RESUMEN

As one of rare high-value ocotillol (OCT)-type ginsenosides, pseudoginsenoside Rt5 has been identified with significant pharmacological activities. UDP-glycosyltransferases (UGTs) play pivotal roles in catalyzing the transfer of a glycosyl moiety from a donor to an acceptor. In this study, the novel UGT, PjUGT10, was screened from the transcriptome database of Panax japonicus and identified with the enzymatic activity of transferring a glucosyl group on OCT to produce Rt5. The catalytic efficiency of PjUGT10 was further enhanced by employing site-directed mutation. Notably, the variant M7 exhibited a remarkable 6.16 × 103-fold increase in kcat/Km towards 20S,24R-ocotillol and a significant 2.02 × 103-fold increase to UDP-glucose, respectively. Moreover, molecular dynamics simulations illustrated a reduced distance between 20S,24R-ocotillol and the catalytic residue His15 or UDP-glucose, favoring conformation interactions between the enzyme and substrates. Subsequently, Rt5 was synthesized in an engineered Escherichia coli strain M7 coupled with a UDP-glucose synthetic system. This study not only shed light on the protein engineering that can enhance the catalytic activity of PjUGT10, but also established a whole-cell approach for the production of Rt5.

Asunto(s)

Ginsenósidos , Glicosiltransferasas , Panax , Ingeniería de Proteínas , Panax/enzimología , Panax/genética , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Glicosiltransferasas/química , Ingeniería de Proteínas/métodos , Ginsenósidos/biosíntesis , Ginsenósidos/química , Ginsenósidos/metabolismo , Simulación de Dinámica Molecular , Especificidad por Sustrato , Escherichia coli/genética

RESUMEN

Ginseng, a cornerstone of traditional herbal medicine in Asia, garnered significant attention for its therapeutic potential. Central to its pharmacological effects are ginsenosides, the primary active metabolites, many of which fall within the dammarane-type and share protopanaxadiol as a common precursor. Challenges in extracting protopanaxadiol and ginsenosides from ginseng arise due to their low concentrations in the roots. Emerging solutions involve leveraging microbial cell factories employing genetically engineered yeasts. Here, we optimized the fermentation conditions via the Design of Experiment, realizing 1.2 g/L protopanaxadiol in simple shake flask cultivations. Extrapolating the optimized setup to complex ginsenosides, like compound K, achieved 7.3-fold (0.22 g/L) titer improvements. Our adaptable fermentation conditions enable the production of high-value products, such as sustainable triterpenoids synthesis. Through synthetic biology, microbial engineering, and formulation studies, we pave the way for a scalable and sustainable production of bioactive compounds from ginseng.

Asunto(s)

Fermentación , Ginsenósidos , Triterpenos , Ginsenósidos/biosíntesis , Ginsenósidos/metabolismo , Triterpenos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Panax/metabolismo , Panax/crecimiento & desarrollo , Panax/química , Ingeniería Metabólica , Sapogeninas

RESUMEN

Panax ginseng is a perennial herb with the main active compounds of ginsenosides. Among the reported ginsenosides, ginsenoside Rg_1 not only has a wide range of medicinal functions and abundant content but also is one of the major ginsenoside for the quality evaluation of this herb in the Chinese Pharmacopoeia. The main biosynthesis pathway of ginsenoside Rg_1 in P. ginseng has been clarified, which lays a foundation for the comprehensive and in-depth analysis of the biosynthesis and regulatory mechanism of ginseno-side Rg_1. However, the biosynthesis of ginsenoside Rg_1 is associated with other complex processes involving a variety of regulatory genes and catalyzing enzyme genes, which remain to be studied comprehensively. With the transcriptome data of 344 root samples from 4-year-old P. ginseng plants and their corresponding ginsenoside Rg_1 content obtained in the previous study, this study screened out 217 differentially expressed genes(DEGs) with Rg_1 content changes by DEseq2 analysis in R language. Furthermore, the weighted gene co-expression network analysis(WGCNA) revealed 40 hub genes among the DEGs.Pearsoncorrelation analysis was further perforned to yield 20 candidate genes significantly correlated with ginsenoside Rg_1 content, and these genes were annotated to multiple metabolic processes including primary metabolism and secondary metabolism. Finally, the treatment of P. ginseng adventitious roots with methyl jasmonate indicated that 16 of these genes promoted the biosynthesis of ginsenoside Rg_1 in response to methyl jasmonate induction. Finally, one of the 16 genes was randomly selected to verify the function of the gene by genetic transformation and qRT-PCR and to confirm the rationality of the methodology of this study. The above results lay a foundation for studying the mechanism for regulation on the synthesis of ginsenoside Rg_1 and provide genetic resources for the industrial production of ginsenoside Rg_1.

Asunto(s)

Regulación de la Expresión Génica de las Plantas , Ginsenósidos , Panax , Ginsenósidos/biosíntesis , Panax/genética , Panax/metabolismo , Panax/química , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Perfilación de la Expresión Génica

RESUMEN

Ginseng (Panax ginseng C. A. Meyer) is an ancient and valuable Chinese herbal medicine, and ginsenoside, as the main active ingredient of ginseng, has received wide attention because of its various pharmacological active effects. Cytochrome P450 is the largest family of enzymes in plant metabolism and is involved in the biosynthesis of terpenoids, alkaloids, lipids, and other primary and secondary plant metabolites. It is significant to explore more PgCYP450 genes with unknown functions and reveal their roles in ginsenoside synthesis. In this study, based on the five PgCYP450 genes screened in the pre-laboratory, through the correlation analysis with the content of ginsenosides and the analysis of the interactions network of the key enzyme genes for ginsenoside synthesis, we screened out those highly correlated with ginsenosides, PgCYP309, as the target gene from among the five PgCYP450 genes. Methyl jasmonate-induced treatment of ginseng adventitious roots showed that the PgCYP309 gene responded to methyl jasmonate induction and was involved in the synthesis of ginsenosides. The PgCYP309 gene was cloned and the overexpression vector pBI121-PgCYP309 and the interference vector pART27-PgCYP309 were constructed. Transformation of ginseng adventitious roots by the Agrobacterium fermentum-mediated method and successful induction of transgenic ginseng hairy roots were achieved. The transformation rate of ginseng hairy roots with overexpression of the PgCYP309 gene was 22.7%, and the transformation rate of ginseng hairy roots with interference of the PgCYP309 gene was 40%. Analysis of ginseng saponin content and relative gene expression levels in positive ginseng hairy root asexual lines revealed a significant increase in PPD, PPT, and PPT-type monomeric saponins Re and Rg2. The relative expression levels of PgCYP309 and PgCYP716A53v2 genes were also significantly increased. PgCYP309 gene promotes the synthesis of ginsenosides, and it was preliminarily verified that PgCYP309 gene can promote the synthesis of dammarane-type ginsenosides.

Asunto(s)

Sistema Enzimático del Citocromo P-450 , Ginsenósidos , Panax , Panax/genética , Panax/metabolismo , Panax/enzimología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Ginsenósidos/metabolismo , Ginsenósidos/biosíntesis , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Oxilipinas/farmacología , Oxilipinas/metabolismo , Acetatos/farmacología , Acetatos/metabolismo , Ciclopentanos/farmacología , Ciclopentanos/metabolismo

RESUMEN

Saponins are bioactive components of many medicinal plants, possessing complicated chemical structures and extensive pharmacological activities, but the production of high-value saponins remains challenging. In this study, a 6'-O-glucosyltransferase PpUGT7 (PpUGT91AH7) was functionally characterized from Paris polyphylla Smith var. yunnanensis (Franch.) Hand. -Mazz., which can transfer a glucosyl group to the C-6' position of diosgenin-3-O-rhamnosyl-(1 â†’ 2)-glucoside (1), pennogenin-3-O-rhamnosyl-(1 â†’ 2)-glucoside (2), and diosgenin-3-O-glucoside (5). The KM and Kcat values of PpUGT7 towards the substrate 2 were 8.4 µM and 2 × 10-3 s-1, respectively. Through molecular docking and site-directed mutagenesis, eight residues were identified to interact with the sugar acceptor 2 and be crucial for enzyme activity. Moreover, four rare ophiopogonins and ginsenosides were obtained by combinatorial biosynthesis, including an undescribed compound ruscogenin-3-O-glucosyl-(1 â†’ 6)-glucoside (10). Firstly, two monoglycosides 9 and 11 were generated using a known sterol 3-O-ß-glucosyltransferase PpUGT80A40 with ruscogenin (7) and 20(S)-protopanaxadiol (8) as substrates, which were further glycosylated to the corresponding diglycosides 10 and 12 under the catalysis of PpUGT7. In addition, compounds 7-11 were found to show inhibitory effects on the secretion of TNF-α and IL-6 in macrophages RAW264.7. The findings provide valuable insights into the enzymatic glycosylation processes in the biosynthesis of bioactive saponins in P. polyphylla var. yunnanensis, and also serve as a reference for utilizing UDP-glycosyltransferases to construct high-value or rare saponins for development of new therapeutic agents.

Asunto(s)

Ginsenósidos , Glicosiltransferasas , Saponinas , Glicosiltransferasas/metabolismo , Glicosiltransferasas/química , Saponinas/química , Saponinas/biosíntesis , Saponinas/metabolismo , Ginsenósidos/química , Ginsenósidos/biosíntesis , Ginsenósidos/metabolismo , Animales , Ratones , Estructura Molecular , Células RAW 264.7 , Melanthiaceae/química , Melanthiaceae/enzimología , Melanthiaceae/metabolismo , Simulación del Acoplamiento Molecular , Liliaceae/química

RESUMEN

Panax ginseng C.A. Mey., known for its medicinal and dietary supplement properties, primarily contains pharmacologically active ginsenosides. However, the regulatory mechanisms linking ginseng root development with ginsenoside biosynthesis are still unclear. Root meristem growth factors (RGFs) are crucial for regulating plant root growth. In our study, we identified five ginseng RGF peptide sequences from the ginseng genome and transcriptome libraries. We treated Arabidopsis and ginseng adventitious roots with exogenous Panax ginseng RGFs (PgRGFs) to assess their activities. Our results demonstrate that PgRGF1 influences gravitropic responses and reduces lateral root formation in Arabidopsis. PgRGF1 has been found to restrict the number and length of ginseng adventitious root branches in ginseng. Given the medicinal properties of ginseng, We determined the ginsenoside content and performed transcriptomic analysis of PgRGF1-treated ginseng adventitious roots. Specifically, the total ginsenoside content in ginseng adventitious roots decreased by 19.98 % and 63.71 % following treatments with 1 µM and 10 µM PgRGF1, respectively, compared to the control. The results revealed that PgRGF1 affects the accumulation of ginsenosides by regulating the expression of genes associated with auxin transportation and ginsenoside biosynthesis. These findings suggest that PgRGF1, as a peptide hormone regulator in ginseng, can modulate adventitious root growth and ginsenoside accumulation.

Asunto(s)

Regulación de la Expresión Génica de las Plantas , Ginsenósidos , Meristema , Panax , Raíces de Plantas , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Ginsenósidos/biosíntesis , Ácidos Indolacéticos/metabolismo , Meristema/genética , Meristema/crecimiento & desarrollo , Meristema/metabolismo , Panax/genética , Panax/crecimiento & desarrollo , Panax/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo

RESUMEN

Ginsenoside F1 has high medicinal values, which is a kind of rare triterpene saponin isolated from Panax plants. The extremely low content of ginsenoside F1 in herbs has limited its research and application in medical field. In this work, we constructed a pathway in tobacco for the biosynthesis of ginsenoside F1 by metabolic engineering. Four enzyme genes (PnDDS, CYP716A47, CYP716S1 and UGT71A56) isolated from Panax notoginseng were introduced into tobacco. Thus, a biosynthetic pathway for ginsenoside F1 synthesis was artificially constructed in tobacco cells; moreover, the four exogenous genes could be expressed in the roots, stems and leaves of transgenic plants. Consequently, ginsenoside F1 and its precursors were successfully synthesized in the transgenic tobacco, compared with Panax plants, the content of ginsenoside F1 in transgenic tobacco was doubled. In addition, accumulation of ginsenoside F1 and its precursors in transgenic tobacco shows organ specificity. Based on these results, a new approach was established to produce rare ginsenoside F1; meanwhile, such strategy could also be employed in plant hosts for the heterologous synthesis of other important or rare natural products.

Asunto(s)

Ginsenósidos , Nicotiana , Plantas Modificadas Genéticamente , Ginsenósidos/biosíntesis , Ginsenósidos/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Plantas Modificadas Genéticamente/genética , Ingeniería Metabólica/métodos , Vías Biosintéticas/genética

RESUMEN

Ginsenosides, the primary bioactive constituents in ginseng (Panax ginseng), possess substantial pharmacological potential and are in high demand in the market. The plant hormone methyl jasmonate (MeJA) effectively elicits ginsenoside biosynthesis in P. ginseng, though the regulatory mechanism remains largely unexplored. NAC transcription factors are critical in intricate plant regulatory networks and participate in numerous plant physiological activities. In this study, we identified a MeJA-responsive NAC transcription factor gene, PgNAC72, from a transcriptome library produced from MeJA-treated P. ginseng callus. Predominantly expressed in P. ginseng flowers, PgNAC72 localizes to the nucleus. Overexpressing PgNAC72 (OE-PgNAC72) in P. ginseng callus notably elevated total saponin levels, particularly dammarane-type ginsenosides, by upregulating dammarenediol synthase (PgDDS), encoding a key enzyme in the ginsenoside biosynthesis pathway. Electrophoretic mobility shift assays and dual-luciferase assays confirmed that PgNAC72 binds to the NAC-binding elements in the PgDDS promoter, thereby activating its transcription. Further RNA-seq and terpenoid metabolomic data in the OE-PgNAC72 line confirmed that PgNAC72 enhances ginsenoside biosynthesis. These findings uncover a regulatory role of PgNAC72 in MeJA-mediated ginsenoside biosynthesis, providing insights into the ginsenoside regulatory network and presenting a valuable target gene for metabolic engineering.

Asunto(s)

Acetatos , Regulación de la Expresión Génica de las Plantas , Oxilipinas , Panax , Proteínas de Plantas , Saponinas , Factores de Transcripción , Panax/genética , Panax/metabolismo , Saponinas/biosíntesis , Saponinas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Oxilipinas/metabolismo , Oxilipinas/farmacología , Acetatos/farmacología , Ciclopentanos/metabolismo , Ciclopentanos/farmacología , Ginsenósidos/biosíntesis , Ginsenósidos/metabolismo , Regiones Promotoras Genéticas/genética , Transferasas Alquil y Aril

RESUMEN

Ginsenosides, the primary pharmacologically active constituents of the Panax genus, have demonstrated a variety of medicinal properties, including anticardiovascular disease, cytotoxic, antiaging, and antidiabetes effects. However, the low concentration of ginsenosides in plants and the challenges associated with their extraction impede the advancement and application of ginsenosides. Heterologous biosynthesis represents a promising strategy for the targeted production of these natural active compounds. As representative triterpenoids, the biosynthetic pathway of the aglycone skeletons of ginsenosides has been successfully decoded. While the sugar moiety is vital for the structural diversity and pharmacological activity of ginsenosides, the mining of uridine diphosphate-dependent glycosyltransferases (UGTs) involved in ginsenoside biosynthesis has attracted a lot of attention and made great progress in recent years. In this paper, we summarize the identification and functional study of UGTs responsible for ginsenoside synthesis in both plants, such as Panax ginseng and Gynostemma pentaphyllum, and microorganisms including Bacillus subtilis and Saccharomyces cerevisiae. The UGT-related microbial cell factories for large-scale ginsenoside production are also mentioned. Additionally, we delve into strategies for UGT mining, particularly potential rapid screening or identification methods, providing insights and prospects. This review provides insights into the study of other unknown glycosyltransferases as candidate genetic elements for the heterologous biosynthesis of rare ginsenosides.

Asunto(s)

Ginsenósidos , Glicosiltransferasas , Ginsenósidos/biosíntesis , Ginsenósidos/química , Ginsenósidos/metabolismo , Glicosiltransferasas/metabolismo , Saccharomyces cerevisiae , Estructura Molecular , Panax/química , Uridina Difosfato/metabolismo , Bacillus subtilis/enzimología , Vías Biosintéticas

RESUMEN

Panax root is an important material used in food and medicine. Its cultivation and production usually depend on root shape and ginsenoside content. There is limited understanding about the synergistic regulatory mechanisms underlying root development and ginsenoside accumulation in Panax. MADS-box transcription factors possibly play a significant role in regulation of root growth and secondary metabolites. In this study, we identified MADS-box transcription factors of Panax, and found high expression levels of SVP, ANR1 and SOC1-like clade genes in its roots. We confirmed that two SOC1-like genes, PgMADS41 and PgMADS44, bind to expansion gene promoters (PgEXLB5 and PgEXPA13), which contribute to root growth, and to SE-4, CYP716A52v2-4, and ß-AS-13 promoters, which participate in ginsenoside Ro biosynthesis. These two genes were found to increase lateral root number and main root length in transgenic Arabidopsis thaliana by improving AtEXLA1, AtEXLA3, AtEXPA5, and AtEXPA6 gene expression. As a non-phytohormone regulatory tool, Ro can stimulate adventitious root growth by influencing their expression and ginsenoside accumulation. Our study provides new insights into the coordinated regulatory function of SOC1-like clade genes in Panax root development and triterpenoid accumulation, paving the way towards understanding root formation and genetic improvement in Panax.

Asunto(s)

Ginsenósidos , Panax , Proteínas de Plantas , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Ginsenósidos/biosíntesis , Panax/genética , Panax/metabolismo , Raíces de Plantas/metabolismo , Factores de Transcripción/genética , Proteínas de Plantas/metabolismo

RESUMEN

Ginsenosides are glycosylated dammarene-type triterpenes that have been identified in distantly related Panax ginseng and Gynostemma pentaphyllum. The phylogenetic relatedness of the ginsenoside biosynthetic genes in the two species was previously unknown. The final steps of ginsenoside biosynthesis are the glycosylations of hydroxylated triterpenes, protopanaxadiol (PPD) and protopanaxatriol (PPT), and their glycosylated forms by UDP-glycosyltransferases (UGTs). Ginsenoside biosynthetic UGTs have been identified in Panax but not in Gynostemma. Through a biochemical screening of Gynostemma UGTs (GpUGTs), we herein identified three groups of ginsenoside biosynthetic GpUGTs. These groups comprise: two GpUGTs that belong to the UGT71 family and glucosylate the C20-OH positions of PPD- and PPT-type ginsenosides; one GpUGT that belongs to the UGT74 family and glucosylates the C3-OH position of PPD-type ginsenosides; and two GpUGTs that belong to the UGT94 family and add a glucose to the C3-O-glucosides of PPD-type ginsenosides. These GpUGTs belong to the same UGT families as the ginsenoside biosynthetic Panax UGTs (PgUGTs). However, GpUGTs and PgUGTs belong to different subfamilies. Furthermore, cucumber UGTs orthologous to GpUGTs do not glucosylate ginsenosides. These results collectively suggest that, during evolution, P. ginseng and G. pentaphyllum independently opted to use the same UGT families to synthesize ginsenosides.

Asunto(s)

Vías Biosintéticas/genética , Ginsenósidos/biosíntesis , Ginsenósidos/genética , Glicosiltransferasas/metabolismo , Gynostemma/genética , Gynostemma/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas

RESUMEN

Among rare earth elements, cerium has the unique ability of regulating the growth of plant cells and the biosynthesis of metabolites at different stages of plant development. The signal pathways of Ce3+-mediated ginsenosides biosynthesis in ginseng hairy roots were investigated. At a low concentration, Ce3+ improved the elongation and biomass of hairy roots. The Ce3+-induced accumulation of ginsenosides showed a high correlation with the reactive oxygen species (ROS), as well as the biosynthesis of endogenous methyl jasmonate (MeJA) and ginsenoside key enzyme genes (PgSS, PgSE and PgDDS). At a Ce3+ concentration of 20 mg L-1, the total ginsenoside content was 1.7-fold, and the total ginsenosides yield was 2.7-fold that of the control. Malondialdehyde (MDA) content and the ROS production rate were significantly higher than those of the control. The activity of superoxide dismutase (SOD) was significantly activated within the Ce3+ concentration range of 10 to 30 mg L-1. The activity of catalase (CAT) and peroxidase (POD) strengthened with the increasing concentration of Ce3+ in the range of 20-40 mg L-1. The Ce3+ exposure induced transient production of superoxide anion (O2•-) and hydrogen peroxide (H2O2). Together with the increase in the intracellular MeJA level and enzyme activity for lipoxygenase (LOX), there was an increase in the gene expression level of MeJA biosynthesis including PgLOX, PgAOS and PgJMT. Our results also revealed that Ce3+ did not directly influence PgSS, PgSE and PgDDS activity. We speculated that Ce3+-induced ROS production could enhance the accumulation of ginsenosides in ginseng hairy roots via the direct stimulation of enzyme genes for MeJA biosynthesis. This study demonstrates a potential approach for understanding and improving ginsenoside biosynthesis that is regulated by Ce3+-mediated signal transduction.

Asunto(s)

Acetatos/metabolismo , Cerio/farmacología , Ciclopentanos/metabolismo , Ginsenósidos/biosíntesis , Oxilipinas/metabolismo , Panax/química , Panax/metabolismo , Raíces de Plantas/química , Raíces de Plantas/metabolismo , Ginsenósidos/análisis , Panax/efectos de los fármacos , Panax/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

RNA alternative splicing (AS) is prevalent in higher organisms and plays a paramount role in biology; therefore, it is crucial to have comprehensive knowledge on AS to understand biology. However, knowledge is limited about how AS activates in a single plant and functions in a biological process. Ginseng is one of the most widely used medicinal herbs that is abundant in a number of medicinal bioactive components, especially ginsenosides. In this study, we sequenced the transcripts of 14 organs from a 4-year-old ginseng plant and quantified their ginsenoside contents. We identified AS genes by analyzing their transcripts with the ginseng genome and verified their AS events by PCR. The plant had a total of 13,863 AS genes subjected to 30,801 AS events with five mechanisms: skipped exon, retained intron, alternative 5'splice site, alternative 3' splice site, and mutually exclusive exon. The genes that were more conserved, had more exons, and/or expressed across organs were more likely to be subjected to AS. AS genes were enriched in over 500 GO terms in the plant even though the number of AS gene-enriched GO terms varied across organs. At least 24 AS genes were found to be involved in ginsenoside biosynthesis. These AS genes were significantly up-enriched and more likely to form a co-expression network, thus suggesting the functions of AS and correlations of the AS genes in the process. This study provides comprehensive insights into the molecular characteristics and biological functions of AS in a single plant; thus, helping better understand biology.

Asunto(s)

Empalme Alternativo/genética , Ginsenósidos/biosíntesis , Panax , Secuencia de Bases , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Ginsenósidos/genética , Redes y Vías Metabólicas/genética , Panax/genética , Panax/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcriptoma

RESUMEN

MAIN CONCLUSION: Short-term cold stress can induce the increased expression of key enzyme-encoding genes involved in secondary metabolite synthesis, thereby increasing secondary metabolite concentration. Cold stress is an ecologically limiting factor that strongly affects the physiological and biochemical properties of medicinal plants often resulting in changes of the secondary metabolic process. Ginsenosides are the main active ingredients in medicinal ginseng yet few studies exist on the effect of cold stress on the expression of ginsenosides or the molecular mechanism underlying its regulation. Here, we evaluated the effects of cold stress on the physiological characteristics and secondary metabolism of P. ginseng embryogenic calli. Physiological measurements and RNA-Seq analysis were used to dissect the metabolic and molecular responses of P. ginseng to cold conditions. We found that the dynamic accumulation of ginsenoside and various physiological indicators leads to homogenous adaptation to cold stress. Secondary metabolism of ginseng could be a compensation mechanism to facilitate its adaptation to cold stress. Combined with the changes in the endogenous hormone content, 9-cis-epoxycarotenoid dioxygenase (NCED), zeaxanthin epoxidase (ZEP), and short chain dehydrogenase (SDR) from the abscisic acid (ABA) synthesis pathway were identified as key mediators of this response. Thus, an appropriate degree of cold stress may promote accumulation of ginsenosides. Moreover, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR2), squalene epoxidase (SE1), squalene synthase (SS), dammarenediol synthase (DS-II), and ß-alanine C-28 hydroxylase (CYP716A52v2) should be considered key mediators of the cold stress response and ginsenoside biosynthesis. During industrial production, short-term cold stress should be carried out on ginseng calli to improve the quality of its medicinal materials.

Asunto(s)

Respuesta al Choque por Frío , Ginsenósidos/biosíntesis , Panax/fisiología , Metabolismo Secundario , Regulación de la Expresión Génica de las Plantas

RESUMEN

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.

Asunto(s)

Mapeo Cromosómico , Genoma de Planta , Ginsenósidos/biosíntesis , Ginsenósidos/genética , Panax notoginseng/genética , Panax notoginseng/metabolismo , China , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Extractos Vegetales/biosíntesis , Extractos Vegetales/genética , Plantas Medicinales/genética , Plantas Medicinales/metabolismo , Transcriptoma

RESUMEN

Multiple enzyme coimmobilizations mimicking nature cascade enzymatic pathways have potential applications in diverse fields. We have developed a strategy for orderly coimmobilizing multienzymes by combining hierarchically self-assembled multimeric enzymes with specifically abundant polyhistidine tag affinity-mediated immobilization. Using this strategy, an ordered coimmobilization of the glycosyltransferase UGT51 mutant and sucrose synthase was constructed to realize the regeneration of costly sugar donor UDP-glucose that was used in the biosynthesis of the rare ginsenoside Rh2. The ordered coimmobilization array not only significantly boosted the immobilization and catalysis efficiency but also improved UDP-glucose regeneration, storage stability, and reusability compared to those of random coimmobilization and free enzyme-assembly systems. This study provides a great promise for fabricating enzyme arrays and highlights the synergistic benefits of nanocomplexes in enhancing biocatalytic cascade performance.

Asunto(s)

Materiales Biocompatibles/metabolismo , Ginsenósidos/biosíntesis , Glicosiltransferasas/metabolismo , Biocatálisis , Materiales Biocompatibles/química , Conformación de Carbohidratos , Ginsenósidos/química , Ensayo de Materiales , Modelos Moleculares , Tamaño de la Partícula

RESUMEN

Ginseng, also known as the king of herbs, has been regarded as an important traditional medicine for several millennia. Ginsenosides, a group of triterpenoid saponins, have been characterized as bioactive compounds of ginseng. The complexity of ginsenosides hindered ginseng research and development both in cultivation and clinical research. Therefore, deciphering the ginsenoside biosynthesis pathway has been a focus of interest for researchers worldwide. The new emergence of biological research tools consisting of omics and bioinformatic tools or computational biology tools are the research trend in the new century. Ginseng is one of the main subjects analyzed using these new quantification tools, including tools of genomics, transcriptomics, and proteomics. Here, we review the current progress of ginseng omics research and provide results for the ginsenoside biosynthesis pathway. Organization and expression of the entire pathway, including the upstream MVA pathway, the cyclization of ginsenoside precursors, and the glycosylation process, are illustrated. Regulatory gene families such as transcriptional factors and transporters are also discussed in this review.

Asunto(s)

Ginsenósidos/biosíntesis , Panax/metabolismo , Animales , Ginsenósidos/química , Ginsenósidos/genética , Humanos , Panax/química , Panax/genética , Transcriptoma

RESUMEN

Panax notoginseng is one of the most widely used traditional Chinese herbs with particularly valued roots. Triterpenoid saponins are mainly specialized secondary metabolites, which medically act as bioactive components. Knowledge of the ginsenoside biosynthesis in P. notoginseng, which is of great importance in the industrial biosynthesis and genetic breeding program, remains largely undetermined. Here we combined single molecular real time (SMRT) and Second-Generation Sequencing (SGS) technologies to generate a widespread transcriptome atlas of P. notoginseng. We mapped 2,383 full-length non-chimeric (FLNC) reads to adjacently annotated genes, corrected 1,925 mis-annotated genes and merged into 927 new genes. We identified 8,111 novel transcript isoforms that have improved the annotation of the current genome assembly, of which we found 2,664 novel lncRNAs. We characterized more alternative splicing (AS) events from SMRT reads (20,015 AS in 6,324 genes) than Illumina reads (18,498 AS in 9,550 genes), which contained a number of AS events associated with the ginsenoside biosynthesis. The comprehensive transcriptome landscape reveals that the ginsenoside biosynthesis predominantly occurs in flowers compared to leaves and roots, substantiated by levels of gene expression, which is supported by tissue-specific abundance of isoforms in flowers compared to roots and rhizomes. Comparative metabolic analyses further show that a total of 17 characteristic ginsenosides increasingly accumulated, and roots contained the most ginsenosides with variable contents, which are extraordinarily abundant in roots of the three-year old plants. We observed that roots were rich in protopanaxatriol- and protopanaxadiol-type saponins, whereas protopanaxadiol-type saponins predominated in aerial parts (leaves, stems and flowers). The obtained results will greatly enhance our understanding about the ginsenoside biosynthetic machinery in the genus Panax.

Asunto(s)

Ginsenósidos/biosíntesis , Ginsenósidos/genética , Panax notoginseng/genética , Transcriptoma/genética , Empalme Alternativo/genética , Flores/genética , Flores/metabolismo , Flores/fisiología , Perfilación de la Expresión Génica/métodos , Genes de Plantas/genética , Ginsenósidos/metabolismo , Anotación de Secuencia Molecular/métodos , Panax/genética , Panax/metabolismo , Panax notoginseng/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Raíces de Plantas/fisiología , RNA-Seq/métodos , Rizoma/genética , Rizoma/metabolismo , Rizoma/fisiología , Sapogeninas/metabolismo , Saponinas/genética , Saponinas/metabolismo , Secuenciación del Exoma/métodos

RESUMEN

More than 150 ginsenosides have been isolated and identified from Panax plants. Ginsenosides with different glycosylation degrees have demonstrated different chemical properties and bioactivity. In this study, we systematically cloned and characterized 46 UGT94 family UDP-glycosyltransferases (UGT94s) from a mixed Panax ginseng/callus cDNA sample with high amino acid identity. These UGT94s were found to catalyze sugar chain elongation at C3-O-Glc and/or C20-O-Glc of protopanaxadiol (PPD)-type, C20-O-Glc or C6-O-Glc of protopanaxatriol (PPT)-type or both C3-O-Glc of PPD-type and C6-O-Glc of PPT-type or C20-O-Glc of PPD-type and PPT-type ginsenosides with different efficiencies. We also cloned 26 and 51 UGT94s from individual P. ginseng and P. notoginseng plants, respectively; our characterization results suggest that there is a group of UGT94s with high amino acid identity but diverse functions or catalyzing activities even within individual plants. These UGT94s were classified into three clades of the phylogenetic tree and consistent with their catalytic function. Based on these UGT94s, we elucidated the biosynthetic pathway of a group of ginsenosides. Our present results reveal a series of UGTs involved in second sugar chain elongation of saponins in Panax plants, and provide a scientific basis for understanding the diverse evolution mechanisms of UGT94s among plants.

Asunto(s)

Ginsenósidos/biosíntesis , Glicosiltransferasas/genética , Panax/enzimología , Vías Biosintéticas , Ginsenósidos/metabolismo , Glicosilación , Glicosiltransferasas/metabolismo , Panax/genética , Panax/metabolismo , Filogenia , Uridina Difosfato/metabolismo

RESUMEN

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

Asunto(s)

Ginsenósidos/biosíntesis , Glicosiltransferasas/biosíntesis , Sapogeninas/metabolismo , Ginsenósidos/química , Glicosiltransferasas/química , Panax/química , Ingeniería de Proteínas/métodos , Sapogeninas/química , Biología Sintética/métodos