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1.
Nat Commun ; 12(1): 1760, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741955

RESUMO

Plant natural products (PNPs) and their derivatives are important but underexplored sources of pharmaceutical molecules. To access this untapped potential, the reconstitution of heterologous PNP biosynthesis pathways in engineered microbes provides a valuable starting point to explore and produce novel PNP derivatives. Here, we introduce a computational workflow to systematically screen the biochemical vicinity of a biosynthetic pathway for pharmaceutical compounds that could be produced by derivatizing pathway intermediates. We apply our workflow to the biosynthetic pathway of noscapine, a benzylisoquinoline alkaloid (BIA) with a long history of medicinal use. Our workflow identifies pathways and enzyme candidates for the production of (S)-tetrahydropalmatine, a known analgesic and anxiolytic, and three additional derivatives. We then construct pathways for these compounds in yeast, resulting in platforms for de novo biosynthesis of BIA derivatives and demonstrating the value of cheminformatic tools to predict reactions, pathways, and enzymes in synthetic biology and metabolic engineering.


Assuntos
Produtos Biológicos/metabolismo , Vias Biossintéticas/genética , Biologia Computacional/métodos , Engenharia Metabólica/métodos , Noscapina/metabolismo , Saccharomyces cerevisiae/metabolismo , Alcaloides/biossíntese , Benzilisoquinolinas/metabolismo , Noscapina/química , Plantas/genética , Plantas/metabolismo , Saccharomyces cerevisiae/genética , Software
2.
Molecules ; 26(4)2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33567664

RESUMO

As the population ages globally, there seem to be more people with Alzheimer's disease. Unfortunately, there is currently no specific treatment for the disease. At present, Huperzine A (HupA) is one of the best drugs used for the treatment of Alzheimer's disease and has been used in clinical trials for several years in China. HupA was first separated from Huperzia serrata, a traditional medicinal herb that is used to cure fever, contusions, strains, hematuria, schizophrenia, and snakebite for several hundreds of years in China, and has been confirmed to have acetylcholinesterase inhibitory activity. With the very slow growth of H. serrata, resources are becoming too scarce to meet the need for clinical treatment. Some endophytic fungal strains that produce HupA were isolated from H. serrate in previous studies. In this article, the diversity of the endophytic fungal community within H. serrata was observed and the relevance to the production of HupA by the host plant was further analyzed. A total of 1167 strains were obtained from the leaves of H. serrata followed by the stems (1045) and roots (824). The richness as well as diversity of endophytic fungi within the leaf and stem were higher than in the root. The endophytic fungal community was similar within stems as well as in leaves at all taxonomic levels. The 11 genera (Derxomyces, Lophiostoma, Cyphellophora, Devriesia, Serendipita, Kurtzmanomyces, Mycosphaerella, Conoideocrella, Brevicellicium, Piskurozyma, and Trichomerium) were positively correlated with HupA content. The correlation index of Derxomyces with HupA contents displayed the highest value (CI = 0.92), whereas Trichomerium showed the lowest value (CI = 0.02). Through electrospray ionization mass spectrometry (ESI-MS), it was confirmed that the HS7-1 strain could produce HupA and the total alkaloid concentration was 3.7 ug/g. This study will enable us to screen and isolate the strain that can produce HupA and to figure out the correlation between endophytic fungal diversity with HupA content in different plant organs. This can provide new insights into the screening of strains that can produce HupA more effectively.


Assuntos
Alcaloides/biossíntese , Biodiversidade , Endófitos/classificação , Endófitos/metabolismo , Fungos/classificação , Fungos/metabolismo , Huperzia/microbiologia , Endófitos/isolamento & purificação , Endófitos/fisiologia , Fungos/isolamento & purificação , Fungos/fisiologia , Sesquiterpenos
3.
Nature ; 585(7826): 614-619, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879484

RESUMO

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.


Assuntos
Alcaloides/biossíntese , Alcaloides/provisão & distribuição , Hiosciamina/biossíntese , Saccharomyces cerevisiae/metabolismo , Escopolamina/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Atropa belladonna/enzimologia , Derivados da Atropina/metabolismo , Transporte Biológico , Datura/enzimologia , Glucosídeos/biossíntese , Glucosídeos/metabolismo , Hiosciamina/provisão & distribuição , Lactatos/metabolismo , Ligases/genética , Ligases/metabolismo , Modelos Moleculares , Doenças do Sistema Nervoso/tratamento farmacológico , Oxirredutases/genética , Oxirredutases/metabolismo , Engenharia de Proteínas , Saccharomyces cerevisiae/genética , Escopolamina/provisão & distribuição , Vacúolos/metabolismo
4.
Nat Commun ; 11(1): 3337, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620756

RESUMO

The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogs. Plants manufacture more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a yeast THIQ platform by increasing production of the central BIA intermediate (S)-reticuline to 4.6 g L-1, a 57,000-fold improvement over our first-generation strain. We show that gains in BIA output coincide with the formation of several substituted THIQs derived from amino acid catabolism. We use these insights to repurpose the Ehrlich pathway and synthesize an array of THIQ structures. This work provides a blueprint for building diverse alkaloid scaffolds and enables the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.


Assuntos
Alcaloides/biossíntese , Benzilisoquinolinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Tetra-Hidroisoquinolinas/metabolismo , Alcaloides/química , Analgésicos Opioides/química , Analgésicos Opioides/metabolismo , Benzilisoquinolinas/química , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Vias Biossintéticas/genética , Engenharia Genética , Modelos Químicos , Estrutura Molecular , Saccharomyces cerevisiae/genética , Tetra-Hidroisoquinolinas/química
5.
Nat Commun ; 11(1): 1867, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313070

RESUMO

Plant halogenated natural products are rare and harbor various interesting bioactivities, yet the biochemical basis for the involved halogenation chemistry is unknown. While a handful of Fe(II)- and 2-oxoglutarate-dependent halogenases (2ODHs) have been found to catalyze regioselective halogenation of unactivated C-H bonds in bacteria, they remain uncharacterized in the plant kingdom. Here, we report the discovery of dechloroacutumine halogenase (DAH) from Menispermaceae plants known to produce the tetracyclic chloroalkaloid (-)-acutumine. DAH is a 2ODH of plant origin and catalyzes the terminal chlorination step in the biosynthesis of (-)-acutumine. Phylogenetic analyses reveal that DAH evolved independently in Menispermaceae plants and in bacteria, illustrating an exemplary case of parallel evolution in specialized metabolism across domains of life. We show that at the presence of azide anion, DAH also exhibits promiscuous azidation activity against dechloroacutumine. This study opens avenues for expanding plant chemodiversity through halogenation and azidation biochemistry.


Assuntos
Alcaloides/biossíntese , Compostos Ferrosos/metabolismo , Hidrolases/metabolismo , Ácidos Cetoglutáricos/metabolismo , Menispermaceae/metabolismo , Compostos de Espiro/metabolismo , Alcaloides/química , Alcaloides/genética , Bactérias/metabolismo , Biocatálise , Genes de Plantas/genética , Halogenação , Menispermaceae/embriologia , Menispermaceae/genética , Mutagênese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes , Metabolismo Secundário/genética , Alinhamento de Sequência , Compostos de Espiro/química , Transcriptoma
6.
J Appl Microbiol ; 129(2): 345-355, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32091657

RESUMO

AIMS: Paclitaxel is a type of broad-spectrum anticancer drug in short supply. The price of acetyl-CoA (17 709 677·4 USD mol-1 ), which is the acetyl group donor for the enzymatic synthesis of the intermediate, baccatin Ⅲ, is still the bottleneck of the mass production of paclitaxel. This study reports a novel acetyl group donor, which could substantially reduce the cost of production. METHODS AND RESULTS: In this study, a substrate spectrum with 14 kinds of representative acetyl-donor substitutes predicted by computer-aided methods was tested in a 10-deacetylbaccatin Ⅲ-10-O-acetyltransferase (DBAT) heterogeneous-expressed open-whole-cell catalytic system. The results of computer prediction and experimental analysis revealed the rule of the acetyl-donor compounds based on this substrate spectrum. N-acetyl-d-glucosamine (30·95 USD mol-1 , about 572 202-fold cheaper than acetyl-CoA) is selected as a suitable substitute under the rule. The yield when using N-acetyl-d-glucosamine as acetyl donor in open-whole-cell catalytic system was 2·13-fold of that when using acetyl-CoA. In the in vivo system, the yield increased 24·17%, which may indicate its cooperation with acetyl-CoA. CONCLUSION: The success of open-whole-cell synthesis and in vivo synthesis of baccatin Ⅲ by adding N-acetyl-d-glucosamine as acetyl substrate demonstrates that it is a useful substrate to improve the yield of baccatin Ⅲ. SIGNIFICANCE AND IMPACT OF THE STUDY: All these findings provided a potential acetyl-donor substitute for acetyl-CoA, as well as a low cost and efficient method of preparing paclitaxel through baccatin Ⅲ semi-synthesis.


Assuntos
Acetilglucosamina/metabolismo , Alcaloides/biossíntese , Acetilcoenzima A/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Alcaloides/economia , Antineoplásicos Fitogênicos/biossíntese , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/economia , Biocatálise , Paclitaxel/biossíntese , Paclitaxel/química , Paclitaxel/economia , Especificidade por Substrato , Taxoides/economia
7.
Nat Chem Biol ; 16(4): 383-386, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32066966

RESUMO

Cycloaddition reactions generate chemical complexity in a single step. Here we report the crystal structures of three homologous plant-derived cyclases involved in the biosynthesis of iboga and aspidosperma alkaloids. These enzymes act on the same substrate, named angryline, to generate three distinct scaffolds. Mutational analysis reveals how these highly similar enzymes control regio- and stereo-selectivity.


Assuntos
Alcaloides/biossíntese , Aspidosperma/química , Tabernaemontana/química , Alcaloides/química , Carbazóis/química , Reação de Cicloadição/métodos , Alcaloides Indólicos/química , Plantas/química
8.
Plant Mol Biol ; 102(4-5): 477-499, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31902069

RESUMO

KEY MESSAGE: The study carry out comprehensive transcriptome analysis of C. deltoidea and exploration of BIAs biosynthesis and accumulation based on UHPLC-MS/MS and combined sequencing platforms. Coptis deltoidea is an important medicinal plant with a long history of medicinal use, which is rich in benzylisoquinoline alkaloids (BIAs). In this study, Ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) and combined sequencing platforms were performed for exploration of BIAs biosynthesis, accumulation and comprehensive transcriptome analysis of C. deltoidea. By metabolism profiling, the accumulation of ten BIAs was analyzed using UHPLC-MS/MS and different contents were observed in different organs. From transcriptome sequencing result, we applied single-molecule real-time (SMRT) sequencing to C. deltoidea and generated a total of 75,438 full-length transcripts. We proposed the candidate biosynthetic pathway of tyrosine, precursor of BIAs, and identified 64 full length-transcripts encoding enzymes putatively involved in BIAs biosynthesis. RNA-Seq data indicated that the majority of genes exhibited relatively high expression level in roots. Transport of BIAs was also important for their accumulation. Here, 9 ABC transporters and 2 MATE transporters highly homologous to known alkaloid transporters related with BIAs transport in roots and rhizomes were identified. These findings based on the combined sequencing platforms provide valuable genetic information for C. deltoidea and the results of transcriptome combined with metabolome analysis can help us better understand BIAs biosynthesis and transport in this medicinal plant. The information will be critical for further characterization of C. deltoidea transcriptome and molecular-assisted breeding for this medicinal plant with scarce resources.


Assuntos
Alcaloides/biossíntese , Benzilisoquinolinas/química , Coptis/genética , Coptis/metabolismo , Transcriptoma , Cromatografia Líquida de Alta Pressão , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Filogenia , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Plantas Medicinais/metabolismo , Rizoma/metabolismo , Análise de Sequência de DNA , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
9.
Proc Natl Acad Sci U S A ; 117(2): 1174-1180, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31882449

RESUMO

Indolizidine alkaloids such as anticancer drugs vinblastine and vincristine are exceptionally attractive due to their widespread occurrence, prominent bioactivity, complex structure, and sophisticated involvement in the chemical defense for the producing organisms. However, the versatility of the indolizidine alkaloid biosynthesis remains incompletely addressed since the knowledge about such biosynthetic machineries is only limited to several representatives. Herein, we describe the biosynthetic gene cluster (BGC) for the biosynthesis of curvulamine, a skeletally unprecedented antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10. The molecular architecture of curvulamine results from the functional collaboration of a highly reducing polyketide synthase (CuaA), a pyridoxal-5'-phosphate (PLP)-dependent aminotransferase (CuaB), an NADPH-dependent dehydrogenase (CuaC), and a FAD-dependent monooxygenase (CuaD), with its transportation and abundance regulated by a major facilitator superfamily permease (CuaE) and a Zn(II)Cys6 transcription factor (CuaF), respectively. In contrast to expectations, CuaB is bifunctional and capable of catalyzing the Claisen condensation to form a new C-C bond and the α-hydroxylation of the alanine moiety in exposure to dioxygen. Inspired and guided by the distinct function of CuaB, our genome mining effort discovers bipolamines A-I (bipolamine G is more antibacterial than curvulamine), which represent a collection of previously undescribed polyketide alkaloids from a silent BGC in Bipolaris maydis ATCC48331. The work provides insight into nature's arsenal for the indolizidine-coined skeletal formation and adds evidence in support of the functional versatility of PLP-dependent enzymes in fungi.


Assuntos
Alcaloides/biossíntese , Ascomicetos/enzimologia , Ascomicetos/metabolismo , Indolizidinas/metabolismo , Policetídeo Sintases/metabolismo , Fosfato de Piridoxal/metabolismo , Alcaloides/genética , Alcaloides/isolamento & purificação , Antibacterianos/metabolismo , Ascomicetos/genética , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Catálise , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Hidroxilação , Alcaloides Indólicos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Família Multigênica , Filogenia , Policetídeo Sintases/classificação , Policetídeo Sintases/genética , Policetídeos , Fosfato de Piridoxal/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transaminases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Fitoterapia ; 140: 104412, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31698060

RESUMO

Aconitum carmichaelii has been used in traditional Chinese medicine for treating various diseases for several thousand years. Based on the biosynthetic pathway of some alkaloids such as C19-diterpenoid alkaloids and obvious differences in alkaloid content between leaves of two A. carmichaelii varieties has been reported, we performed leaves transcriptome analysis of two A. carmichaelii varieties. Besides we characterized the biosynthetic pathway of salsolinol. A total of 56 million raw reads (8.28 G) and 55 million clean reads (8.24 G) were obtained from two varieties (Z175 and R184) leaves transcriptome, respectively, and 176,793 unigenes were annotated. 281 and 843 unigenes are involved in the salsolinol biosynthetic pathway and the formation of C19-diterpenoid alkaloids respectively. And including 34 and 24 unigenes are the differentially expressed genes (DEGs) in the biosynthesis pathway for C19-diterpenoid alkaloids and salsolinol between Z175 and R184 respectively, which were target genes to explore differences in C19-diterpenoid alkaloid and salsolinol biosynthesis in Z175 and R184. Thus genes involved in alkaloid biosynthesis and accumulation differ between varieties leaves. The mechanisms underlying the differences and their relevance require further exploration. The results expand our knowledge of alkaloids biosynthesis in A. carmichaelii leaves, and provide a theoretical basis for analysis differences in alkaloids biosynthesis patterns in different varieties.


Assuntos
Aconitum/química , Vias Biossintéticas , Isoquinolinas/química , Transcriptoma , Alcaloides/biossíntese , China , Diterpenos/química , Perfilação da Expressão Gênica , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Folhas de Planta/química , Plantas Medicinais/química
11.
Plant Sci ; 290: 110293, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31779893

RESUMO

Class I TGA transcription factors (TFs) are known to participate in plant resistance responses, however, their regulatory functions in the biosynthesis of secondary metabolites were rarely revealed. In this study, a class I TGA TF, TwTGA1, from Tripterygium wilfordii Hook.f. was cloned and characterized. Overexpression of TwTGA1 in T. wilfordii Hook.f. cells increased the production of triptolide and two sesquiterpene pyridine alkaloids, which was further enhanced by methyl jasmonate (MeJA) treatment. RNA interference of TwTGA1 showed no significant effects on the production of these metabolites, indicating the existence of other TGA partner(s) with overlapping functions. Heterologous expression of TwTGA1 in tobacco By-2 cells promoted the biosynthesis of pyridine alkaloids. Under the elicitation of MeJA, the contents of nonpyrrolidine alkaloids further increased but not for nicotine. TwTGA1 could induce the expression of Putrescine N-methyltransferase (PMT) and N-methylputrescine oxidase 1 (MPO1) through binding to their promoters. Finally, transient expression of TwTGA1 in leaves of Catharanthus roseus changed both the profiles of vinca alkaloids (increased contents of serpentine and catharanthine, but decreased that of vinblastine) and the expressions of biosynthesis-related genes. The metabolic and transcriptional data indicated a relationship between jasmonic acid signaling pathway and the functions of TwTGA1.


Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Tripterygium/genética , Alcaloides/biossíntese , Sequência de Aminoácidos , Catharanthus/metabolismo , Diterpenos/metabolismo , Compostos de Epóxi/metabolismo , Fenantrenos/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Metabolismo Secundário , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Tripterygium/metabolismo
12.
Appl Biochem Biotechnol ; 190(1): 73-89, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31301009

RESUMO

Curvulamine, a novel scaffold alkaloid with remarkable selective antibacterial activity, is produced by marine fungus Curvularia sp. IFB-Z10. However, its deep pharmaceutical research and application are severely restricted by the low yield, which needs to be solved urgently. The purpose of this study was to improve curvulamine production via precursors co-addition strategy and further reveal the regulation mechanism. In this work, the optimal precursors co-addition conditions were firstly obtained, and curvulamine production achieved 166.74 mg/L with the supply of 250 mg/L alanine and 200 mg/L proline at 60 h, which was 4.08 times that of control. It was observed that under alanine and proline stimulation, fungus exhibited the morphology of a small-diameter compact pellet. Furthermore, the organic acid levels in central carbon metabolism (CCM) were declined with precursors supplement. Besides, precursors also induced the critical biosynthetic gene transcriptions. The above findings collectively promoted curvulamine synthesis. Finally, Curvularia sp. IFB-Z10 fermentation process was successfully established by feeding alanine and proline at 0.021 g/L/h and 0.017 g/L/h rate from 60 to 72 h, and curvulamine production reached 133.58 mg/L in a 5-L bioreactor. The information acquired would facilitate the enhancement of curvulamine yield in submerged fermentation and the research on synthesis regulation of other alkaloids.


Assuntos
Alcaloides/biossíntese , Ascomicetos/metabolismo , Aminoácidos/metabolismo , Ascomicetos/genética , Ascomicetos/crescimento & desenvolvimento , Reatores Biológicos , Metabolismo dos Carboidratos , Divisão Celular , Fermentação , Genes Fúngicos , Alcaloides Indólicos , Nitrogênio/metabolismo , Transcrição Genética
13.
BMC Plant Biol ; 19(1): 560, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852435

RESUMO

BACKGROUND: Croton draco is an arboreal species and its latex as well as some other parts of the plant, are traditionally used in the treatment of a wide range of ailments and diseases. Alkaloids, such as magnoflorine, prevent early atherosclerosis progression while taspine, an abundant constituent of latex, has been described as a wound-healer and antitumor-agent. Despite the great interest for these and other secondary metabolites, no omics resources existed for the species and the biosynthetic pathways of these alkaloids remain largely unknown. RESULTS: To gain insights into the pathways involved in magnoflorine and taspine biosynthesis by C. draco and identify the key enzymes in these processes, we performed an integrated analysis of the transcriptome and metabolome in the major organs (roots, stem, leaves, inflorescences, and flowers) of this species. Transcript profiles were generated through high-throughput RNA-sequencing analysis while targeted and high resolution untargeted metabolomic profiling was also performed. The biosynthesis of these compounds appears to occur in the plant organs examined, but intermediaries may be translocated from the cells in which they are produced to other cells in which they accumulate. CONCLUSIONS: Our results provide a framework to better understand magnoflorine and taspine biosynthesis in C. draco. In addition, we demonstrate the potential of multi-omics approaches to identify candidate genes involved in the biosynthetic pathways of interest.


Assuntos
Alcaloides/biossíntese , Aporfinas/metabolismo , Croton/metabolismo , Metaboloma , Transcriptoma , Vias Biossintéticas
14.
Genes (Basel) ; 10(11)2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31739571

RESUMO

Nicotine, the most abundant pyridine alkaloid in cultivated tobacco (Nicotiana tabacum L.), is a potent inhibitor of insect and animal herbivory and a neurostimulator of human brain function. Nicotine biosynthesis is controlled developmentally and can be induced by abiotic and biotic stressors via a jasmonic acid (JA)-mediated signal transduction mechanism involving members of the APETALA 2/ethylene-responsive factor (AP2/ERF) and basic helix-loop-helix (bHLH) transcription factor (TF) families. AP2/ERF and bHLH TFs work combinatorically to control nicotine biosynthesis and its subsequent accumulation in tobacco leaves. Here, we demonstrate that overexpression of the tobacco NtERF32, NtERF221/ORC1, and NtMYC2a TFs leads to significant increases in nicotine accumulation in T2 transgenic K326 tobacco plants before topping. Up to 9-fold higher nicotine production was achieved in transgenics overexpressing NtERF221/ORC1 under the control of a constitutive GmUBI3 gene promoter compared to wild-type plants. The constitutive 2XCaMV35S promoter and a novel JA-inducible 4XGAG promoter were less effective in driving high-level nicotine formation. Methyljasmonic acid (MeJA) treatment further elevated nicotine production in all transgenic lines. Our results show that targeted manipulation of NtERF221/ORC1 is an effective strategy for elevating leaf nicotine levels in commercial tobacco for use in the preparation of reduced risk tobacco products for smoking replacement therapeutics.


Assuntos
Nicotina/biossíntese , Reguladores de Crescimento de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Tabaco/metabolismo , Fatores de Transcrição/genética , Acetatos/metabolismo , Alcaloides/biossíntese , Alcaloides/toxicidade , Anabasina/biossíntese , Anabasina/toxicidade , Ciclopentanos/metabolismo , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Sequências Hélice-Alça-Hélice/genética , Nicotina/análogos & derivados , Nicotina/economia , Nicotina/toxicidade , Oxilipinas/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas/genética , Piridinas/toxicidade , Tabaco/genética , Produtos do Tabaco/economia , Produtos do Tabaco/toxicidade , Fatores de Transcrição/metabolismo
15.
Sci Rep ; 9(1): 16152, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31695105

RESUMO

Huperzine A (HupA), a natural Lycopodium alkaloid derived from Huperzia serrata (Thunb. ex Murray) Trev. plants, is a highly active acetylcholinesterase inhibitor and a key compound used for treating Alzheimer's disease (AD). Recently, HupA has been reported in various endophytic fungi isolated from H. serrata. In the present study, 153 endophytic fungi were isolated from healthy tissues of H. serrata collected from natural populations in Lam Dong province of Central Vietnam. The endophytic fungi were identified based on morphological characteristics and Internal Transcribed Spacer sequences. Among them, 34 strains were classified into seven genera belonging to Ascomycota, including Alternaria, Fusarium, Trichoderma, Penicillium, Paecilomyces, and Phoma, and eight strains belonging to the genus Mucor (Zygomycota). The other strains remained unidentified. According to the results of thin-layer chromatography and high-performance liquid chromatography, only one of the 153 strains, Penicillium sp. LDL4.4, could produce HupA, with a yield 1.38 mg l-1 (168.9 µg g-1 dried mycelium) when cultured in potato dextrose broth, which was considerably higher than that of other reported endophytic fungi. Such a fungus is a promising candidate and alternative to presently available HupA production techniques for treating AD and preventing further memory decline.


Assuntos
Alcaloides/biossíntese , Ascomicetos/isolamento & purificação , Inibidores da Colinesterase/farmacologia , Endófitos/isolamento & purificação , Huperzia/microbiologia , Mucor/isolamento & purificação , Acetilcolinesterase/efeitos dos fármacos , Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Inibidores da Colinesterase/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Delgada , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Endófitos/química , Endófitos/classificação , Endófitos/crescimento & desenvolvimento , Huperzia/metabolismo , Técnicas de Tipagem Micológica , Filogenia , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologia , Especificidade da Espécie , Vietnã
16.
Nat Commun ; 10(1): 4702, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619678

RESUMO

Black pepper (Piper nigrum), dubbed the 'King of Spices' and 'Black Gold', is one of the most widely used spices. Here, we present its reference genome assembly by integrating PacBio, 10x Chromium, BioNano DLS optical mapping, and Hi-C mapping technologies. The 761.2 Mb sequences (45 scaffolds with an N50 of 29.8 Mb) are assembled into 26 pseudochromosomes. A phylogenomic analysis of representative plant genomes places magnoliids as sister to the monocots-eudicots clade and indicates that black pepper has diverged from the shared Laurales-Magnoliales lineage approximately 180 million years ago. Comparative genomic analyses reveal specific gene expansions in the glycosyltransferase, cytochrome P450, shikimate hydroxycinnamoyl transferase, lysine decarboxylase, and acyltransferase gene families. Comparative transcriptomic analyses disclose berry-specific upregulated expression in representative genes in each of these gene families. These data provide an evolutionary perspective and shed light on the metabolic processes relevant to the molecular basis of species-specific piperine biosynthesis.


Assuntos
Alcaloides/biossíntese , Genoma de Planta , Piper nigrum/genética , Aciltransferases/genética , Benzodioxóis , Carboxiliases/genética , Mapeamento Cromossômico , Cromossomos , Sistema Enzimático do Citocromo P-450/genética , Perfilação da Expressão Gênica , Genômica , Glicosiltransferases/genética , Filogenia , Piperidinas , Alcamidas Poli-Insaturadas
17.
Chemistry ; 25(49): 11574-11580, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31407847

RESUMO

The so-called Securinega alkaloids constitute a class of tetracyclic biologically active specialised metabolites isolated principally from subtropical plants belonging to the Phyllanthaceae family. Following a strategy based on alternative hypotheses for their biosynthesis, an easy and time-efficient divergent synthesis enabled access to twelve of those alkaloids featuring (neo)(nor)securinane skeletons. Moreover, this work permitted to reassign the absolute configurations of (+)-virosine B and (-)-episecurinol A.


Assuntos
Alcaloides/biossíntese , Alcaloides/química , Euphorbiaceae/química , Euphorbiaceae/metabolismo , Mesilatos/química , Conformação Molecular , Estereoisomerismo
18.
Food Chem ; 301: 125285, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31387039

RESUMO

A method was developed and validated for determination of tryptoquialanines A and C in orange samples on epicarp (exterior peel), mesocarp (white peel), and endocarp (fruit juice) based on QuEChERS extraction and LC-MS/MS analysis. The method showed an excellent linearity over a range of 5-400 µg kg-1, with r2 ≥ 0.998. The limits of detection (LOD) and quantification (LOQ) were 5 and 10 µg kg-1, respectively. Recoveries showed values between 57 and 101%, with RSD ≤ 12%. Analysis of infected oranges showed diffusion of the alkaloids between the orange layers after 4 days post infection in concentrations > LOQ. Mycotoxin diffusion to healthy oranges after direct contact with infected oranges for 48 h, showed alkaloid concentrations ≥10 µg kg-1 on epicarp layer. The developed method can be easily applied for quality control in routine analysis of orange fruit due to the high risk that these tremorgenic alkaloids represent to human health.


Assuntos
Alcaloides/análise , Cromatografia Líquida/métodos , Citrus sinensis/microbiologia , Frutas/microbiologia , Penicillium/metabolismo , Espectrometria de Massas em Tandem/métodos , Alcaloides/biossíntese , Citrus sinensis/química , Frutas/química , Limite de Detecção , Micotoxinas/análise
19.
Chem Commun (Camb) ; 55(58): 8390-8393, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31257394

RESUMO

Diverse bioactive alkaloids with a tryptophan 2,5-diketopiperazine (DKP) core and an annulated structure forming a methylated pyrroloindoline-DKP assembly have been isolated from various microbial sources. However, little is known about their biosynthesis. In this study, a novel indole C3 methyltransferase from Streptomyces sp. HPH0547 was discovered and characterized. Structural elucidation of the products revealed that this enzyme catalyzed unique pyrroloindoline cyclization in tryptophan-containing cyclodipeptides. This is the first C3 methyltransferase reported to catalyze pyrroloindoline cyclization in cyclic dipeptides, which provides a feasible and simple method to access diverse alkaloids.


Assuntos
Alcaloides/biossíntese , Proteínas de Bactérias/metabolismo , Dipeptídeos/biossíntese , Metiltransferases/metabolismo , Peptídeos Cíclicos/biossíntese , Streptomyces/enzimologia , Ciclização , Dicetopiperazinas/metabolismo , Modelos Químicos , Especificidade por Substrato , Triptofano/química , Triptofano/metabolismo
20.
Gene ; 712: 143962, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31288057

RESUMO

Veratrum nigrum is protected plant of Melanthiaceae family, able to synthetize unique steroidal alkaloids important for pharmacy. Transcriptomes from leaves, stems and rhizomes of in vitro maintained V. nigrum plants were sequenced and annotated for genes and markers discovery. Sequencing of samples derived from the different organs resulted in a total of 108,511 contigs with a mean length of 596 bp. Transcripts derived from leaf and stalk were annotated at 28%, and 38% in Nr nucleotide database, respectively. The sequencing revealed 949 unigenes related with lipid metabolism, including 73 transcripts involved in steroids and genus-specific steroid alkaloids biosynthesis. Additionally, 3203 candidate SSRs markers we identified in unigenes with average density of one SSR locus every 6.2 kb sequence. Unraveling of biochemical machinery of the pathway responsible for steroidal alkaloids will open possibility to design and optimize biotechnological process. The transcriptomic data provide valuable resources for biochemical, molecular genetics, comparative transcriptomics, functional genomics, ecological and evolutionary studies of V. nigrum.


Assuntos
Alcaloides/biossíntese , Regulação da Expressão Gênica de Plantas , Esteroides/biossíntese , Transcriptoma , Veratrum/metabolismo , Mapeamento de Sequências Contíguas , DNA Complementar/metabolismo , Biblioteca Gênica , Ontologia Genética , Marcadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Repetições de Microssatélites , Anotação de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Análise de Sequência de RNA
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