Application of multiple strategies to enhance oleanolic acid biosynthesis by engineered Saccharomyces cerevisiae.

Oleanolic acid and its derivatives are widely used in the pharmaceutical, agricultural, cosmetic and food industries. Previous studies have shown that oleanolic acid production levels in engineered cell factories are low, which is why oleanolic acid is still widely extracted from traditional medicinal plants. To construct a highly efficient oleanolic acid production strain, rate-limiting steps were regulated by inducible promoters and the expression of key genes in the oleanolic acid synthetic pathway was enhanced. Subsequently, precursor pool expansion, pathway refactoring and diploid construction were considered to harmonize cell growth and oleanolic acid production. The multi-strategy combination promoted oleanolic acid production of up to 4.07 g/L in a 100 L bioreactor, which was the highest level reported.

Engineering Saccharomyces cerevisiae for Hyperproduction of ß-Amyrin by Mitigating the Inhibition Effect of Squalene on ß-Amyrin Synthase.

The study aims to enhance ß-amyrin production in Saccharomyces cerevisiae by peroxisome compartmentalization. First, overaccumulated squalene was determined as a key limiting factor for the production of ß-amyrin since it could inhibit the activity of ß-amyrin synthase GgbAs1. Second, to mitigate the inhibition effect, the enhanced squalene synthesis pathway was compartmentalized into peroxisomes to insulate overaccumulated squalene from GgbAs1, and thus the specific titer of ß-amyrin reached 57.8 mg/g dry cell weight (DCW), which was 2.6-fold higher than that of the cytosol engineering strain. Third, by combining peroxisome compartmentalization with the "push-pull-restrain" strategy (ERG1 and GgbAs1 overexpression and ERG7 weakening), the production of ß-amyrin was further increased to 81.0 mg/g DCW (347.0 mg/L). Finally, through fed-batch fermentation in a 5 L fermenter, the titer of ß-amyrin reached 2.6 g/L, which is the highest reported to date. The study provides a new perspective to engineering yeasts as a platform for triterpene production.

The Methionine 549 and Leucine 552 Residues of Friedelin Synthase from Maytenus ilicifolia Are Important for Substrate Binding Specificity.

Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and ß-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.

De Novo Biosynthesis of the Oleanane-Type Triterpenoids of Tunicosaponins in Yeast.

Tunicosaponins are natural products extracted from Psammosilene tunicoides, which is an important ingredient of Yunnan Baiyao Powder, an ancient and famous Asian herbal medicine. The representative aglycones of tunicosaponins are the oleanane-type triterpenoids of gypsogenin and quillaic acid, which were found to manipulate a broad range of virus-host fusion via wrapping the heptad repeat-2 (HR2) domain prevalent in viral envelopes. However, the unknown biosynthetic pathway and difficulty in chemical synthesis hinder the therapeutic use of tunicosaponins. Here, two novel cytochrome P450-dependent monooxygenases that take part in the biosynthesis of tunicosaponins, CYP716A262 (CYP091) and CYP72A567 (CYP099), were identified from P. tunicoides. In addition, the whole biosynthesis pathway of the tunicosaponin aglycones was reconstituted in yeast by transforming the platform strain BY-bAS with the CYP716A262 and CYP716A567 genes, the resulting strain could produce 146.84 and 314.01 mg/L of gypsogenin and quillaic acid, respectively. This synthetic biology platform for complicated metabolic pathways elucidation and microbial cell factories construction can provide alternative sources of important natural products, helping conserve natural plant resources.

Recent Progress in Saikosaponin Biosynthesis in Bupleurum.

BACKGROUND: Chaihu is a popular traditional Chinese medicine that has been used for centuries. It is traditionally used to treat cold fever and liver-related diseases. Saikosaponins (SSs) are one of the main active components of chaihu, in addition to essential oils, flavonoids, and polysaccharides. Considerable effort is needed to reveal the biosynthesis and regulation of SSs on the basis of current progress. OBJECTIVE: The aim of this study is to provide a reference for further studies and arouse attention by summarizing the recent achievements of SS biosynthesis. METHODS: All the data compiled and presented here were obtained from various online resources, such as PubMed Scopus and Baidu Scholar in Chinese, up to October 2019. RESULTS: A few genes of the enzymes of SSs participating in the biosynthesis of SSs were isolated. Among these genes, only the P450 gene was verified to catalyze the SS skeleton ß-amyrin synthase. Several UDP-glycosyltransferase genes were predicted to be involved in the biosynthesis of SSs. SSs could be largely biosynthesized in the phloem and then transported from the protoplasm, which is the biosynthetic site, to the vacuoles to avoid self-poisoning. As for the other secondary metabolites, the biosynthesis of SSs was strongly affected by environmental factors and the different species belonging to the genus of Bupleurum. Transcriptional regulation was studied at the molecular level. CONCLUSION: Profound discoveries in SSs may elucidate the mechanism of diverse the monomer formation of SSs and provide a reference for maintaining the stability of SS content in Radix Bupleuri.

Genome-Wide Identification of OSC Gene Family and Potential Function in the Synthesis of Ursane- and Oleanane-Type Triterpene in Momordica charantia.

The triterpenes in bitter gourd (Momordica charantia) show a variety of medicinal activities. Oxidosqualene cyclase (OSC) plays an indispensable role in the formation of triterpene skeletons during triterpene biosynthesis. In this study, we identified nine genes encoding OSCs from bitter gourd (McOSC1-9). Analyses of their expression patterns in different tissues suggested that characteristic triterpenoids may be biosynthesized in different tissues and then transported. We constructed a hairy root system in which McOSC7 overexpression led to an increased accumulation of camaldulenic acid, enoxolone, and quinovic acid. Thus, the overexpression of McOSC7 increased the active components content in bitter gourd. Our data provide an important foundation for understanding the roles of McOSCs in triterpenoid synthesis.

Molecular cloning and functional characterization of multiple ApOSCs from Andrographis paniculata.

Triterpenoids have been described in Andrographis paniculata. Oleanolic acid exhibits high biological activity and is widely used in the clinic, and ß-sitosterol not only has good biological activity but also plays an important physiological role in plants. However, analysis of the biosynthetic pathway of triterpenoids in Andrographis paniculata has not been reported. Here, we provide the first report of the isolation and identification of nine 2, 3-oxidosqualene cyclases (ApOSC3 to ApOSC11) from A. paniculata. The results showed that ApOSC4 represented a monofunctional synthase that could convert 2, 3-oxidosqualene to ß-amyrin. ApOSC5 as a bifunctional 2, 3-oxidosqualene cyclases, could transfer 2, 3-oxidosqualene to ß-amyrin and α-amyrin. ApOSC6 to ApOSC8 composed the multifunctional 2, 3-oxidosqualene cyclases that could convert 2, 3-oxidosqualene to ß-amyrin, α-amyrin and one or two undetermined triterpenoids. This study provides a better understanding of the biosynthetic pathway of triterpenoids in A. paniculata, and the discovery of multifunctional 2, 3-oxidosqualene cyclases ApOSC5 to ApOSC8 of the facilitates knowledge of the compounds diversity in A. paniculata.

A systematic comparison of triterpenoid biosynthetic enzymes for the production of oleanolic acid in Saccharomyces cerevisiae.

Triterpenoids are high-value plant metabolites with numerous applications in medicine, agriculture, food, and home and personal care products. However, plants produce triterpenoids in low abundance, and their complex structures make their chemical synthesis prohibitively expensive and often impossible. As such, the yeast Saccharomyces cerevisiae has been explored as an alternative means of production. An important triterpenoid is oleanolic acid because it is the precursor to many bioactive triterpenoids of commercial interest, such as QS-21 which is being evaluated as a vaccine adjuvant in clinical trials against HIV and malaria. Oleanolic acid is derived from 2,3-oxidosqualene (natively produced by yeast) via a cyclisation and a multi-step oxidation reaction, catalysed by a ß-amyrin synthase and a cytochrome P450 of the CYP716A subfamily, respectively. Although many homologues have been characterised, previous studies have used arbitrarily chosen ß-amyrin synthases and CYP716As to produce oleanolic acid and its derivatives in yeast. This study presents the first comprehensive comparison of ß-amyrin synthase and CYP716A enzyme activities in yeast. Strains expressing different homologues are compared for production, revealing 6.3- and 4.5-fold differences in ß-amyrin and oleanolic acid productivities and varying CYP716A product profiles, which are important to consider when engineering strains for the production of bioactive oleanolic acid derivatives.

NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in Achyranthes bidentata Bl.

Application of plant growth regulators has become one of the most important means of improving yield and quality of medicinal plants. To understand the molecular basis of phytohormone-regulated oleanolic acid metabolism, RNA-seq was used to analyze global gene expression in Achyranthes bidentata treated with 2.0 mg/L 1-naphthaleneacetic acid (NAA) and 1.0 mg/L 6-benzyladenine (6-BA). Compared with untreated controls, the expression levels of 20,896 genes were significantly altered with phytohormone treatment. We found that 13071 (62.5%) unigenes were up-regulated, and a lot of differentially expressed genes involved in hormone or terpenoid biosynthesis, or transcription factors were significantly up-regulated. These results suggest that oleanolic acid biosynthesis induced by NAA and 6-BA occurs due to the expression of key genes involved in jasmonic acid signal transduction. This study is the first to analyze the production and hormonal regulation of medicinal A. bidentata metabolites at the molecular level. The results herein contribute to a better understanding of the regulation of oleanane-type triterpenoid saponins accumulation and define strategies to improve the yield of these useful metabolites.

Triglyceride deficiency and diacylglycerol kinase1 activity lead to the upregulation of mevalonate pathway in yeast: A study for the development of potential yeast platform for improved production of triterpenoid.

Besides energy storage and membrane biogenesis, lipids are known for their numerous biological functions. The two essential lipids, diacylglycerol (DG) and phosphatidic acid (PA), are shown to be associated with cell signalling processes. In this study, we examined whether triglyceride-deficient yeast mutants (tgΔ), dga1Δ and dga1Δlro1Δ, may play an important role in mevalonate (MEV) pathway regulation. Our metabolite analyses revealed that tgΔ cells showed high levels of squalene (SQ) and ergosterol (ERG), which are key indicators of MEV pathway activity. In addition, gene expression studies indicated that the MEV pathway genes in tgΔ cells were significantly upregulated. Interestingly, tgΔ cells exhibited high diacylglycerol kinase1 (DGK1) expression. Furthermore, DGK1 overexpression in WT and tgΔ phenotypes causes a substantial elevation in SQ and ERG levels, and we also found a significant increase in transcript levels of MEV pathway genes, confirming the new role of DGK1 in MEV pathway regulation. This suggests that high DG phosphorylation activity increases the PA pool that may induce the upregulation of MEV pathway in tgΔ cells. The induced MEV pathway is one of the key strategies in the field of synthetic biology for improved production of terpenoids in yeast. Thus, to examine whether increased endogenous MEV pathway flux can be redirected to triterpenoid, ß-Amyrin synthase gene was heterologously expressed in DGK1 overexpressing tgΔ cells that led to significant production of ß-Amyrin, a natural triterpenoid. In conclusion, our findings provide a novel strategy to increase MEV pathway precursors by modulating endogenous signal lipids for improved production of terpenoids.

De novo transcriptome of Gymnema sylvestre identified putative lncRNA and genes regulating terpenoid biosynthesis pathway.

Gymnema sylvestre is a highly valuable medicinal plant in traditional Indian system of medicine and used in many polyherbal formulations especially in treating diabetes. However, the lack of genomic resources has impeded its research at molecular level. The present study investigated functional gene profile of G. sylvestre via RNA sequencing technology. The de novo assembly of 88.9 million high quality reads yielded 23,126 unigenes, of which 18116 were annotated against databases such as NCBI nr database, gene ontology (GO), KEGG, Pfam, CDD, PlantTFcat, UniProt & GreeNC. Total 808 unigenes mapped to 78 different Transcription Factor families, whereas 39 unigenes assigned to CYP450 and 111 unigenes coding for enzymes involved in the biosynthesis of terpenoids including transcripts for synthesis of important compounds like Vitamin E, beta-amyrin and squalene. Among them, presence of six important enzyme coding transcripts were validated using qRT-PCR, which showed high expression of enzymes involved in methyl-erythritol phosphate (MEP) pathway. This study also revealed 1428 simple sequence repeats (SSRs), which may aid in molecular breeding studies. Besides this, 8 putative long non-coding RNAs (lncRNAs) were predicted from un-annotated sequences, which may hold key role in regulation of essential biological processes in G. sylvestre. The study provides an opportunity for future functional genomic studies and to uncover functions of the lncRNAs in G. sylvestre.

Effects of drought-re-watering-drought on the photosynthesis physiology and secondary metabolite production of Bupleurum chinense DC.

KEY MESSAGE: Drastic changes in soil water content can activate the short-term high expression of key enzyme-encoding genes involved in secondary metabolite synthesis thereby increasing the content of secondary metabolites. Bupleurum chinense DC. is a traditional medicinal herb that is famous for its abundant saikosaponins. In the current study, the effects of drought-re-watering-drought on the photosynthesis physiology and biosynthesis of saikosaponins were investigated in 1-year-old B. chinense. The results showed that alterations in soil moisture altered the photosynthesis physiological process of B. chinense. The dry weight and fresh weight of the roots, photosynthesis capacity, chlorophyll fluorescence parameters, and SOD, POD and CAT activities were significantly reduced, and the contents of SP, soluble sugars, PRO and MDA increased. There were strong correlations between different physiological stress indices. All indices promoted and restricted each other, responded to soil moisture changes synergistically, maintained plant homeostasis and guaranteed normal biological activities. It was found that RW and RD_1 were the key stages of the water-control experiment affecting the expression of saikosaponin-related genes. At these two stages, the expression of multiple genes was affected by changes in soil moisture, with their expression levels reaching several-fold higher than those at the previous stage. We noticed that the expression of saikosaponin synthesis genes (which were rapidly upregulated at the RW and RD_1 stages) did not coincide with the rapid accumulation of saikosaponins (at the RD-2 stage), which were found to correspond to each other at the later stages of the water-control experiment. This finding indicates that there is a time lag between gene expression and the final product synthesis. Rapid changes in the external environment (RW to RD_1) have a short-term promoting effect on gene expression. This study reveals that short-term stress regulation may be an effective way to improve the quality of medicinal materials.

[Study of heterologous efficient synthesis of ß-amyrin and high-density fermentation].

In this study, the synthetic pathway of ß-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing ß-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced ß-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of ß-amyrin synthesis for further improving ß-amyrin production, resulting the strain Y2-C2-4 which produced ß-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of ß-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of ß-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of ß-amyrin-based triterpenoids.

Cloning and Characterization of Oxidosqualene Cyclases Involved in Taraxasterol, Taraxerol and Bauerenol Triterpene Biosynthesis in Taraxacum coreanum.

Triterpenes, consisting of six isoprene units, are one of the largest classes of natural compounds in plants. The genus Taraxacum is in the family Asteraceae and is widely distributed in the Northern Hemisphere. Various triterpenes, especially taraxerol and taraxasterol, are present in Taraxacum plants. Triterpene biosynthesis occurs through the action of oxidosqualene cyclase (OSC), which generates various types of triterpenes from 2,3-oxidosqualene after the rearrangement of the triterpene skeleton. However, no functional characterization of the OSC genes involved in triterpene biosynthesis, except for a lupeol synthase in Taraxacum officinale, has been performed. Taraxacum coreanum, or Korean dandelion, grows in Korea and China. Putative OSC genes in T. coreanum plants were isolated by transcriptome analysis, and four of these (TcOSC1, TcOSC2, TcOSC3 and TcOSC4) were functionally characterized by heterologous expression in yeast. Both TcOSC1 and TcOSC2 were closely related to dammarenediol-II synthases. TcOSC3 and TcOSC4 were strongly grouped with ß-amyrin synthases. Functional analysis revealed that TcOSC1 produced several triterpenes, including taraxasterol; Ψ-taraxasterol; α-, ß- and δ-amyrin; and dammarenediol-II. TcOSC2 catalyzed the production of bauerenol and another unknown triterpene, TcOSC3 catalyzed the production of ß-amyrin. TcOSC4 catalyzed the production of taraxerol. Moreover, we identified taraxasterol, ψ-taraxasterol, taraxerol, lupeol, δ-amyrin, α-amyrin, ß-amyrin and bauerenol in the roots and leaves of T. coreanum. Our results suggest that TcOSC1, TcOSC2, TcOSC3 and TcOSC4 are key triterpene biosynthetic enzymes in T. coreanum. These enzymes are novel triterpene synthases involved in the production of taraxasterol, bauerenol and taraxerol.

Tuscan Varieties of Sweet Cherry Are Rich Sources of Ursolic and Oleanolic Acid: Protein Modeling Coupled to Targeted Gene Expression and Metabolite Analyses.

The potential of six ancient Tuscan sweet cherry (Prunus avium L.) varieties as a source of health-promoting pentacyclic triterpenes is here evaluated by means of a targeted gene expression and metabolite analysis. By using a sequence homology criterion, we identify five oxidosqualene cyclase genes (OSCs) and three cytochrome P450s (CYP85s) that are putatively involved in the triterpene production pathway in sweet cherries. We performed 3D structure prediction and induced-fit docking using cation intermediates and reaction products for some OSCs to predict their function. We show that the Tuscan varieties have different amounts of ursolic and oleanolic acids and that these variations are related to different gene expression profiles. This study stresses the interest of valorizing ancient fruits as alternative sources of functional molecules with nutraceutical value. It also provides information on sweet cherry triterpene biosynthetic genes, which could be the object of follow-up functional studies.

Enhanced ß-Amyrin Synthesis in Saccharomyces cerevisiae by Coupling An Optimal Acetyl-CoA Supply Pathway.

ß-Amyrin is a plant-derived triterpenoid skeleton with wide applications in food and medical industry. ß-Amyrin biosynthesis in Saccharomyces cerevisiae is derived from the mevalonate pathway with cytosolic acetyl-CoA as a precursor. In this work, endogenous and several heterologous acetyl-CoA synthesis pathways were coupled to ß-amyrin production and a combinational acetyl-CoA supply route was demonstrated to be optimal due to more balanced redox cofactors, much lower energy consumption, and glucose utilization as well as significantly enhanced ß-amyrin production (a 200% increase compared to the original ß-amyrin-producing strain). Further disruption of an acetyl-CoA competing pathway led to a 330% increase in ß-amyrin production as compared to the original strain. Finally, the engineered strain harboring the optimal pathway configuration achieved a final ß-amyrin production of 279.0 ± 13.0 mg/L in glucose fed-batch fermentation, which is the highest as ever reported. This work provides an efficient platform for triterpenoid biosynthesis in Saccharomyces cerevisiae.

Oleanane-Type Saponins Biosynthesis in Panax notoginseng via Transformation of ß-Amyrin Synthase Gene from Panax japonicus.

Oleanane-type saponins considered as the main medicinal ingredients in Panax japonicus are not found in Panax notoginseng. ß-Amyrin synthase (ßAS) was recognized as the first key enzyme in the biosynthetic branch of oleanane-type saponins. In this study, ßAS gene from P. japonicus ( PjßAS) was transferred into P. notoginseng cells. Along with PjßAS expression in the transgenic cells, the expression levels of several key enzyme genes related to triterpenoid saponins biosynthesis and the content of P. notoginseng saponins were also increased. Two oleanane-type saponins, chikusetsusaponin IV and chikusetsusaponin IVa, contained in P. japonicus were first discovered in transgenic P. notoginseng cells. This study successfully constructed a biosynthetic pathway of oleanane-type saponins in P. notoginseng by introducing just one gene into the species. On the basis of this discovery and previous studies, the common biosynthetic pathway of triterpenoid saponins in Panax genus may be unified to some extent.

The cytochrome P450 CYP72A552 is key to production of hederagenin-based saponins that mediate plant defense against herbivores.

Plants continuously evolve new defense compounds. One class of such compounds is triterpenoid saponins. A few species in the Barbarea genus produce saponins as the only ones in the large crucifer family. However, the molecular mechanism behind saponin biosynthesis and their role in plant defense remains unclear. We used pathway reconstitution in planta, enzymatic production of saponins in vitro, insect feeding assays, and bioinformatics to identify a missing gene involved in saponin biosynthesis and saponin-based herbivore defense. A tandem repeat of eight CYP72A cytochromes P450 colocalise with a quantitative trait locus (QTL) for saponin accumulation and flea beetle resistance in Barbarea vulgaris. We found that CYP72A552 oxidises oleanolic acid at position C-23 to hederagenin. In vitro-produced hederagenin monoglucosides reduced larval feeding by up to 90% and caused 75% larval mortality of the major crucifer pest diamondback moth and the tobacco hornworm. Sequence analysis indicated that CYP72A552 evolved through gene duplication and has been under strong selection pressure. In conclusion, CYP72A552 has evolved to catalyse the formation of hederagenin-based saponins that mediate plant defense against herbivores. Our study highlights the evolution of chemical novelties by gene duplication and selection for enzyme innovations, and the importance of chemical modification in plant defense evolution.

Increased synthesis of a new oleanane-type saponin in hairy roots of marigold (Calendula officinalis) after treatment with jasmonic acid.

Native plant of marigold (Calendula officinalis L.) synthesizes oleanolic acid saponins classified as glucosides or glucuronides according to the first residue in sugar chain bound to C-3 hydroxyl group. Hairy root culture, obtained by transformation with Agrobacterium rhizogenes strain 15834, exhibit a potent ability of synthesis of oleanolic acid glycosides. The HPLC profile of saponin fraction obtained from C. officinalis hairy roots treated with plant stress hormone, jasmonic acid, showed the 10-times increase of the content of one particular compound, determined by NMR and MALDI TOF as a new bisdesmoside saponin, 3-O-ß-d-glucuronopyranosyl-28-O-ß-d-galactopyranosyl-oleanolic acid. Such a diglycoside does not occur in native C. officinalis plant. It is a glucuronide, whereas in the native plant glucuronides are mainly accumulated in flowers, while glucosides are the most abundant saponins in roots. Thus, our results revealed that the pathways of saponin biosynthesis, particularly reactions of glycosylation, are altered in C. officinalis hairy root culture.

Transcriptome analysis of Panax zingiberensis identifies genes encoding oleanolic acid glucuronosyltransferase involved in the biosynthesis of oleanane-type ginsenosides.

MAIN CONCLUSION: Oleanolic acid glucuronosyltransferase (OAGT) genes synthesizing the direct precursor of oleanane-type ginsenosides were discovered. The four recombinant proteins of OAGT were able to transfer glucuronic acid at C-3 of oleanolic acid that yields oleanolic acid 3-O-ß-glucuronide. Ginsenosides are the primary active components in the genus Panax, and great efforts have been made to elucidate the mechanisms underlying dammarane-type ginsenoside biosynthesis. However, there is limited information on oleanane-type ginsenosides. Here, high-performance liquid chromatography analysis demonstrated that oleanane-type ginsenosides (particularly ginsenoside Ro and chikusetsusaponin IV and IVa) are the abundant ginsenosides in Panax zingiberensis, an extremely endangered Panax species in southwest China. These ginsenosides are derived from oleanolic acid 3-O-ß-glucuronide, which may be formed from oleanolic acid catalyzed by an unknown oleanolic acid glucuronosyltransferase (OAGT). Transcriptomic analysis of leaves, stems, main roots, and fibrous roots of P. zingiberensis was performed, and a total of 46,098 unigenes were obtained, including all the identified homologous genes involved in ginsenoside biosynthesis. The most upstream genes were highly expressed in the leaves, and the UDP-glucosyltransferase genes were highly expressed in the roots. This finding indicated that the precursors of ginsenosides are mainly synthesized in the leaves and transported to different parts for the formation of particular ginsenosides. For the first time, enzyme activity assay characterized four genes (three from P. zingiberensis and one from P. japonicus var. major, another Panax species with oleanane-type ginsenosides) encoding OAGT, which particularly transfer glucuronic acid at C-3 of oleanolic acid to form oleanolic acid 3-O-ß-glucuronide. Taken together, our study provides valuable genetic information for P. zingiberensis and the genes responsible for synthesizing the direct precursor of oleanane-type ginsenosides.