RESUMO
By limiting the nitrogen source to glutamic acid, we isolated cyclic peptides from Euglena gracilis containing asparagine and non-proteinogenic amino acids. Structure elucidation was accomplished through spectroscopic methods, mass spectrometry and chemical degradation. The euglenatides potently inhibit pathogenic fungi and cancer cell lines e.g., euglenatide B exhibiting IC50 values of 4.3â µM in Aspergillus fumigatus and 0.29â µM in MCF-7 breast cancer cells. In an unprecedented convergence of non-ribosomal peptide synthetase and polyketide synthase assembly-line biosynthesis between unicellular species and the metazoan kingdom, euglenatides bear resemblance to nemamides from Caenorhabditis elegans and inhibited both producing organisms E.â gracilis and C.â elegans. By molecular network analysis, we detected over forty euglenatide-like metabolites in E.â gracilis, E.â sanguinea and E.â mutabilis, suggesting an important biological role for these natural products.
Assuntos
Euglena gracilis , Microalgas , Animais , Caenorhabditis elegans , Euglena gracilis/metabolismo , Água Doce , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologiaRESUMO
Eukaryotic algae are an extremely diverse category of photosynthetic organisms and some species produce highly potent bioactive compounds poisonous to humans or other animals, most notably observed during harmful algal blooms. These natural products include some of the most poisonous small molecules known and unique cyclic polyethers. However, the diversity and complexity of algal genomes means that sequencing-based research has lagged behind research into more readily sequenced microbes, such as bacteria and fungi. Applying informatics techniques to the algal genomes that are now available reveals new natural product biosynthetic pathways, with different groups of algae containing different types of pathways. There is some evidence for gene clusters and the biosynthetic logic of polyketides enables some prediction of these final products. For other pathways, it is much more challenging to predict the products and there may be many gene clusters that are not identified with the automated tools. These results suggest that there is a great diversity of biosynthetic capacity for natural products encoded in the genomes of algae and suggest areas for future research focus.
Assuntos
Produtos Biológicos/isolamento & purificação , Microalgas/metabolismo , Animais , Humanos , Informática/métodos , Microalgas/genética , Família Multigênica , Policetídeos/metabolismoRESUMO
BACKGROUND: Photosynthetic euglenids are major contributors to fresh water ecosystems. Euglena gracilis in particular has noted metabolic flexibility, reflected by an ability to thrive in a range of harsh environments. E. gracilis has been a popular model organism and of considerable biotechnological interest, but the absence of a gene catalogue has hampered both basic research and translational efforts. RESULTS: We report a detailed transcriptome and partial genome for E. gracilis Z1. The nuclear genome is estimated to be around 500 Mb in size, and the transcriptome encodes over 36,000 proteins and the genome possesses less than 1% coding sequence. Annotation of coding sequences indicates a highly sophisticated endomembrane system, RNA processing mechanisms and nuclear genome contributions from several photosynthetic lineages. Multiple gene families, including likely signal transduction components, have been massively expanded. Alterations in protein abundance are controlled post-transcriptionally between light and dark conditions, surprisingly similar to trypanosomatids. CONCLUSIONS: Our data provide evidence that a range of photosynthetic eukaryotes contributed to the Euglena nuclear genome, evidence in support of the 'shopping bag' hypothesis for plastid acquisition. We also suggest that euglenids possess unique regulatory mechanisms for achieving extreme adaptability, through mechanisms of paralog expansion and gene acquisition.
Assuntos
Euglena gracilis/genética , Genoma , Proteoma , Transcriptoma , Núcleo Celular , Euglena gracilis/metabolismo , PlastídeosRESUMO
Intense competition between microbes in the environment has directed the evolution of antibiotic production in bacteria. Humans have harnessed these natural molecules for medicinal purposes, magnifying them from environmental concentrations to industrial scale. This increased exposure to antibiotics has amplified antibiotic resistance across bacteria, spurring a global antimicrobial crisis and a search for antibiotics with new modes of action. Genetic insights into these antibiotic-producing microbes reveal that they have evolved several resistance strategies to avoid self-toxicity, including product modification, substrate transport and binding, and target duplication or modification. Of these mechanisms, target duplication or modification will be highlighted in this review, as it uniquely links an antibiotic to its mode of action. We will further discuss and propose a strategy to mine microbial genomes for these genes and their associated biosynthetic gene clusters to discover novel antibiotics using target directed genome mining.
Assuntos
Antibacterianos/farmacologia , Bactérias/genética , Farmacorresistência Bacteriana , Genoma Bacteriano , Animais , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Descoberta de Drogas , HumanosRESUMO
Prymnesium parvum is a toxin-producing microalga that causes harmful algal blooms globally, which often result in large-scale fish kills that have severe ecological and economic implications. Although many toxins have previously been isolated from P. parvum, ambiguity still surrounds the responsible ichthyotoxins in P. parvum blooms and the biotic and abiotic factors that promote bloom toxicity. A major fish kill attributed to P. parvum occurred in Spring 2015 on the Norfolk Broads, a low-lying set of channels and lakes (Broads) found on the East of England. Here, we discuss how water samples taken during this bloom have led to diverse scientific advances ranging from toxin analysis to discovery of a new lytic virus of P. parvum, P. parvum DNA virus (PpDNAV-BW1). Taking recent literature into account, we propose key roles for sialic acids in this type of viral infection. Finally, we discuss recent practical detection and management strategies for controlling these devastating blooms.
Assuntos
Haptófitas/crescimento & desenvolvimento , Proliferação Nociva de Algas , Açúcares , Animais , DNA/genética , Inglaterra , Peixes , Haptófitas/genética , Haptófitas/metabolismo , Haptófitas/virologia , Toxinas Biológicas/metabolismoRESUMO
The photosynthetic, autotrophic lifestyle of plants and algae position them as ideal platform organisms for sustainable production of biomolecules. However, their use in industrial biotechnology is limited in comparison to heterotrophic organisms, such as bacteria and yeast. This usage gap is in part due to the challenges in generating genetically modified plants and algae and in part due to the difficulty in the development of synthetic biology tools for manipulating gene expression in these systems. Plant and algal metabolism, pre-installed with multiple biosynthetic modules for precursor compounds, bypasses the requirement to install these pathways in conventional production organisms, and creates new opportunities for the industrial production of complex molecules. This review provides a broad overview of the successes, challenges and future prospects for genetic engineering in plants and algae for enhanced or de novo production of biomolecules. The toolbox of technologies and strategies that have been used to engineer metabolism are discussed, and the potential use of engineered plants for industrial manufacturing of large quantities of high-value compounds is explored. This review also discusses the routes that have been taken to modify the profiles of primary metabolites for increasing the nutritional quality of foods as well as the production of specialized metabolites, cosmetics, pharmaceuticals and industrial chemicals. As the universe of high-value biosynthetic pathways continues to expand, and the tools to engineer these pathways continue to develop, it is likely plants and algae will become increasingly valuable for the biomanufacturing of high-value compounds.
Assuntos
Fotossíntese , Biotecnologia , Engenharia Genética , Engenharia Metabólica , Plantas , Saccharomyces cerevisiaeRESUMO
In order to expedite the rapid and efficient discovery and isolation of novel specialized metabolites, while minimizing the waste of resources on rediscovery of known compounds, it is crucial to develop efficient approaches for strain prioritization, rapid dereplication, and the assessment of favored cultivation and extraction conditions. Herein we interrogated bacterial strains by systematically evaluating cultivation and extraction parameters with LC-MS/MS analysis and subsequent dereplication through the Global Natural Product Social Molecular Networking (GNPS) platform. The developed method is fast, requiring minimal time and sample material, and is compatible with high-throughput extract analysis, thereby streamlining strain prioritization and evaluation of culturing parameters. With this approach, we analyzed 146 marine Salinispora and Streptomyces strains that were grown and extracted using multiple different protocols. In total, 603 samples were analyzed, generating approximately 1.8 million mass spectra. We constructed a comprehensive molecular network and identified 15 molecular families of diverse natural products and their analogues. The size and breadth of this network shows statistically supported trends in molecular diversity when comparing growth and extraction conditions. The network provides an extensive survey of the biosynthetic capacity of the strain collection and a method to compare strains based on the variety and novelty of their metabolites. This approach allows us to quickly identify patterns in metabolite production that can be linked to taxonomy, culture conditions, and extraction methods, as well as informing the most valuable growth and extraction conditions.
Assuntos
Bactérias/genética , Produtos Biológicos , Variação Genética , Bactérias/química , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Metabolômica , Estrutura Molecular , Salinidade , Streptomyces/química , Streptomyces/genéticaRESUMO
The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The "gate" is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms.
Assuntos
Arabidopsis/enzimologia , Enzimas/metabolismo , Plastídeos/enzimologia , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Enzimas/química , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
The crystal structure of the GH78 family α-rhamnosidase from Klebsiella oxytoca (KoRha) has been determined at 2.7 Å resolution with rhamnose bound in the active site of the catalytic domain. Curiously, the putative catalytic acid, Asp 222, is preceded by an unusual non-proline cis-peptide bond which helps to project the carboxyl group into the active centre. This KoRha homodimeric structure is significantly smaller than those of the other previously determined GH78 structures. Nevertheless, the enzyme displays α-rhamnosidase activity when assayed in vitro, suggesting that the additional structural domains found in the related enzymes are dispensible for function.
Assuntos
Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Klebsiella oxytoca/enzimologia , Estrutura Terciária de Proteína , Ramnose/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Biocatálise , Configuração de Carboidratos , Cristalografia por Raios X , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Klebsiella oxytoca/genética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Ramnose/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Eukaryotic microalgae are a diverse group of organisms that can be used for the sustainable production of a wide range of high value compounds, including lipids, flavours and dyes, bioplastics, and cosmetics. Optimising total biomass production often does not lead to optimal product yield and more sophisticated biphasic growth strategies are needed, introducing specific stresses to induce product synthesis. Genetic tools have been used to increase yields of natural products or to introduce new pathways to algae, and wider deployment of these tools offers promising routes for commercial production of high value compounds utilising minimal inputs.
Assuntos
Microalgas , Microalgas/metabolismo , Biomassa , Produtos Biológicos/metabolismoRESUMO
In connection with prospective (18)F-PET imaging studies, the potential for enzymatic synthesis of fluorine-labelled glycosides of small molecules was investigated. Approaches to the enzymatic synthesis of anomeric phosphates of d-gluco-configured fluorosugars proved ineffective. In contrast, starting in the d-galacto series and relying on the consecutive action of Escherichia coli galactokinase (GalK), galactose-1-phosphate uridylyltransferase (GalPUT), uridine-5'-diphosphogalactose 4-epimerase (GalE) and oat root glucosyltransferase (SAD10), a quick and effective synthesis of 6-deoxy-6-fluoro-d-glucosyl N-methylanthranilate ester was achieved.
Assuntos
Proteínas de Escherichia coli/metabolismo , Galactoquinase/metabolismo , Glucosídeos/química , Glucosiltransferases/metabolismo , Proteínas de Plantas/metabolismo , UTP-Hexose-1-Fosfato Uridililtransferase/metabolismo , ortoaminobenzoatos/química , ortoaminobenzoatos/metabolismo , Avena/enzimologia , Escherichia coli/enzimologia , Glucosídeos/metabolismoRESUMO
Protein glycosylation, and in particular N-linked glycans, is a hallmark of eukaryotic cells and has been well-studied in mammalian cells and parasites. However, little research has been conducted to investigate the conservation and variation of protein glycosylation pathways in other eukaryotic organisms. Euglena gracilis is an industrially important microalga, used in the production of biofuels and nutritional supplements. It is evolutionarily highly divergent from green algae and more related to kinetoplastid pathogens. It was recently shown that E. gracilis possesses the machinery for producing a range of protein glycosylations and make simple N-glycans, but the modified proteins were not identified. This study identifies the glycosylated proteins, including transporters, extracellular proteases, and those involved in cell surface signalling. Notably, many of the most highly expressed and glycosylated proteins are not related to any known sequences and are, therefore, likely to be involved in important novel functions in Euglena.
Assuntos
Euglena gracilis , Proteômica , Animais , Euglena gracilis/metabolismo , Células Eucarióticas/metabolismo , Glicoproteínas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Mamíferos/metabolismoRESUMO
Euglenoids (Euglenida) are unicellular flagellates possessing exceptionally wide geographical and ecological distribution. Euglenoids combine a biotechnological potential with a unique position in the eukaryotic tree of life. In large part these microbes owe this success to diverse genetics including secondary endosymbiosis and likely additional sources of genes. Multiple euglenoid species have translational applications and show great promise in production of biofuels, nutraceuticals, bioremediation, cancer treatments and more exotically as robotics design simulators. An absence of reference genomes currently limits these applications, including development of efficient tools for identification of critical factors in regulation, growth or optimization of metabolic pathways. The Euglena International Network (EIN) seeks to provide a forum to overcome these challenges. EIN has agreed specific goals, mobilized scientists, established a clear roadmap (Grand Challenges), connected academic and industry stakeholders and is currently formulating policy and partnership principles to propel these efforts in a coordinated and efficient manner.
Assuntos
Euglena , Euglena/fisiologia , Biotecnologia , SimbioseRESUMO
Euglenids are a group of algae of great interest for biotechnology, with a large and complex metabolic capability. To study the metabolic network, it is necessary to know where the component enzymes are in the cell, but despite a long history of research into Euglena, the subcellular locations of many major pathways are only poorly defined. Euglena is phylogenetically distant from other commonly studied algae, they have secondary plastids bounded by three membranes, and they can survive after destruction of their plastids. These unusual features make it difficult to assume that the subcellular organization of the metabolic network will be equivalent to that of other photosynthetic organisms. We analysed bioinformatic, biochemical, and proteomic information from a variety of sources to assess the subcellular location of the enzymes of the central metabolic pathways, and we use these assignments to propose a model of the metabolic network of Euglena. Other than photosynthesis, all major pathways present in the chloroplast are also present elsewhere in the cell. Our model demonstrates how Euglena can synthesise all the metabolites required for growth from simple carbon inputs, and can survive in the absence of chloroplasts.
RESUMO
Synthetic hexynyl α-D-mannopyranoside and its α-1,6-linked disaccharide counterpart were fluorescently labelled through CuAAC click chemistry with 3-azido-7-hydroxycoumarin. The resulting triazolyl-coumarin adducts, which were amenable to analysis by TLC, HPLC and mass spectrometry, proved to be acceptor substrates for α-1,6-ManT activities in mycobacterial membranes, as well as α- and ß-GalT activities in trypanosomal membranes, benchmarking the potential of the fluorescent acceptor approach against earlier radiochemical assays. Following on to explore the glycobiology of the benign protozoan alga Euglena gracilis, α-1,3- and α-1,2-ManT activities were detected in membrane preparations, along with GlcT, Glc-P-T and GlcNAc-P-T activities. These studies serve to demonstrate the potential of readily accessible fluorescent glycans as substrates for exploring carbohydrate active enzymes.
Assuntos
Membrana Celular/metabolismo , Euglena gracilis/enzimologia , Fluorescência , Glicosiltransferases/metabolismo , Manosídeos/metabolismo , Microssomos/metabolismo , Cinética , Manosídeos/química , Especificidade por SubstratoRESUMO
The GH94 glycoside hydrolase cellodextrin phosphorylase (CDP, EC 2.4.1.49) produces cellodextrin oligomers from short ß-1â4-glucans and α-D-glucose 1-phosphate. Compared to cellobiose phosphorylase (CBP), which produces cellobiose from glucose and α-D-glucose 1-phosphate, CDP is biochemically less well characterised. Herein, we investigate the donor and acceptor substrate specificity of recombinant CDP from Ruminiclostridium thermocellum and we isolate and characterise a glucosamine addition product to the cellobiose acceptor with the non-natural donor α-D-glucosamine 1-phosphate. In addition, we report the first X-ray crystal structure of CDP, along with comparison to the available structures from CBPs and other closely related enzymes, which contributes to understanding of the key structural features necessary to discriminate between monosaccharide (CBP) and oligosaccharide (CDP) acceptor substrates.
Assuntos
Glucosiltransferases/metabolismo , Cristalografia por Raios X , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glucofosfatos/metabolismo , Monossacarídeos/química , Oligossacarídeos/química , Especificidade por SubstratoRESUMO
There are hundreds of Trypanosoma species that live in the blood and tissue spaces of their vertebrate hosts. The vast majority of these do not have the ornate system of antigenic variation that has evolved in the small number of African trypanosome species, but can still maintain long-term infections in the face of the vertebrate adaptive immune system. Trypanosoma theileri is a typical example, has a restricted host range of cattle and other Bovinae, and is only occasionally reported to cause patent disease although no systematic survey of the effect of infection on agricultural productivity has been performed. Here, a detailed genome sequence and a transcriptome analysis of gene expression in bloodstream form T. theileri have been performed. Analysis of the genome sequence and expression showed that T. theileri has a typical kinetoplastid genome structure and allowed a prediction that it is capable of meiotic exchange, gene silencing via RNA interference and, potentially, density-dependent growth control. In particular, the transcriptome analysis has allowed a comparison of two distinct trypanosome cell surfaces, T. brucei and T. theileri, that have each evolved to enable the maintenance of a long-term extracellular infection in cattle. The T. theileri cell surface can be modeled to contain a mixture of proteins encoded by four novel large and divergent gene families and by members of a major surface protease gene family. This surface composition is distinct from the uniform variant surface glycoprotein coat on African trypanosomes providing an insight into a second mechanism used by trypanosome species that proliferate in an extracellular milieu in vertebrate hosts to avoid the adaptive immune response.
Assuntos
Trypanosoma/genética , Trypanosoma/patogenicidade , Tripanossomíase Bovina/parasitologia , Animais , Sangue/parasitologia , Bovinos , Ciclo Celular/genética , Perfilação da Expressão Gênica , Inativação Gênica , Genoma de Protozoário , Interações Hospedeiro-Parasita/genética , Filogenia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interferência de RNA , RNA Mensageiro/sangue , Sacarose/metabolismo , Tripanossomíase Bovina/sangueRESUMO
Euglena gracilis is an alga of great biotechnological interest and extensive metabolic capacity, able to make high levels of bioactive compounds, such as polyunsaturated fatty acids, vitamins and ß-glucan. Previous work has shown that Euglena expresses a wide range of carbohydrate-active enzymes, suggesting an unexpectedly high capacity for the synthesis of complex carbohydrates for a single-celled organism. Here, we present an analysis of some of the carbohydrates synthesised by Euglena gracilis. Analysis of the sugar nucleotide pool showed that there are the substrates necessary for synthesis of complex polysaccharides, including the unusual sugar galactofuranose. Lectin- and antibody-based profiling of whole cells and extracted carbohydrates revealed a complex galactan, xylan and aminosugar based surface. Protein N-glycan profiling, however, indicated that just simple high mannose-type glycans are present and that they are partially modified with putative aminoethylphosphonate moieties. Together, these data indicate that Euglena possesses a complex glycan surface, unrelated to plant cell walls, while its protein glycosylation is simple. Taken together, these findings suggest that Euglena gracilis may lend itself to the production of pharmaceutical glycoproteins.
RESUMO
Starch makes up more than half of the calories in the human diet and is also a valuable bulk commodity that is used across the food, brewing and distilling, medicines and renewable materials sectors. Despite its importance, our understanding of how plants make starch, and what controls the deposition of this insoluble, polymeric, liquid crystalline material, remains rather limited. Advances are hampered by the challenges inherent in analyzing enzymes that operate across the solid-liquid interface. Glyconanotechnology, in the form of glucan-coated sensor chips and metal nanoparticles, present novel opportunities to address this problem. Herein, we review recent developments aimed at the bottom-up generation and self-assembly of starch-like materials, in order to better understand which enzymes are required for starch granule biogenesis and metabolism.
RESUMO
Carbohydrate phosphorylases are readily accessible but under-explored catalysts for glycoside synthesis. Their use of accessible and relatively stable sugar phosphates as donor substrates underlies their potential. A wide range of these enzymes has been reported of late, displaying a range of preferences for sugar donors, acceptors and glycosidic linkages. This has allowed this class of enzymes to be used in the synthesis of diverse carbohydrate structures, including at the industrial scale. As more phosphorylase enzymes are discovered, access to further difficult to synthesise glycosides will be enabled. Herein we review reported phosphorylase enzymes and the glycoside products that they have been used to synthesise.