Gene excavation and expression analysis of CYP and UGT related to the post modifying stage of gypenoside biosynthesis in Gynostemma pentaphyllum (Thunb.) Makino by comprehensive analysis of RNA and proteome sequencing.

Previous studies have revealed that gypenosides produced from Gynostemma pentaphyllum (Thunb.) Makino are mainly dammarane-type triterpenoid saponins with diverse structures and important biological activities, but the mechanism of diversity for gypenoside biosynthesis is still unclear. In this study, a combination of isobaric tags for relative and absolute quantification (iTRAQ) proteome analysis and RNA sequencing transcriptome analysis was performed to identify the proteins and genes related to gypenoside biosynthesis. A total of 3925 proteins were identified by proteomic sequencing, of which 2537 were quantified. Seventeen cytochrome P450 (CYP) and 11 uridine 5'-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT) candidate genes involved in the side chain synthesis and modification of gypenosides were found. Seven putative CYPs (CYP71B19, CYP77A3, CYP86A7, CYP86A8, CYP89A2, CYP90A1, CYP94A1) and five putative UGTs (UGT73B4, UGT76B1, UGT74F2, UGT91C1 and UGT91A1) were selected as candidate structural modifiers of triterpenoid saponins, which were cloned for gene expression analysis. Comprehensive analysis of RNA sequencing and proteome sequencing showed that some CYPs and UGTs were found at both the transcription and translation levels. In this study, an expression analysis of 7 CYPs and 5 UGTs that contributed to gypenoside biosynthesis and distribution in G. pentaphyllum was performed, providing consistent results that will inspire more future research on vital genes/proteins involved in gypenoside biosynthesis.

A comparative appraisal of three important oil yielding plants for their biodiesel potential.

In the present scenario, alternative energy sources are required to achieve the future economic prosperity where shortage of fossil fuels will be a limiting factor and hamper the global economic growth. Therefore, interest in biofuel is increasing continuously. The best way of sustainable development is fossil fuel supplementation with biodiesel to reduce the fossil fuel demand. Biodiesel is a clean burning, ester-based, oxygenated fuel derived from natural and renewable sources. Till now, majority of the people have worked on the biodiesel derived from edible oil. Instead of using edible oil, non-edible oil needs to be explored as feedstock for biofuel because half of the world's population is unable to afford the food oil as feedstock for fuel production. Looking at the significance of biodiesel and the resources of biofuel, in this paper, a comparative exhaustive study has been reported with for three important plants, namely Jatropha curcas, Pongemia pinnata and Balanites aegyptiaca. These plants were selected based on their biodiesel potential, availability, cultivation practices and general information available. The present study involves scientometric publications, comparison of fatty acid composition and biodiesel parameters. We have also compared climatic conditions for the growth of the plants, economic feasibility of biodiesel production and other ecological services. The study paves a way for sustainable solution to policy makers and foresters looking for selection of plant species as bioenergy resource.

Recent Research Progress in Taxol Biosynthetic Pathway and Acylation Reactions Mediated by Taxus Acyltransferases.

Taxol is one of the most effective anticancer drugs in the world that is widely used in the treatments of breast, lung and ovarian cancer. The elucidation of the taxol biosynthetic pathway is the key to solve the problem of taxol supply. So far, the taxol biosynthetic pathway has been reported to require an estimated 20 steps of enzymatic reactions, and sixteen enzymes involved in the taxol pathway have been well characterized, including a novel taxane-10ß-hydroxylase (T10ßOH) and a newly putative ß-phenylalanyl-CoA ligase (PCL). Moreover, the source and formation of the taxane core and the details of the downstream synthetic pathway have been basically depicted, while the modification of the core taxane skeleton has not been fully reported, mainly concerning the developments from diol intermediates to 2-debenzoyltaxane. The acylation reaction mediated by specialized Taxus BAHD family acyltransferases (ACTs) is recognized as one of the most important steps in the modification of core taxane skeleton that contribute to the increase of taxol yield. Recently, the influence of acylation on the functional and structural diversity of taxanes has also been continuously revealed. This review summarizes the latest research advances of the taxol biosynthetic pathway and systematically discusses the acylation reactions supported by Taxus ACTs. The underlying mechanism could improve the understanding of taxol biosynthesis, and provide a theoretical basis for the mass production of taxol.

Biosynthesis of gold nanoparticles for the treatment of osteoarthritis alone or in combination with Diacerein® in a rat model.

Gold (Au) compounds were used as an effective therapeutic agent for various inflammatory diseases; however, the use of Au compounds becomes limited because of its association with several side effects. Hence, gold nanoparticles (AuNPs) were developed as a new option for the medical proposes. However, the safety evaluation of gold nanoparticles (AuNPs) in osteoarthritis (OA) treatment remains vague. This study aimed to biosynthesize, characterize and evaluate the therapeutic effects of biosynthesized AuNPs and/or Diacerein® (DIA) in experimental OA. OA was induced by a single injection of monosodium iodoacetate (3 mg/joint) in the intra-articular knee of female rats. Normal rats (N-rats) and OA-rats were treated orally for 5 weeks as follow: untreated N-rats; untreated OA-rats; N-rats received DIA (50 mg/kg b.w); N-rats received AuNPs (30 µg/kg b.w.); N-rats received AuNPs plus DIA; OA-rats received DIA; OA-rats received AuNPs, and OA-rats received AuNPs plus DIA. Blood, knee cartilage, liver and kidney samples were collected for biochemical and histological analysis. The synthesized AuNPs were nearly spherical with average size of 20 nm and zeta potential of 33 mV. AuNPs and DIA induced a significant improvement in serum inflammatory cytokines, biochemical parameters, estrogen level, hepatic and renal oxidative markers, hepatic DNA fragmentation, genomic template stability and cartilage joint histology of OA-rats. AuNPs were more effective than DIA and the combined treatment was more effective than the single treatment. It could be concluded that AuNPs are promising for the treatment of OA alone or in combination with DIA.

Improve the Biosynthesis of Baicalein and Scutellarein via Manufacturing Self-Assembly Enzyme Reactor In Vivo.

Baicalein and scutellarein are bioactive flavonoids isolated from the traditional Chinese medicine Scutellaria baicalensis Georgi; however, there is a lack of effective strategies for producing baicalein and scutellarein. In this study, we developed a sequential self-assembly enzyme reactor involving two enzymes in the baicalein pathway with a pair of protein-peptide interactions in E. coli. These domains enabled us to optimize the stoichiometry of two baicalein biosynthetic enzymes recruited to be an enzymes complex. This strategy reduces the accumulation of intermediates and removes the pathway bottleneck. With this strategy, we successfully promoted the titer of baicalein by 6.6-fold (from 21.6 to 143.5 mg/L) and that of scutellarein by 1.4-fold (from 84.3 to 120.4 mg/L) in a flask fermentation, respectively. Furthermore, we first achieved the de novo biosynthesis of baicalein directly from glucose, and the strain was capable of producing 214.1 mg/L baicalein by fed-batch fermentation. This work provides novel insights for future optimization and large-scale fermentation of baicalein and scutellarein.

Black rice cultivar from Java Island of Indonesia revealed genomic, proteomic, and anthocyanin nutritional value.

Black rice is considered to be functional food containing anthocyanins as bioactive compounds. This study examined the genomic and proteomic patterns in local black rice from Java Island, Indonesia, with attention to the mechanism of anthocyanin synthesis. Three kinds of black rice from Java Island, including black rice from East Java (BREJ), black rice from Central Java (BRCJ), and black rice from West Java (BRWJ), were studied in comparison to white rice (WREJ) and red rice (RREJ). Genomic profiling was done by simple sequence repeat (SSR) analysis, and sequencing of red coleoptile (Rc) and glycosyltransferase (GT) genes, followed by in silico analysis. Total anthocyanin was investigated by ultra-high performance liquid chromatography- diode array detector (UHPLC-DAD). The proteomic profiles were determined by liquid-chromatography and mass spectrometry of tryptic peptides. The SSR profiles showed a specific band in each black rice variant. The Rc gene exon-2 sequences were similar in the three black rice cultivars. The GT gene sequence was identified as a new variant that correlates with the purple stem, leaf, bran, and whole grain morphology seen exclusively in the BRWJ cultivar. The anthocyanin composition in Java black rice is diverse. The highest cyanidin level was seen in BRWJ and the highest level of peonidin-3-O-glucoside in BREJ. Proteomic profiling of the black rice cultivars demonstrated that the expression of proteins that might be related to the levels of anthocyanin synthesis varied. These studies conclude that the genomic, proteomic and anthocyanins composition of Java black rice cultivars may be used the improvement of their functional nutrition values.

Medicinal Inula Species: Phytochemistry, Biosynthesis, and Bioactivities.

As a genus of the Asteraceae, Inula is widely distributed all over the world, and several of them are being used in traditional medicines. A number of metabolites were isolated from Inula species, and some of these have shown to possess ranges of pharmacological activities. The genus Inula contains abundant sesquiterpenoids, such as eudesmanes, xanthanes, and sesquiterpenoid dimers and trimers. In addition, other types of terpenoids, flavonoids, and lignins also exist in the genus Inula. Since 2010, more than 300 new secondary metabolites, including several known natural products that were isolated for the first time from the genus Inula. Most of them exhibited potential bioactivities in various diseases. The review aimed to summarize the advance of recent researches (2010-2020) on phytochemical constituents, biosynthesis, and pharmacological properties of the genus Inula for providing a scientific basis and supporting its application and exploitation for new drug development.

Unusual derivatives from Hypericum scabrum.

Three new compounds (1-3) with unusual skeletons were isolated from the n-hexane extract of the air-dried aerial parts of Hypericum scabrum. Compound 1 represents the first example of an esterified polycyclic polyprenylated acylphloroglucinol that features a unique tricyclo-[4.3.1.11,4]-undecane skeleton. Compound 2 is a fairly simple MPAP, but with an unexpected cycloheptane ring decorated with prenyl substituents, and compound 3 has an unusual 5,5-spiroketal lactone core. Their structures were determined by extensive spectroscopic and spectrometric techniques (1D and 2D NMR, HRESI-TOFMS). Absolute configurations were established by ECD calculations, and the absolute structure of 2 was confirmed by a single crystal determination. Plausible biogenetic pathways of compounds 1-3 were also proposed. The in vitro antiprotozoal activity of the compounds against Trypanosoma brucei rhodesiense and Plasmodium falciparum and cytotoxicity against rat myoblast (L6) cells were determined. Compound 1 showed a moderate activity against T. brucei and P. falciparum, with IC50 values of 3.07 and 2.25 µM, respectively.

The chromosome-level reference genome assembly for Panax notoginseng and insights into ginsenoside biosynthesis.

Panax notoginseng, a perennial herb of the genus Panax in the family Araliaceae, has played an important role in clinical treatment in China for thousands of years because of its extensive pharmacological effects. Here, we report a high-quality reference genome of P. notoginseng, with a genome size up to 2.66 Gb and a contig N50 of 1.12 Mb, produced with third-generation PacBio sequencing technology. This is the first chromosome-level genome assembly for the genus Panax. Through genome evolution analysis, we explored phylogenetic and whole-genome duplication events and examined their impact on saponin biosynthesis. We performed a detailed transcriptional analysis of P. notoginseng and explored gene-level mechanisms that regulate the formation of characteristic tubercles. Next, we studied the biosynthesis and regulation of saponins at temporal and spatial levels. We combined multi-omics data to identify genes that encode key enzymes in the P. notoginseng terpenoid biosynthetic pathway. Finally, we identified five glycosyltransferase genes whose products catalyzed the formation of different ginsenosides in P. notoginseng. The genetic information obtained in this study provides a resource for further exploration of the growth characteristics, cultivation, breeding, and saponin biosynthesis of P. notoginseng.

The Bioavailability, Extraction, Biosynthesis and Distribution of Natural Dihydrochalcone: Phloridzin.

Phloridzin is an important phytochemical which was first isolated from the bark of apple trees. It is a member of the dihydrochalcones and mainly distributed in the plants of the Malus genus, therefore, the extraction method of phloridzin was similar to those of other phenolic substances. High-speed countercurrent chromatography (HSCCC), resin adsorption technology and preparative high-performance liquid chromatography (HPLC) were used to separate and purify phloridzin. Many studies showed that phloridzin had multiple pharmacological effects, such as antidiabetic, anti-inflammatory, antihyperglycaemic, anticancer and antibacterial activities. Besides, the physiological activities of phloridzin are cardioprotective, neuroprotective, hepatoprotective, immunomodulatory, antiobesity, antioxidant and so on. The present review summarizes the biosynthesis, distribution, extraction and bioavailability of the natural compound phloridzin and discusses its applications in food and medicine.

Transcriptome-wide analysis of the AP2/ERF transcription factor gene family involved in the regulation of gypenoside biosynthesis in Gynostemma pentaphyllum.

Gynostemma pentaphyllum is a traditional Chinese medicinal herb, serving as natural source of gypenosides (triterpene saponins). The APETALA2/ethylene response factor (AP2/ERF) transcription factors, playing essential regulation roles in plant biotic and abiotic stress responses and secondary metabolism biosynthesis. However, the regulation roles of AP2/ERF transcription factors in gypenosides biosynthesis in G. pentaphyllum remains little understood. In the present study, 125 AP2/ERF genes were identified from G. pentaphyllum transcriptome datasets. Phylogenetic, conserved motifs and expression pattern were employed to comprehensively analyze the 125 GpAP2/ERF genes. Based on the sequence similarity and phylogeny tree, the 125 GpAP2/ERF genes can be classified into 10 groups. Moreover, the distribution of conserved motifs among GpAP2/ERF proteins in phylogenetic trees was consistent with previous studies, thus supporting the classification. Expression profiling indicated that the 125 GpAP2/ERF genes exhibited distinct tissue-specific expression patterns. As confirmed by qRT-PCR, the four candidate GpAP2/ERF genes and gypenoside biosynthetic genes were highly expressed in leaves and/or flowers, and show similar expression patterns in response to MeJA. Base on the expression patterns and phylogenetic relationships, two GpAP2/ERF genes were considered as potential regulatory genes for gypenoside biosynthesis. Our study enhances understanding roles of GpAP2/ERF genes in regulation of gypenosides biosynthesis.

Single components of botanicals and nature-identical compounds as a non-antibiotic strategy to ameliorate health status and improve performance in poultry and pigs.

In the current post-antibiotic era, botanicals represent one of the most employed nutritional strategies to sustain antibiotic-free and no-antibiotic-ever production. Botanicals can be classified either as plant extracts, meaning the direct products derived by extraction from the raw plant materials (essential oils (EO) and oleoresins (OR)), or as nature-identical compounds (NIC), such as the chemically synthesised counterparts of the pure bioactive compounds of EO/OR. In the literature, differences between the use of EO/OR or NIC are often unclear, so it is difficult to attribute certain effects to specific bioactive compounds. The aim of the present review was to provide an overview of the effects exerted by botanicals on the health status and growth performance of poultry and pigs, focusing attention on those studies where only NIC were employed or those where the composition of the EO/OR was defined. In particular, phenolic compounds (apigenin, quercetin, curcumin and resveratrol), organosulfur compounds (allicin), terpenes (eugenol, thymol, carvacrol, capsaicin and artemisinin) and aldehydes (cinnamaldehyde and vanillin) were considered. These molecules have different properties such as antimicrobial (including antibacterial, antifungal, antiviral and antiprotozoal), anti-inflammatory, antioxidant, immunomodulatory, as well as the improvement of intestinal morphology and integrity of the intestinal mucosa. The use of NIC allows us to properly combine pure compounds, according to the target to achieve. Thus, they represent a promising non-antibiotic tool to allow better intestinal health and a general health status, thereby leading to improved growth performance.

Journey in the Polyphenol Research World with Ragai Ibrahim.

Dr. Ragai K. Ibrahim, Professor Emeritus at Concordia University, Montréal, Canada, passed away on the November 19, 2017 at the age of 88 years. Dr. Ibrahim dedicated his entire professional life to polyphenols and spent most of his academic career (1967-1997) at the Department of Biology of Concordia University in Montréal. He has been an active member of the Groupe Polyphénols since the beginning. This paper is a tribute to Dr. Ibrahim from some of his former students. An overview of the evolution of polyphenol research since the late 1950s and the outstanding contribution that Dr. Ibrahim had to this topic is given. The input of Dr. Ibrahim's research to the enzymology and genetics of polyphenol biosynthesis is discussed. Furthermore, the links between Dr. Ibrahim's work and some aspects of modern studies on the health benefits of polyphenols are presented.

Metabolic Profiling and Transcriptome Analysis of Mulberry Leaves Provide Insights into Flavonoid Biosynthesis.

Flavonoids are widely distributed in mulberry leaves and have been recognized for their beneficial physiological effects on the human health. Here, we analyzed variations in 44 flavonoid compounds among 91 mulberry resources. Metabolic profiling revealed that O-rhamnosylated flavonols and malonylated flavonol glycosides, including rutin and quercetin 3-O-(6″-O-malonylglucoside) (Q3MG), were absent from Morus notabilis and multiple mulberry (Morus alba L.) resources. Transcriptome and phylogenetic analyses of flavonoid-related UDP-glycosyltransferases (UGTs) suggested that the flavonol 3-O-glucoside-O-rhamnosyltransferase (FGRT) KT324624 is a key enzyme involved in rutin synthesis. A recombinant FGRT protein was able to convert kaempferol/quercetin 3-O-glucoside to kaempferol 3-O-rutinoside (K3G6″Rha) and rutin. The recombinant FGRT was able to use 3-O-glucosylated flavonols but not flavonoid aglycones or 7-O-glycosylated flavonoids as substrates. The enzyme preferentially used UDP-rhamnose as the sugar donor, indicating that it was a flavonol 3-O-glucoside: 6″-O-rhamnosyltransferase. This study provided insights into the biosynthesis of rutin in mulberry.

Enhanced Cordycepin Production in Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), Mutated by a Multifunctional Plasma Mutagenesis System.

A multifunctional plasma mutation system (MPMS) method was used to create high cordycepin-yielding mutations from wild Cordyceps militaris, which yielded many viable mutants, many of which produced more cordycepin compared to the wild strain. One particular mutant strain (GYS60) produced 7.883 mg/mL, which is much higher than those reported to date and is more than 20 times higher than that of the wild strain, whereas the cordycepin production of another viable mutant (GYS80) was almost zero. The extraction and purification of cordycepin, using the fermentation broth of C. militaris GYS60, was also investigated. Cordycepin was extracted by using AB-8 macroporous resin and purified by using reversed-phase column chromatography. When the sample was adsorbed onto the macroporous resin, 20% ethanol was used as the desorption solvent yielding various fractions. The fractions containing cordycepin were loaded onto a reversed-phase chromatography column packed with octadecyl bonded silica as the stationary phase and ethanol (95%)/acetic acid solution (5%) at pH 6.0 as the mobile phase. The combination of this two-step extraction-purification process yielded cordycepin at 95% purity with a total recovery rate of 90%.

An Efficient Strategy for Enhancement of Bioactive Compounds in the Fruit Body of Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), by Spraying Biotic Elicitors.

Cordyceps militaris is a mushroom species with high nutritive and medicinal values based on diverse bioactive metabolites. The contents of bioactive ingredients are indicative of the quality of commercially available fruit body of this fungus. Although the application of biotic elicitors has been an efficient strategy to induce the accumulation of valuable bioactive compounds in vivo, related research in C. militaris is rarely reported. In this study, five biotic elicitors in different concentrations (0.05, 0.5, 1, and 2 mg/mL), including chitosan (CHT), 2,4-dichlorophenoxyacetic acid (2,4-D), methyl jasmonate (MeJA), gibberellic acid (GA), and triacontanol (TRIA), were first introduced to enhance the production of 10 kinds of major bioactive components in the fruit body of C. militaris. Results showed that the effect of biotic elicitors on bioactive compounds in the fruit body of C. militaris was elicitor-specific and concentration-dependent. Overall, 1 mg/L CHT was considered the most favorable for the production of 10 bioactive ingredients in C. militaris fruit body, which could increase the content of protein, polysaccharides, polyphenol, triterpenoids, flavonoids, cordyceps acid, cordycepin, and anthocyanins by 20.38-, 1.41-, 0.7-, 0.47-, 11.90-, 1.09-, 0.34-, and 2.64-fold, respectively, compared with the control. The results of this study would provide an efficient strategy for the production of a superior quality fruit body of and contribute to further elucidation of the effects of biotic elicitors on metabolite accumulation in C. militaris.

Transcriptomic and metabolomic analyses reveal several critical metabolic pathways and candidate genes involved in resin biosynthesis in Pinus massoniana.

Pine resin, which typically consists of terpenoids, is a natural product used in various industrial applications. Oleoresin can be obtained from the xylem tissue by wounding the stem bark. Pinus massoniana (masson pine) is an important resin-tapping tree species that originated in southern China. Masson pines with different genetic backgrounds typically have different resin-yielding capacities (RYCs). However, the mechanisms underlying high resin yield in masson pines are unclear. The aim of this study was to identify the possible genetic regulation pathways and functional genes that influence the resin yield. In this study, we conducted transcriptomic and metabolomic studies of masson pine secondary xylem with high, medium, and low RYCs. A total of 230,068 unigenes and 3894 metabolites were identified from the tissue of the secondary xylem. Several differentially expressed regulation factors, including WRKY, bHLH, and ERF, and functional genes such as PKc and LRR-RLKs, were identified among these masson pines. The Kyoto Encyclopedia of Genes and Genomes pathways were mainly focused on diterpenoid biosynthesis, plant hormone signal transduction, and ABC transporters. Furthermore, integration of the transcriptomic and metabolomic data indicated that the PKc- and LRR-RLK-related regulatory and metabolic pathways may play critical roles in the biosynthesis of terpenoids. These above results improve our understanding of the biosynthesis mechanism of oleoresin in P. massoniana and facilitate further research work into the functional analysis of these candidate genes.

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade.

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.

Hybrid sequencing of the Gynostemma pentaphyllum transcriptome provides new insights into gypenoside biosynthesis.

BACKGROUND: Gypenosides are a group of triterpene saponins from Gynostemma pentaphyllum that are the same as or very similar to ginsenosides from the Panax species. Several enzymes involved in ginsenoside biosynthesis have been characterized, which provide important clues for elucidating the gypenoside biosynthetic pathway. We suppose that gypenosides and ginsenosides may have a similar biosynthetic mechanism and that the corresponding enzymes in the two pathways may have considerable similarity in their sequences. To further understand gypenoside biosynthesis, we sequenced the G. pentaphyllum transcriptome with a hybrid sequencing-based strategy and then determined the candidate genes involved in this pathway using phylogenetic tree construction and gene expression analysis. RESULTS: Following the PacBio standard analysis pipeline, 66,046 polished consensus sequences were obtained, while Illumina data were assembled into 140,601 unigenes with Trinity software. Then, these output sequences from the two analytical routes were merged. After removing redundant data with CD-HIT software, a total of 140,157 final unigenes were obtained. After functional annotation, five 2,3-oxidosqualene cyclase genes, 145 cytochrome P450 genes and 254 UDP-glycosyltransferase genes were selected for the screening of genes involved in gypenoside biosynthesis. Using phylogenetic analysis, several genes were divided into the same subfamilies or closely related evolutionary branches with characterized enzymes involved in ginsenoside biosynthesis. Using real-time PCR technology, their expression patterns were investigated in different tissues and at different times after methyl jasmonate induction. Since the genes in the same biosynthetic pathway are generally coexpressed, we speculated that GpOSC1, GpCYP89, and GpUGT35 were the leading candidates for gypenoside biosynthesis. In addition, six GpWRKYs and one GpbHLH might play a possible role in regulating gypenoside biosynthesis. CONCLUSIONS: We developed a hybrid sequencing strategy to obtain longer length transcriptomes with increased accuracy, which will greatly contribute to downstream gene screening and characterization, thus improving our ability to elucidate secondary metabolite biosynthetic pathways. With this strategy, we found several candidate genes that may be involved in gypenoside biosynthesis, which laid an important foundation for the elucidation of this biosynthetic pathway, thus greatly contributing to further research in metabolic regulation, synthetic biology and molecular breeding in this species.