensis) is a leguminous plant species whose roots and rhizomes have been widely used as a herbal medicine and natural sweetener. Whole-genome sequencing is essential for gene discovery studies and molecular breeding in liquorice. Here, we report a draft assembly of the approximately 379-Mb whole-genome sequence of strain 308-19 of G. uralensis; this assembly contains 34 445 predicted protein-coding genes. Comparative analyses suggested well-conserved genomic components and collinearity of gene loci (synteny) between the genome of liquorice and those of other legumes such as Medicago and chickpea. We observed that three genes involved in isoflavonoid biosynthesis, namely, 2-hydroxyisoflavanone synthase (CYP93C), 2,7,4'-trihydroxyisoflavanone 4'-O-methyltransferase/isoflavone 4'-O-methyltransferase (HI4OMT) and isoflavone-7-O-methyltransferase (7-IOMT) formed a cluster on the scaffold of the liquorice genome and showed conserved microsynteny with Medicago and chickpea. Based on the liquorice genome annotation, we predicted genes in the P450 and UDP-dependent glycosyltransferase (UGT) superfamilies, some of which are involved in triterpenoid saponin biosynthesis, and characterised their gene expression with the reference genome sequence. The genome sequencing and its annotations provide an essential resource for liquorice improvement through molecular breeding and the discovery of useful genes for engineering bioactive components through synthetic biology approaches.

α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry.

Dimunito/Dwarf1 (DWF1) is an oxidoreductase enzyme that is responsible for the conversion of C28- and C29-Δ24(28)-olefinic sterols to 24-methyl- and 24-ethylcholesterols. Generally, the reaction proceeds in two steps via the Δ24(25) intermediate. In this study, we characterized the ArDWF1 gene from an expression sequence tag library of Ajuga reptans var. atropurpurea hairy roots. The gene was functionally expressed in the yeast T21 strain. The in vivo and in vitro study of the transformed yeast indicated that ArDWF1 catalyzes the conversion of 24-methylenecholesterol to campesterol. A labeling study followed by GC-MS analysis suggested that the reaction proceeded with retention of the C-25 hydrogen. The 25-H retention was established by the incubation of the enzyme with (23,23,25-2H3,28-13C)-24-methylenecholesterol, followed by 13C NMR analysis of the resulting campesterol. Thus, it has been concluded that ArDWF1 directly reduces 24-methylenecholesterol to produce campesterol without passing through a Δ24(25) intermediate. This is the first characterization of such a unique DWF1 enzyme. For comparison purposes, Oryza sativa DWF1 (OsDWF1) was similarly expressed in yeast. An in vivo assay of OsDWF1 supported the generally accepted two-step mechanism because the C-25 hydrogen of 24-methylenecholesterol was eliminated during its conversion to 24-methylcholesterol. As expected, the 24-methylcholesterol produced by OsDWF1 was a mixture of campesterol and dihydrobrassicasterol. Furthermore, the 24-methylcholesterol contained in the Ajuga hairy roots was determined to be solely campesterol through its analysis using chiral GC-MS. Therefore, ArDWF1 has another unique property in that only campesterol is formed by the direct reduction catalyzed by the enzyme.

Lotus japonicus is an important model legume plant in several fields of research, such as secondary (specialized) metabolism and symbiotic nodulation. This plant accumulates triterpenoids; however, less information regarding its composition, content and biosynthesis is available compared with Medicago truncatula and Glycine max. In this study, we analyzed the triterpenoid content and composition of L. japonicus. Lotus japonicus accumulated C-28-oxidized triterpenoids (ursolic, betulinic and oleanolic acids) and soyasapogenols (soyasapogenol B, A and E) in a tissue-dependent manner. We identified an oxidosqualene cyclase (OSC) and two cytochrome P450 enzymes (P450s) involved in triterpenoid biosynthesis using a yeast heterologous expression system. OSC9 was the first enzyme derived from L. japonicus that showed α-amyrin (a precursor of ursolic acid)-producing activity. CYP716A51 showed triterpenoid C-28 oxidation activity. LjCYP93E1 converted β-amyrin into 24-hydroxy-β-amyrin, a metabolic intermediate of soyasapogenols. The involvement of the identified genes in triterpenoid biosynthesis in L. japonicus plants was evaluated by quantitative real-time PCR analysis. Furthermore, gene loss-of-function analysis of CYP716A51 and LjCYP93E1 was conducted. The cyp716a51-mutant L. japonicus hairy roots generated by the genome-editing technique produced no C-28 oxidized triterpenoids. Likewise, the complete abolition of soyasapogenols and soyasaponin I was observed in mutant plants harboring Lotus retrotransposon 1 (LORE1) in LjCYP93E1. These results indicate that the activities of these P450 enzymes are essential for triterpenoid biosynthesis in L. japonicus. This study increases our understanding of triterpenoid biosynthesis in leguminous plants and provides information that will facilitate further studies of the physiological functions of triterpenoids using L. japonicus.

Grey mangrove (Avicennia marina) is a traditional medicine used for the treatment of various diseases, including rheumatism and ulcers; however, the compounds responsible for its curative effects remain largely unknown. Triterpenoids are a diverse group of plant-specialized metabolites derived from a common precursor, 2,3-oxidosqualene. Triterpenoids are potentially responsible for the beneficial effects of A. marina; however, the chemical profiles of triterpenoids in A. marina and their biosynthetic genes have not been identified. Cytochrome P450 monooxygenases (P450s) have key roles in the structural diversification of plant triterpenoids by catalyzing site-specific oxidation of triterpene scaffolds. Recent studies have revealed that the CYP716 family represents the most common clade of P450s involved in triterpenoid biosynthesis. In this study, we performed triterpenoid profiling and RNA sequencing of A. marina leaves. Mining of CYP716 family genes and enzymatic activity assays of encoded proteins revealed that CYP716A259 catalyzed oxidation at the C-28 position of the pentacyclic triterpene skeletons of β-amyrin, α-amyrin, and lupeol to produce oleanolic acid, ursolic acid, and betulinic acid, respectively. The other functionally defined P450, CYP716C53, catalyzed the C-2α hydroxylation of oleanolic acid and ursolic acid to produce maslinic acid and corosolic acid, respectively. The possible involvement of CYP716A259 and CYP716C53 in the biosynthesis of these health-benefiting compounds in A. marina leaves, and the possible contribution of the resulting compounds to the reported bioactivities of A. marina leaf extract, are discussed.

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.

Increased public awareness of negative health effects associated with excess sugar consumption has triggered increasing interest in plant-derived natural sweeteners. Steviol glycosides are a group of highly sweet diterpene glycosides contained in the leaves of stevia (Stevia rebaudiana). Mogrosides, extracted from monk fruit (Siraitia grosvenorii), are a group of cucurbitane-type triterpenoid glycosides. Glycyrrhizin is an oleanane-type triterpenoid glycoside derived from the underground parts of Glycyrrhiza plants (licorice). This review focuses on the natural isoprenoid sweetening agents steviol glycosides, mogrosides, and glycyrrhizin, and describes recent progress in gene discovery and elucidation of the catalytic functions of their biosynthetic enzymes. Recently, remarkable progress has been made in engineering the production of various plant-specialized metabolites in microbial hosts such as Saccharomyces cerevisiae via the introduction of biosynthetic enzyme genes. Perspectives on the microbial production of plant-derived natural sweeteners are also discussed.

Lignans are a class of phenylpropanoids that are widely distributed in the plant kingdom and some lignans are known to be present as glycosides. In the model plant Arabidopsis thaliana, pinoresinol and lariciresinol as well as their glucosides are found to be accumulated in the roots, but the enzymes involved in the glucosylation of lignans remain to be characterized. UGT71C1 showed activity towards several phenylpropanoids in previous studies, although its activity towards lignans has not been investigated. In the present study, the involvement of UGT71C1 in lignan glucosylation was examined. Quantification of lignans in a T-DNA knockout line of the UGT71C1 gene, ugt71c1, by an ultra performance liquid chromatography-tandem mass spectrometry showed that the content of pinoresinol glucoside decreased in parallel with an increase of pinoresinol at the corresponding degree. Two major peaks corresponding to lariciresinol glucosides were detected in the mass chromatogram of the extract from the wild type and one of the peaks decreased in the ugt71c1 line suggesting that the amount of lariciersinol glucoside also decreased in the mutant. UGT71C1 expressed in Escherichia coli showed glucosyltransferase activity towards pinoresinol and lariciresinol. The present results suggest that UGT71C1 is involved in lignan glucosylation in A. thaliana.

Triterpene saponins are widely distributed in higher plants [1,2] and belong to a class of natural products that includes various bioactive compounds found in medicinal plants [3]. The family Leguminosae, which includes plants such as licorice (Glycyrrhiza uralensis) [4], soybean (Glycine max) [5] and alfalfa (Medicago sativa) [6], are well-known triterpene saponin-producing plants. The roots and stolons of licorice accumulate glycyrrhizin as a major bioactive triterpene saponin, while the hypocotyls of soybean accumulate soyasaponin I. Glycyrrhizin and soyasaponin I are synthesized from a common biosynthetic intermediate, the triterpene aglycone b-amyrin, derived from the initial cyclization of 2,3-oxidosqualene. Licorice b-amyrin 11-hydroxylase (CYP88D6) [7] and b-amyrin 30-hydroxylase (CYP72A154) [8] catalyze the oxidation of b-amyrin at positions C-11 (two-step oxidation) and C-30 (three-step oxidation), respectively, to produce glycyrrhetinic acid, the triterpene aglycone of glycyrrhizin. In contrast, soybean b-amyrin 22-hydroxylase [unpublished] and b-amyrin 24-hydroxylase (CYP93E1) [9] catalyze the oxidation of b-amyrin at positions C-22 (one-step oxidation) and C-24 (one-step oxidation), respectively, to produce soyasapogenol B (22-hydroxy, 24-hydroxyb-amyrin), the triterpene aglycone of soyasaponin I (Figure 12.1). Construction of vector plasmids for multi-gene transformation is an essential technique for pathway engineering of secondary metabolites. The construction of complex plasmids for expressing multiple genes is considered to be a timeconsuming process, but we were able to reduce the difficulty by connecting the gene cassettes in tandem at the restriction sites of homing endonucleases [10], which recognize 18 to 39 specific bases, and using a polymerase chain reaction (PCR) technique based on primer overlap extension [11]. The terminator regulates the level of expression by controlling transcriptional termination and 30-endprocessing of mRNA. Arabidopsis heat shock protein18.2

Triterpenoids are a group of structurally diverse specialized metabolites that frequently show useful bioactivities. These chemicals are biosynthesized from the common precursor 2,3-oxidosqualene in plants. The carbon skeletons produced by oxidosqualene cyclase (OSC) are usually modified by cytochrome P450 monooxygenases (P450s) and UDP-dependent glycosyltransferases. These biosynthetic enzymes contribute to the structural diversification of plant triterpenoids. Until now, many P450 enzymes have been characterized as triterpenoid oxidases. Among them, the CYP716 family P450 enzymes, which have been isolated from a wide range of plant families, seem to contribute to the triterpenoid structural diversification. Many CYP716 family P450 enzymes have been characterized as the multifunctional triterpene C-28 oxidases, which oxidize α-amyrin and β-amyrin to the widely distributed triterpenoids ursolic and oleanolic acids, respectively. Tomato (Solanum lycopersicum) is one of the most important solanaceous crops in the world. However, little information is known regarding its triterpenoid biosynthesis. To understand the mechanism of triterpenoid biosynthesis in tomato, we focused on the function of CYP716 family enzymes as triterpenoid oxidases. We isolated all six CYP716 family genes from the Micro-Tom cultivar of tomato, and functionally characterized them in the heterologous yeast expression system. The in vivo enzymatic assays showed that CYP716A44 and CYP716A46 exhibited the ordinary C-28 oxidation activity against α-amyrin and β-amyrin to produce ursolic and oleanolic acids, respectively. Interestingly, one CYP716E subfamily enzyme, CYP716E26, exhibited the previously unreported C-6β hydroxylation activity against β-amyrin to produce a rare bioactive triterpenoid, daturadiol (olean-12-ene-3β,6β-diol). To determine the roles of the CYP716 family genes in tomato triterpenoid biosynthesis, we analyzed the gene expression and triterpenoid accumulation patterns in different plant tissues by performing the quantitative real-time polymerase chain reaction (qPCR) and gas chromatography-mass spectrometry (GC-MS) analyses, respectively. High levels of the CYP716A44 gene expression and the accumulation of C-28-oxidized triterpenoids, ursolic acid, and oleanolic acid were observed in the roots, indicating a significant contribution of the CYP716A44 gene in the triterpenoid biosynthesis in tomato. Thus, our study partially elucidated the mechanism of triterpenoid biosynthesis in tomato, and identified CYP716E26 as a novel C-6β hydroxylase for its subsequent use in the combinatorial biosynthesis of bioactive triterpenoids.

Artemisinin is an antimalarial sesquiterpenoid isolated from the aerial parts of the plant Artemisia annua. CYP71AV1, a cytochrome P450 monooxygenase was identified in the artemisinin biosynthetic pathway. CYP71AV1 catalyzes three successive oxidation steps at the C12 position of amorpha-4,11-diene to produce artemisinic acid. In this study, we isolated putative CYP71AV1 orthologs in different species of Artemisia. Comparative functional analysis of CYP71AV1 and its putative orthologs, together with homology modeling, enabled us to identify an amino acid residue (Ser479) critical for the second oxidation reaction catalyzed by CYP71AV1. Our results clearly show that a comparative study of natural variants is useful to investigate the structure–function relationships of CYP71AV1.

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.

Glycyrrhizin, a triterpenoid saponin derived from the underground parts of Glycyrrhiza plants (licorice), has several pharmacological activities and is also used worldwide as a natural sweetener. The biosynthesis of glycyrrhizin involves the initial cyclization of 2,3-oxidosqualene to the triterpene skeleton β-amyrin, followed by a series of oxidative reactions at positions C-11 and C-30, and glycosyl transfers to the C-3 hydroxyl group. We previously reported the identification of a cytochrome P450 monooxygenase (P450) gene encoding β-amyrin 11-oxidase (CYP88D6) as the initial P450 gene in glycyrrhizin biosynthesis. In this study, a second relevant P450 (CYP72A154) was identified and shown to be responsible for C-30 oxidation in the glycyrrhizin pathway. CYP72A154 expressed in an engineered yeast strain that endogenously produces 11-oxo-β-amyrin (a possible biosynthetic intermediate between β-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-β-amyrin supplied in situ to produce glycyrrhetinic acid, a glycyrrhizin aglycone. Furthermore, CYP72A63 of Medicago truncatula, which has high sequence similarity to CYP72A154, was able to catalyze C-30 oxidation of β-amyrin. These results reveal a function of CYP72A subfamily proteins as triterpene-oxidizing enzymes and provide a genetic tool for engineering the production of glycyrrhizin.

Triterpenoids are a diverse group of secondary metabolites that are associated with a variety of biological activities. Oleanolic acid, ursolic acid and betulinic acid are common triterpenoids in plants with diverse biological activities, including antifungal, antibacterial, anti-human immunodeficiency virus (HIV) and/or antitumor activities. In the present study, using the gene co-expression analysis tool of Medicago truncatula, we found a strong correlation between CYP716A12 and β-amyrin synthase (bAS), which encodes the enzyme responsible for the initial cyclization of 2,3-oxidosqualene to β-amyrin (the basic structural backbone of most triterpenoid saponins). Through an in vitro assay, we identified CYP716A12 as a β-amyrin 28-oxidase able to modify β-amyrin to oleanolic acid (through erythrodiol and, possibly, oleanolic aldehyde). We also confirmed its activity in vivo, by expressing CYP716A12 in transgenic yeast that endogenously produce β-amyrin. In addition, CYP716A12 was evaluated for its potential α-amyrin- and lupeol-oxidizing activities. Interestingly, CYP716A12 was able to generate ursolic acid (through uvaol and, possibly, ursolic aldehyde) and betulinic acid (through betulin). Hence, CYP716A12 was characterized as a multifunctional enzyme with β-amyrin 28-oxidase, α-amyrin 28-oxidase and lupeol 28-oxidase activities. We also identified homologs of CYP716A12 in grape (CYP716A15 and CYP716A17) that are involved in triterpenoid biosynthesis, which indicates the highly conserved functionality of the CYP716A subfamily among plants. These findings will be useful in the heterologous production of pharmacologically and industrially important triterpenoids, including oleanolic acid, ursolic acid and betulinic acid.

Dried roots and stolons of licorice plants (Glycyrrhiza uralensis) are among the most important drugs (Glycyrrhizae radix) in traditional oriental medicine; they are also important commercial products used worldwide in sweetening and flavoring. Here, we describe the establishment of an in vitro stolon culture system for G. uralensis. Stolon formation was induced in single node stems (with axillary buds) grown in Murashige and Skoog (MS) liquid culture medium, supplemented with 0.01 μM α-naphthaleneacetic acid (NAA). Stolons were cultured at 26°C in the dark on a rotary shaker, gyrating at 100 rpm. The same NAA concentration produced the highest rates of stolon proliferation (6.58-fold in 4 weeks). A 6% sucrose concentration also enhanced stolon proliferation (6.34-fold in 4 weeks). GC/MS analysis confirmed the accumulation of small amounts of glycyrrhizin (14 μg g−1 dry weight) in cultured stolons. Interestingly, betulinic acid and oleanoic acid production in vitro were higher than in field-grown stolons. Adventitious root and shoot regeneration from cultured stolons were readily achieved under illuminated conditions in MS medium containing 0.01 μM of NAA and 0.2% gerlite. Regenerated plants produced glycyrrhizin (7,600 μg g−1 dry weight) in their roots. Our in vitro stolon culture system is suitable for studying glycyrrhizin biosynthesis and for rapid propagation of elite licorice clones.

Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells are rapidly proliferating meristematic cells that require auxin for culture in vitro. We have established several transgenic BY-2 cell lines that carry the T-DNA of Agrobacterium rhizogenes 15834, which harbors an agropine-type root-inducing (Ri) plasmid. Two of these lines, BYHR-3 and BYHR-7, were used to test the role of auxin in the proliferation of plant cells. The lines grew rapidly in Linsmaier-Skoog (LS) medium lacking auxin and other phytohormones. The TR-DNA, containing the aux1 (tryptophan monooxygenase) and aux2 (indoleacetamide hydrolase) genes, was present in the genomes of both transgenic lines, whereas the TL-DNA, containing the rolA, B, C and D genes, was present in the genome of BYHR-7 but not BYHR-3. Since the introduction of the rolABCD genes alone did not affect the auxin requirement of BY-2 cells, the aux1 and aux2 genes, but not the rolABCD genes, appear to be relevant to the auxin autotrophy of these transgenic lines. Furthermore, the overexpression of aux1 allowed BY-2 cells to grow rapidly in the absence of auxin, suggesting the existence in plant cells of an unidentified gene whose product is functionally equivalent or similar to that of aux2 of the Ri plasmid.

Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells can be grown in medium containing indole-3-acetamide (IAM). Based on this finding, the NtAMI1 gene, whose product is functionally equivalent to the AtAMI1 gene of Arabidopsis thaliana and the aux2 gene of Agrobacterium rhizogenes, was isolated from BY-2 cells. Overexpression of the NtAMI1 gene allowed BY-2 cells to proliferate at lower concentrations of IAM, whereas suppression of the NtAMI1 gene by RNA interference (RNAi) caused severe growth inhibition in the medium containing IAM. These results suggest that IAM is incorporated into plant cells and converted to the auxin, indole-3-acetic acid, by NtAMI1.

The hairy root culture system has several desirable features as an experimental model for functional analyses of the genes involved in root metabolism and/or physiology. We developed a binary vector set for efficient target gene overexpression and RNA interference (RNAi) in transformed (hairy) roots. The vectors, pHR-OX and pHR-RNAi, contain a cluster of rol (rooting locus) genes, together with the single GATEWAYTM conversion cassette (in pHR-OX vectors) or inverted repeats of the GATEWAYTM conversion cassette separated by an intron sequence (in pHR-RNAi vectors), flanked by a dual CaMV 35S promoter and nopaline synthase polyadenylation signal, on the same T-DNA. Transformation experiments with pHR-OX vectors using Arabidopsis, potato, and tobacco as model plants revealed that by inoculating Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants in a short period of time, and subsequent establishment of root culture lines was possible. A model experiment that focused on the sterol biosynthetic pathway in Arabidopsis validated the utility of pHR-RNAi vectors for gene functional analysis in cultured roots. Use of our vector system may facilitate identification of regulatory or biosynthetic genes for the production of valuable secondary metabolites in plant roots.