RESUMO
Cytochromes P450 (P450s) are one of the largest enzymatic protein families and play critical roles in the synthesis and metabolism of plant secondary metabolites. Astragaloside IV (AS-IV) is one of the primary active components in Astragalus herbs, exhibiting diverse biological activities and pharmacological effects. However, P450s involved in the astragaloside biosynthesis have not been systematically analyzed in Astragalus mongholicus (A. mongholicus). In this study, we identified 209 P450 genes from the genome of A. mongholicus (AmP450s), which were classified into nine clans and 47 families and performed a systematic overview of their physical and chemical properties, phylogeny, gene structures and conserved motifs. Weighted gene co-expression network analysis (WGCNA) revealed that AmP450s are critical in the astragaloside biosynthesis pathway. The expression levels of these AmP450s were verified by quantitative real-time PCR (qRT-PCR) analysis in the root, stem and leaf, showing that most AmP450s are abundant in the root. Additionally, the correlation analysis between gene expressions and AS-IV content showed that twelve AmP450s, especially CYP71A28, CYP71D16 and CYP72A69, may have significant potential in the biosynthesis of astragaloside. This study systematically investigates the P450s of A. mongholicus and offers valuable insights into further exploring the functions of CYP450s in the astragaloside biosynthesis pathway.
Assuntos
Astrágalo , Sistema Enzimático do Citocromo P-450 , Regulação da Expressão Gênica de Plantas , Filogenia , Saponinas , Triterpenos , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Saponinas/biossíntese , Saponinas/genética , Saponinas/metabolismo , Triterpenos/metabolismo , Astrágalo/genética , Astrágalo/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Perfilação da Expressão GênicaRESUMO
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.
Assuntos
Ginsenosídeos , Glicosiltransferases , Saponinas , Glicosiltransferases/metabolismo , Glicosiltransferases/química , Saponinas/química , Saponinas/biossíntese , Saponinas/metabolismo , Ginsenosídeos/química , Ginsenosídeos/biossíntese , Ginsenosídeos/metabolismo , Animais , Camundongos , Estrutura Molecular , Células RAW 264.7 , Melanthiaceae/química , Melanthiaceae/enzimologia , Melanthiaceae/metabolismo , Simulação de Acoplamento Molecular , Liliaceae/químicaRESUMO
BACKGROUND: Soapberry (Sapindus mukorossi) is an economically important multifunctional tree species. Triterpenoid saponins have many functions in soapberry. However, the types of uridine diphosphate (UDP) glucosyltransferases (UGTs) involved in the synthesis of triterpenoid saponins in soapberry have not been clarified. RESULTS: In this study, 42 SmUGTs were identified in soapberry, which were unevenly distributed on 12 chromosomes and had sequence lengths of 450 bp to 1638 bp, with an average of 1388 bp. The number of amino acids in SmUGTs was 149 to 545, with an average of 462. Most SmUGTs were acidic and hydrophilic unstable proteins, and their secondary structures were mainly α-helices and random coils. All had conserved UDPGT and PSPG-box domains. Phylogenetic analysis divided them into four subclasses, which glycosylated different carbon atoms. Prediction of cis-acting elements suggested roles of SmUGTs in plant development and responses to environmental stresses. The expression patterns of SmUGTs differed according to the developmental stage of fruits, as determined by transcriptomics and RT-qPCR. Co-expression network analysis of SmUGTs and related genes/transcription factors in the triterpenoid saponin synthesis pathway was also performed. The results indicated potential roles for many transcription factors, such as SmERFs, SmGATAs and SmMYBs. A correlation analysis showed that 42 SmUGTs were crucial in saponin synthesis in soapberry. CONCLUSIONS: Our findings suggest optimal targets for manipulating glycosylation in soapberry triterpenoid saponin biosynthesis; they also provide a theoretical foundation for further evaluation of the functions of SmUGTs and analyses of their biosynthetic mechanisms.
Assuntos
Glucosiltransferases , Filogenia , Sapindus , Saponinas , Triterpenos , Saponinas/biossíntese , Saponinas/metabolismo , Sapindus/genética , Sapindus/metabolismo , Triterpenos/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Genes de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
BACKGROUND: The formation of pharmacologically active components in medicinal plants is significantly impacted by DNA methylation. However, the exact mechanisms through which DNA methylation regulates secondary metabolism remain incompletely understood. Research in model species has demonstrated that DNA methylation at the transcription factor binding site within functional gene promoters can impact the binding of transcription factors to target DNA, subsequently influencing gene expression. These findings suggest that the interaction between transcription factors and target DNA could be a significant mechanism through which DNA methylation regulates secondary metabolism in medicinal plants. RESULTS: This research conducted a comprehensive analysis of the NAC family in E. senticosus, encompassing genome-wide characterization and functional analysis. A total of 117 EsNAC genes were identified and phylogenetically divided into 15 subfamilies. Tandem duplications and chromosome segment duplications were found to be the primary replication modes of these genes. Motif 2 was identified as the core conserved motif of the genes, and the cis-acting elements, gene structures, and expression patterns of each EsNAC gene were different. EsJUB1, EsNAC047, EsNAC098, and EsNAC005 were significantly associated with the DNA methylation ratio in E. senticosus. These four genes were located in the nucleus or cytoplasm and exhibited transcriptional self-activation activity. DNA methylation in EsFPS, EsSS, and EsSE promoters significantly reduced their activity. The methyl groups added to cytosine directly hindered the binding of the promoters to EsJUB1, EsNAC047, EsNAC098, and EsNAC005 and altered the expression of EsFPS, EsSS, and EsSE genes, eventually leading to changes in saponin synthesis in E. senticosus. CONCLUSIONS: NAC transcription factors that are hindered from binding by methylated DNA are found in E. senticosus. The incapacity of these NACs to bind to the promoter of the methylated saponin synthase gene leads to subsequent alterations in gene expression and saponin synthesis. This research is the initial evidence showcasing the involvement of EsNAC in governing the impact of DNA methylation on saponin production in E. senticosus.
Assuntos
Metilação de DNA , Eleutherococcus , Proteínas de Plantas , Regiões Promotoras Genéticas , Saponinas , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Eleutherococcus/genética , Eleutherococcus/metabolismo , Saponinas/biossíntese , Saponinas/genética , Regulação da Expressão Gênica de Plantas , FilogeniaRESUMO
QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.
Assuntos
Adjuvantes Imunológicos , Engenharia Metabólica , Saccharomyces cerevisiae , Saponinas , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/metabolismo , Vias Biossintéticas/genética , Desenho de Fármacos , Enzimas/genética , Enzimas/metabolismo , Engenharia Metabólica/métodos , Plantas/enzimologia , Plantas/genética , Plantas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saponinas/biossíntese , Saponinas/química , Saponinas/genética , Saponinas/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Acetatos , Regulação da Expressão Gênica de Plantas , Oxilipinas , Panax , Proteínas de Plantas , Saponinas , Fatores de Transcrição , Panax/genética , Panax/metabolismo , Saponinas/biossíntese , Saponinas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Oxilipinas/metabolismo , Oxilipinas/farmacologia , Acetatos/farmacologia , Ciclopentanos/metabolismo , Ciclopentanos/farmacologia , Ginsenosídeos/biossíntese , Ginsenosídeos/metabolismo , Regiões Promotoras Genéticas/genética , Alquil e Aril TransferasesRESUMO
Climate change may result in a drier climate and increased salinization, threatening agricultural productivity worldwide. Quinoa (Chenopodium quinoa) produces highly nutritious seeds and tolerates abiotic stresses such as drought and high salinity, making it a promising future food source. However, the presence of antinutritional saponins in their seeds is an undesirable trait. We mapped genes controlling seed saponin content to a genomic region that includes TSARL1. We isolated desired genetic variation in this gene by producing a large mutant library of a commercial quinoa cultivar and screening the library for specific nucleotide substitutions using droplet digital PCR. We were able to rapidly isolate two independent tsarl1 mutants, which retained saponins in the leaves and roots for defence, but saponins were undetectable in the seed coat. We further could show that TSARL1 specifically controls seed saponin biosynthesis in the committed step after 2,3-oxidosqualene. Our work provides new important knowledge on the function of TSARL1 and represents a breakthrough for quinoa breeding.
Assuntos
Chenopodium quinoa , Genótipo , Saponinas , Sementes , Chenopodium quinoa/genética , Chenopodium quinoa/metabolismo , Saponinas/biossíntese , Saponinas/metabolismo , Sementes/genética , Sementes/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
The leaves and roots of Liriope muscari (Decne.) Baily were subjected to high-throughput Illumina transcriptome sequencing. Bioinformatics analysis was used to investigate the enzyme genes and key transcription factors involved in regulating the accumulation of steroidal saponins, which are the main active ingredient in L. muscari. These analyses aimed to reveal the molecular mechanism behind steroidal saponin accumulation. The sequencing results of L. muscari revealed 31 enzymes, including AACT, CAS, DXS and DXR, that are involved in the synthesis of steroidal saponins. Among these enzymes, 16 were in the synthesis of terpenoid skeleton, 3 were involved in the synthesis of sesquiterpene and triterpene, and 12 were involved in the synthesis of steroidal compound. Differential gene expression identified 15 metabolic enzymes coded by 34 differentially expressed genes (DEGs) in the leaves and roots, which were associated with steroidal saponin synthesis. Further analysis using gene co-expression patterns showed that 14 metabolic enzymes coded by 31 DEGs were co-expressed. In addition, analysis using gene co-expression analysis and PlantTFDB's transcription factor analysis tool predicted the involvement of 8 transcription factors, including GAI, PIF4, PIL6, ERF8, SVP, LHCA4, NF-YB3 and DOF2.4, in regulating 6 metabolic enzymes such as DXS, DXR, HMGR, DHCR7, DHCR24, and CAS. These eight transcription factors were predicted to play important roles in regulating steroidal saponin accumulation in L. muscari. Promoter analysis of these transcription factors indicated that their main regulatory mechanisms involve processes such as abscisic acid response, drought-induction stress response and light response, especially abscisic acid responsive elements (ABRE) response and MYB binding site involved in drought-inducibility (MBS) response pathway. Furthermore, qRT-PCR analysis of these eight key transcription factors demonstrated their specific differences in the leaves and roots.
Assuntos
Biologia Computacional , Liriope (Planta) , Folhas de Planta , Saponinas , Fatores de Transcrição , Transcriptoma , Saponinas/metabolismo , Saponinas/biossíntese , Biologia Computacional/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/genética , Liriope (Planta)/genética , Liriope (Planta)/metabolismo , Esteroides/metabolismo , Esteroides/biossíntese , Raízes de Plantas/metabolismo , Raízes de Plantas/genética , Regulação da Expressão Gênica de Plantas , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Cholesterol is the precursor of bioactive plant metabolites such as steroidal saponins. An Australian plant, Dioscorea transversa, produces only two steroidal saponins: 1ß-hydroxyprotoneogracillin and protoneogracillin. Here, we used D. transversa as a model in which to elucidate the biosynthetic pathway to cholesterol, a precursor to these compounds. Preliminary transcriptomes of D. transversa rhizome and leaves were constructed, annotated, and analyzed. We identified a novel sterol side-chain reductase as a key initiator of cholesterol biosynthesis in this plant. By complementation in yeast, we determine that this sterol side-chain reductase reduces Δ24,28 double bonds required for phytosterol biogenesis as well as Δ24,25 double bonds. The latter function is believed to initiate cholesterogenesis by reducing cycloartenol to cycloartanol. Through heterologous expression, purification, and enzymatic reconstitution, we also demonstrate that the D. transversa sterol demethylase (CYP51) effectively demethylates obtusifoliol, an intermediate of phytosterol biosynthesis and 4-desmethyl-24,25-dihydrolanosterol, a postulated downstream intermediate of cholesterol biosynthesis. In summary, we investigated specific steps of the cholesterol biosynthetic pathway, providing further insight into the downstream production of bioactive steroidal saponin metabolites.
Assuntos
Colesterol , Dioscorea , Fitosteróis , Austrália , Colesterol/biossíntese , Família 51 do Citocromo P450/genética , Família 51 do Citocromo P450/isolamento & purificação , Família 51 do Citocromo P450/metabolismo , Dioscorea/classificação , Dioscorea/enzimologia , Dioscorea/genética , Oxirredutases/metabolismo , Fitosteróis/biossíntese , Fitosteróis/química , Fitosteróis/genética , Saccharomyces cerevisiae/genética , Saponinas/biossíntese , Saponinas/genética , TranscriptomaRESUMO
The genus Polygonatum Mill. belongs to the Liliaceae family, which is widely distributed all over the world. Modern studies have found that Polygonatum plants are very rich in chemical compounds such as saponins, polysaccharides and flavonoids. Steroidal saponins are the most commonly studied saponins in the genus Polygonatum and a total of 156 compounds have been isolated from 10 species of the genus. These molecules possess antitumor, immunoregulatory, anti-inflammatory, antibacterial, antiviral, hypoglycemic, lipid-lowering and anti-osteoporotic activities. In this review, we summarize recent advances in studies of the chemical constituents of steroidal saponins from Polygonatum, including their structural characteristics, possible biosynthetic pathways and pharmacological effects. Then, the relationship between the structure and some physiological activities is considered. This review aims to provide reference for further exploitation and utilization of the genus Polygonatum.
Assuntos
Vias Biossintéticas , Polygonatum , Saponinas , Esteroides , Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Antivirais/farmacologia , Hipoglicemiantes/farmacologia , Polygonatum/química , Polygonatum/metabolismo , Saponinas/biossíntese , Saponinas/química , Saponinas/classificação , Saponinas/isolamento & purificação , Saponinas/farmacologia , Esteroides/biossíntese , Esteroides/química , Esteroides/classificação , Esteroides/isolamento & purificação , Esteroides/farmacologia , Relação Estrutura-Atividade , Humanos , AnimaisRESUMO
The Chilean soapbark tree (Quillaja saponaria) produces soap-like molecules called QS saponins that are important vaccine adjuvants. These highly valuable compounds are sourced by extraction from the bark, and their biosynthetic pathway is unknown. Here, we sequenced the Q. saponaria genome. Through genome mining and combinatorial expression in tobacco, we identified 16 pathway enzymes that together enable the production of advanced QS pathway intermediates that represent a bridgehead for adjuvant bioengineering. We further identified the enzymes needed to make QS-7, a saponin with excellent therapeutic properties and low toxicity that is present in low abundance in Q. saponaria bark extract. Our results enable the production of Q. saponaria vaccine adjuvants in tobacco and open the way for new routes to access and engineer natural and new-to-nature immunostimulants.
Assuntos
Adjuvantes de Vacinas , Vias Biossintéticas , Quillaja , Saponinas , Adjuvantes de Vacinas/biossíntese , Adjuvantes de Vacinas/química , Adjuvantes de Vacinas/genética , Quillaja/enzimologia , Quillaja/genética , Saponinas/biossíntese , Saponinas/química , Saponinas/genética , Análise de Sequência de DNA , Genoma de Planta , Vias Biossintéticas/genética , Nicotiana/genética , Nicotiana/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Reconstituting a plant biosynthetic pathway enables a sustainable supply of vaccine adjuvants.
Assuntos
Adjuvantes de Vacinas , Imunização Secundária , Quillaja , Saponinas , Adjuvantes de Vacinas/biossíntese , Vias Biossintéticas , Quillaja/metabolismo , Saponinas/biossíntese , HumanosRESUMO
Bupleurum falcatum L. is frequently used in traditional herbal medicine in Asia. Saikosaponins (SSs) are the main bioactive ingredients of B. falcatum, but the biosynthetic pathway of SSs is unclear, and the biosynthesis of species-specific phytometabolites is little known. Here we resolved the transcriptome profiles of B. falcatum to identify candidate genes that might be involved in the biosynthesis of SSs. By isoform sequencing (Iso-Seq) analyses of the whole plant, a total of 26.98 Gb of nucleotides were obtained and 124,188 unigenes were identified, and 81,594 unigenes were successfully annotated. A total of 1033 unigenes of 20 families related to the mevalonate (MVA) pathway and methylerythritol phosphate (MEP) pathway of the SS biosynthetic pathway were identified. The WGCNA (weighted gene co-expression network analysis) of these unigenes revealed that only the co-expression module of MEmagenta, which contained 343 unigenes, was highly correlated with the biosynthesis of SSs. Comparing differentially expressed gene analysis and the WGCNA indicated that 130 out of 343 genes of the MEmagenta module exhibited differential expression levels, and genes with the most "hubness" within this module were predicted. Manipulation of these genes might improve the biosynthesis of SSs.
Assuntos
Saponinas , Triterpenos , Saponinas/análise , Saponinas/biossíntese , Transcriptoma , Triterpenos/análise , Triterpenos/metabolismo , Bupleurum/metabolismoRESUMO
Engineering the function of triterpene glucosyltransferases (GTs) is challenging due to the large size of the sugar acceptors. In this work, we identified a multifunctional glycosyltransferase AmGT8 catalyzing triterpene 3-/6-/2'-O-glycosylation from the medicinal plant Astragalus membranaceus. To engineer its regiospecificity, a small mutant library was built based on semi-rational design. Variants A394F, A394D, and T131V were found to catalyze specific 6-O, 3-O, and 2'-O glycosylation, respectively. The origin of regioselectivity of AmGT8 and its A394F variant was studied by molecular dynamics and hydrogen deuterium exchange mass spectrometry. Residue 394 is highly conserved as A/G and is critical for the regiospecificity of the C- and O-GTs TcCGT1 and GuGT10/14. Finally, astragalosides III and IV were synthesized by mutants A394F, T131V and P192E. This work reports biocatalysts for saponin synthesis and gives new insights into protein engineering of regioselectivity in plant GTs.
Assuntos
Glicosiltransferases/metabolismo , Engenharia de Proteínas , Saponinas/biossíntese , Triterpenos/metabolismo , Astragalus propinquus/enzimologia , Biocatálise , Glicosiltransferases/química , Conformação Proteica , Saponinas/química , Estereoisomerismo , Triterpenos/químicaRESUMO
Genomic tRNA copy numbers determine cytoplasmic tRNA abundances, which in turn influence translation efficiency, but the underlying mechanism is not well understood. Using the sea cucumber Apostichopus japonicus as a model, we combined genomic sequence, transcriptome expression and ecological food resource data to study its codon usage adaptation. The results showed that, unlike intragenic non-coding RNAs, transfer RNAs (tRNAs) tended to be transcribed independently. This may be attributed to their specific Pol III promoters that lack transcriptional regulation, which may underlie the correlation between genomic copy number and cytoplasmic abundance of tRNAs. Moreover, codon usage optimization was mostly restrained by a gene's amino acid sequence, which might be a compromise between functionality and translation efficiency for stress responses were highly optimized for most echinoderms, while enzymes for saponin biosynthesis (LAS, CYPs and UGTs) were especially optimized in sea cucumbers, which might promote saponin synthesis as a defence strategy. The genomic tRNA content of A. japonicus was positively correlated with amino acid content in its natural food particles, which should promote its efficiency in protein synthesis. We propose that coevolution between genomic tRNA content and codon usage of sea cucumbers facilitates their saponin synthesis and survival using food resources with low nutrient content.
Assuntos
Perfilação da Expressão Gênica/métodos , RNA de Transferência/genética , Saponinas/biossíntese , Pepinos-do-Mar/genética , Animais , Vias Biossintéticas , Uso do Códon , Citoplasma/genética , Variações do Número de Cópias de DNA , Regulação da Expressão Gênica , RNA de Plantas/genética , Pepinos-do-Mar/metabolismoRESUMO
Endophytes are regarded with immense potentials in terms of plant growth promoting (PGP) elicitors and mimicking secondary metabolites of medicinal importance. Here in the present study, we explored Bacopa monnieri plants to isolate, identify fungal endophytes with PGP elicitation potentials, and investigate secretion of secondary metabolites such as bacoside and withanolide content under in vitro conditions. Three fungal endophytes isolated (out of 40 saponin producing isolates) from leaves of B. monnieri were examined for in vitro biosynthesis of bacosides. On morphological, biochemical, and molecular identification (ITS gene sequencing), the isolated strains SUBL33, SUBL51, and SUBL206 were identified as Nigrospora oryzae (MH071153), Alternaria alternata (MH071155), and Aspergillus terreus (MH071154) respectively. Among these strains, SUBL33 produced highest quantity of Bacoside A3 (4093 µg mL-1), Jujubogenin isomer of Bacopasaponin C (65,339 µg mL-1), and Bacopasaponin C (1325 µg mL-1) while Bacopaside II (13,030 µg mL-1) was produced by SUBL51 maximally. Moreover, these aforementioned strains also produced detectable concentration of withanolides-Withaferrin A, Withanolide A (480 µg mL-1), and Withanolide B (1024 µg mL-1) respectively. However, Withanolide A was not detected in the secondary metabolites of strain SUBL51. To best of our knowledge, the present study is first reports of Nigrospora oryzae as an endophyte in B. monnieri with potentials of biosynthesis of economically important phytomolecules under in vitro conditions.
Assuntos
Bacopa , Endófitos , Fungos , Saponinas , Vitanolídeos , Alternaria/genética , Alternaria/isolamento & purificação , Alternaria/metabolismo , Ascomicetos/genética , Ascomicetos/isolamento & purificação , Ascomicetos/metabolismo , Aspergillus/genética , Aspergillus/isolamento & purificação , Aspergillus/metabolismo , Bacopa/microbiologia , Endófitos/genética , Endófitos/isolamento & purificação , Endófitos/metabolismo , Fungos/genética , Fungos/isolamento & purificação , Fungos/metabolismo , Folhas de Planta/microbiologia , Saponinas/biossíntese , Vitanolídeos/metabolismoRESUMO
Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI 498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI 498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.
Assuntos
4-Aminobutirato Transaminase/metabolismo , Solanina/análogos & derivados , Solanum tuberosum/genética , 4-Aminobutirato Transaminase/genética , Edição de Genes , Hidroxilação , Cetocolesteróis/biossíntese , Cetocolesteróis/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubérculos/enzimologia , Tubérculos/genética , Tubérculos/fisiologia , Saponinas/biossíntese , Saponinas/química , Solanina/química , Solanina/metabolismo , Solanum tuberosum/enzimologia , Solanum tuberosum/fisiologiaRESUMO
Panax notoginseng (Bruk.) FH Chen is a valuable traditional herb in China, with saponins being the main medicinal components in its roots. However, leaf diseases are a major factor limiting growth and production of P. notoginseng. Melatonin is a ubiquitous signaling molecule associated with abiotic stress resistance. In this study, we investigated the role of melatonin in leaf disease resistance of P. notoginseng in field conditions. Additionally, saponin concentrations were analyzed to evaluate the suitability of melatonin use in agricultural practice. Our results showed that exogenous application of melatonin promoted the endogenous phytomelatonin accumulation via upregulation of genes involved in its biosynthesis. The application of 10 µM melatonin decreased the incidence of leaf diseases (gray mold, round spot, and black spot) by about 40% compared with the solvent control, which might have been due to the increased expression of genes associated with immunity and disease resistance. Furthermore, concentrations of saponins and expression of their biosynthesis-related genes were significantly increased by melatonin. Taken together, the data presented here suggested that melatonin could be used in agricultural management of P. notoginseng because it increased leaf disease resistance and biosynthesis of saponins.
Assuntos
Resistência à Doença/fisiologia , Melatonina/metabolismo , Panax notoginseng/crescimento & desenvolvimento , Panax notoginseng/metabolismo , Doenças das Plantas , Folhas de Planta/metabolismo , Saponinas/biossíntese , China , Plantas Medicinais/crescimento & desenvolvimento , Plantas Medicinais/metabolismoRESUMO
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.