RESUMEN
Dendrobium species, which are perennial herbs widely distributed in tropical and subtropical regions, are notable for their therapeutic properties attributed to various bioactive compounds, including dendrobine-type sesquiterpenoid alkaloids (DTSAs). The objective of this review article is to provide a comprehensive overview of recent advances in the biosynthesis of DTSAs, including their extraction from Dendrobium species and endophytes, elucidation of associated genes through genomic and transcriptomic sequencing in both Dendrobium spp. and endophytes, exploration of the biosynthetic pathways of DTSAs, and drawing conclusions and outlining future perspectives in this field. Alkaloids, predominantly nitrogen-containing compounds found in medicinal orchids, include over 140 types discovered across more than 50 species. DTSAs, identified in 37 picrotoxane alkaloids, have a distinctive five-membered nitrogen heterocyclic ring. This review highlights endophytic fungi as alternative sources of DTSAs, emphasizing their potential in pharmaceutical applications when plant-derived compounds are scarce or complex. Genomic and transcriptomic sequencing of Dendrobium spp. and their endophytes has identified key genes involved in DTSAs biosynthesis, elucidating pathways such as the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways. Genes encoding enzymes, such as acetyl-CoA C-acetyltransferase and diphosphomevalonate decarboxylase, are positively associated with dendrobine production. Despite significant advancements, the complexity of terpenoid biosynthesis in different subcellular compartments remains a challenge. Future research should focus on leveraging high-quality genomic data and omics technologies to further understand and manipulate the biosynthetic pathways of DTSAs and enhance their medicinal use.
Asunto(s)
Alcaloides , Vías Biosintéticas , Sesquiterpenos , Transcriptoma , Alcaloides/biosíntesis , Sesquiterpenos/metabolismo , Vías Biosintéticas/genética , Dendrobium/genética , Dendrobium/metabolismo , Dendrobium/enzimología , Perfilación de la Expresión Génica , Genómica/métodos , Endófitos/metabolismo , Endófitos/genética , Endófitos/enzimologíaRESUMEN
Endophytic microbial communities colonize plants growing under various abiotic stress conditions. Candelilla (Euphorbia antisyphilitica Zucc.) is a shrub that develops functionally in arid and semi-arid zones of Mexico; these conditions generate an association between the plant and the microorganisms, contributing to the production of enzymes as a defense mechanism for resistance to abiotic stress. The objective of this research was to isolate and identify endophyte fungi of candelilla and bioprospection of these endophytic fungi for enzyme production using candelilla by-products. Fungi were isolated and identified using ITS1/ITS4 sequencing. Their potency index (PI) was evaluated in producing endoglucanase, xylanase, amylase, and laccase. Fermentation was carried out at 30°C for 8 days at 200 rpm, with measurements every 2 days, using candelilla by-products as substrate. All fungi exhibited higher cellulase, amylase, and laccase activities on the 2nd, 6th, and 8th day of fermentation, respectively, of fermentation. The fungus Aspergillus niger ITD-IN4.1 showed the highest amylase activity (246.84 U/mg), the genus Neurospora showed the highest cellulase activity, reaching up to 13.45 FPU/mg, and the strain Neurospora sp. ITD-IN5.2 showed the highest laccase activity (3.46 U/mg). This work provides the first report on the endophytic diversity of E. antisyphilitica and its potential role in enzyme production.
Asunto(s)
Bioprospección , Celulasa , Endófitos , Fermentación , Lacasa , Endófitos/aislamiento & purificación , Endófitos/enzimología , Endófitos/metabolismo , Endófitos/genética , Lacasa/metabolismo , Lacasa/biosíntesis , Celulasa/metabolismo , Celulasa/biosíntesis , Amilasas/metabolismo , Aspergillus niger/aislamiento & purificación , Aspergillus niger/enzimología , México , Neurospora , Hongos/aislamiento & purificación , Hongos/enzimología , Hongos/clasificación , Hongos/genéticaRESUMEN
Fusarium head blight is a devastating disease that causes severe yield loses and mycotoxin contamination in wheat grain. Additionally, balancing the trade-off between wheat production and disease resistance has proved challenging. This study aimed to expand the genetic tools of the endophyte Phomopsis liquidambaris against Fusarium graminearum. Specifically, we engineered a UDP-glucosyltransferase-expressing P. liquidambaris strain (PL-UGT) using ADE1 as a selection marker and obtained a deletion mutant using an inducible promoter that drives Cas9 expression. Our PL-UGT strain converted deoxynivalenol (DON) into DON-3-G in vitro at a rate of 71.4 % after 36 h. DON inactivation can be used to confer tolerance in planta. Wheat seedlings inoculated with endophytic strain PL-UGT showed improved growth compared with those inoculated with wildtype P. liquidambaris. Strain PL-UGT inhibited the growth of Fusarium graminearum and reduced infection rate to 15.7 %. Consistent with this finding, DON levels in wheat grains decreased from 14.25 to 0.56 µg/g when the flowers were pre-inoculated with PL-UGT and then infected with F. graminearum. The expression of UGT in P. liquidambaris was nontoxic and did not inhibit plant growth. Endophytes do not enter the seeds nor induce plant disease, thereby representing a novel approach to fungal disease control.
Asunto(s)
Ascomicetos , Endófitos , Fusarium , Glucosiltransferasas , Enfermedades de las Plantas , Tricotecenos , Triticum , Triticum/microbiología , Triticum/genética , Tricotecenos/metabolismo , Fusarium/genética , Fusarium/efectos de los fármacos , Fusarium/enzimología , Endófitos/genética , Endófitos/enzimología , Endófitos/metabolismo , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Ascomicetos/genética , Ascomicetos/efectos de los fármacos , Ascomicetos/enzimología , Resistencia a la Enfermedad/genética , Micotoxinas/metabolismoRESUMEN
Serratiopeptidase, a proteolytic enzyme serves as an important anti-inflammatory and analgesic medication. Present study reports the production and purification of extracellular serratiopeptidase from an endophyte, Serratia marcescens MES-4, isolated from Morus rubra. Purification of the enzyme by Ion exchange chromatography led to the specific activity of 13,030â¯U/mg protein of serratiopeptidase, showcasing about 3.1 fold enhanced activity. The catalytic domain of the purified serratiopeptidase, composed of Zn coordinated with three histidine residues (His 209, His 213, and His 219), along with glutamate (Glu 210) and tyrosine (Tyr 249). The molecular mass, as determined by SDS-PAGE was â¼51â¯kDa. The purified serratiopeptidase displayed optimal activity at pH 9.0, temperature 50°C. Kinetic studies revealed Vmax and Km values of 33,333â¯U/mL and 1.66â¯mg/mL, respectively. Further, optimized conditions for the production of serratiopeptidase by Taguchi design led to the productivity of 87â¯U/mL/h with 87.9 fold enhanced production as compared to the previous conditions.
Asunto(s)
Endófitos , Péptido Hidrolasas , Serratia marcescens , Serratia marcescens/enzimología , Serratia marcescens/genética , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/aislamiento & purificación , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Endófitos/enzimología , Concentración de Iones de Hidrógeno , Cinética , Temperatura , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificaciónRESUMEN
Enzymes are biocatalysts that are widely used in different industries and generate billions of dollars annually. With the advancement of biotechnology, new enzymatic sources are being evaluated, especially microbial ones, in order to find efficient producers. Endophytic fungi are promising sources of biomolecules; however, Amazonian species are still poorly studied as to their enzymatic production potential. In this sense, the production of hydrolases (amylases, lipases, cellulases and pectinases) was evaluated in endophytic fungi isolated from the leaves, roots and stems of açai palms (Euterpe precatoria). A qualitative test was carried out to detect the enzymatic synthesis in each isolate, and the most promising ones were cultivated using submerged fermentation. The enzyme extracts were quantified to determine those with the greatest activity. Cellulolytic and amylolytic extracts showed the highest enzymatic activities and were partially characterized. Among 50 isolates, 82.9% produced pectinase, 58.5% produced cellulase, 31.7% produced amylase, and 12.2% produced lipase. Penicillium sp. L3 was the best producer of amylase and Colletotrichum sp. S1 was the best producer of cellulase in liquid medium cultivation. The amylolytic extract showed the highest enzymatic activity at pH 8.0 and 45 °C, and the cellulolytic extract at pH 5.0 and 35 °C. The cellulase and amylase produced by the endophytes had their molecular masses estimated between 38 and 76 kDa. These results indicate that endophytic fungi from the açai palm can be used as a new source of hydrolytic enzymes, which can be applied in numerous biotechnological processes.
Asunto(s)
Endófitos/enzimología , Endófitos/metabolismo , Euterpe/microbiología , Hongos/enzimología , Hongos/metabolismo , Amilasas/metabolismo , Biotecnología/métodos , Celulasa/metabolismo , Celulasas/metabolismo , Colletotrichum , Hongos/clasificación , Hidrólisis , Lipasa/metabolismo , Penicillium , Péptido Hidrolasas , Poligalacturonasa/metabolismoRESUMEN
The purpose of this systematic review was to identify the available literature of production, purification, and characterization of proteases by endophytic fungi. There are few complete studies that entirely exhibit the production, characterization, and purification of proteases from endophytic fungi. This study followed the PRISMA, and the search was conducted on five databases: PubMed, PMC, Science Direct, Scopus Articles, and Web of Science up until 18 May 2021, with no time or language restrictions. The methodology of the selected studies was evaluated using GRADE. Protease production, optimization, purification, and characterization were the main evaluated outcomes. Of the 5540 initially gathered studies, 15 met the inclusion criteria after a two-step selection process. Only two studies optimized the protease production using statistical design and two reported enzyme purification and characterization. The genus Penicillium and Aspergillus were the most cited among the eleven different genera of endophytic fungi evaluated in the selected articles. Six studies proved the ability of some endophytic fungi to produce fibrinolytic proteases, demonstrating that endophytic fungi can be exploited for the further production of agents used in thrombolytic therapy. However, further characterization and physicochemical studies are required to evaluate the real potential of endophytic fungi as sources of industrial enzymes.
Asunto(s)
Aspergillus/enzimología , Endófitos/enzimología , Proteínas Fúngicas/biosíntesis , Penicillium/enzimología , Péptido Hidrolasas/biosíntesis , Proteínas Fúngicas/química , Péptido Hidrolasas/químicaRESUMEN
L-asparaginase from endophytic Fusarium proliferatum (isolate CCH, GenBank accession no. MK685139) isolated from the medicinal plant Cymbopogon citratus (Lemon grass), was optimized for its L-asparaginase production and its subsequent cytotoxicity towards Jurkat E6 cell line. The following factors were optimized; carbon source and concentration, nitrogen source and concentration, incubation period, temperature, pH and agitation rate. Optimization of L-asparaginase production was performed using One-Factor-At-A-Time (OFAT) and Response surface methodology (RSM) model. The cytotoxicity of the crude enzyme from isolate CCH was tested on leukemic Jurkat E6 cell line. The optimization exercise revealed that glucose concentration, nitrogen source, L-asparagine concentration and temperature influenced the L-asparaginase production of CCH. The optimum condition suggested using OFAT and RSM results were consistent. As such, the recommended conditions were 0.20% of glucose, 0.99% of L-asparagine and 5.34 days incubation at 30.50 °C. The L-asparaginase production of CCH increased from 16.75 ± 0.76 IU/mL to 22.42 ± 0.20 IU/mL after optimization. The cytotoxicity of the crude enzyme on leukemic Jurkat cell line recorded IC50 value at 33.89 ± 2.63% v/v. To conclude, the enzyme extract produced from Fusarium proliferatum under optimized conditions is a potential alternative resource for L-asparaginase.
Asunto(s)
Asparaginasa/biosíntesis , Citotoxinas/biosíntesis , Endófitos/metabolismo , Fusarium/metabolismo , Antineoplásicos , Asparaginasa/genética , Asparaginasa/aislamiento & purificación , Carbono , Medios de Cultivo/química , Citotoxinas/genética , Bases de Datos de Ácidos Nucleicos , Endófitos/enzimología , Endófitos/genética , Fusarium/enzimología , Fusarium/genética , Concentración de Iones de Hidrógeno , Técnicas Microbiológicas/métodos , Nitrógeno , Plantas Medicinales , TemperaturaRESUMEN
Endophytic bacteria represent microorganisms that live during the whole life cycle within the tissues of healthy plants without causing any obvious signs of disease. In this study, the ability of 128 endophyte bacterial isolates from some cultivated and wild grain plants (Poaceae family) in Van, Turkey, were investigated in terms of producing several extracellular hydrolytic enzymes. It was demonstrated that lipases, proteases, amylases, cellulases, pectinases, and xylanases were produced by the bacteria with relative frequencies of 74.2%, 65.6%, 55.4%, 32%, 21.8%, and 7.8%, respectively. In addition, molecular identification of a certain number of isolates selected according to their enzyme-producing capabilities was performed by 16S rRNA gene sequencing using a next-generation sequencing platform. As a result of the analysis, the isolates yielded certain strains belonging to Pseudomonas, Micrococcus, Paenibacillus, Streptococcus, Curtobacterium, Chryseobacterium, and Bacillus genera. Also, the strain G117Y1T was evaluated as a member of potential novel species based on 16S rRNA sequencing results.
Asunto(s)
Bacterias/enzimología , Endófitos/enzimología , Enzimas/metabolismo , Poaceae/microbiología , Bacterias/genética , ADN Bacteriano/genética , Endófitos/genética , Hidrólisis , ARN Ribosómico 16S , TurquíaRESUMEN
Marine fungi and, particularly, endophytic species have been recognised as one of the most prolific sources of structurally new and diverse bioactive secondary metabolites with multiple biotechnological applications. Despite the increasing number of bioprospecting studies, very few have already evaluated the cosmeceutical potential of marine fungal compounds. Thus, this study focused on a frequent seaweed in the Portuguese coast, Halopteris scoparia, to identify the endophytic marine fungi associated with this host, and assess their ability to biosynthesise secondary metabolites with antioxidative, enzymatic inhibitory (hyaluronidase, collagenase, elastase and tyrosinase), anti-inflammatory, photoprotective, and antimicrobial (Cutibacterium acnes, Staphylococcus epidermidis and Malassezia furfur) activities. The results revealed eight fungal taxa included in the Ascomycota, and in the most representative taxonomic classes in marine ecosystems (Eurotiomycetes, Sordariomycetes and Dothideomycetes). These fungi were reported for the first time in Portugal and in association with H. scoparia, as far as it is known. The screening analyses showed that most of these endophytic fungi were producers of compounds with relevant biological activities, though those biosynthesised by Penicillium sect. Exilicaulis and Aspergillus chevalieri proved to be the most promising ones for being further exploited by dermocosmetic industry. The chemical analysis of the crude extract from an isolate of A. chevalieri revealed the presence of two bioactive compounds, echinulin and neoechinulin A, which might explain the high antioxidant and UV photoprotective capacities exhibited by the extract. These noteworthy results emphasised the importance of screening the secondary metabolites produced by these marine endophytic fungal strains for other potential bioactivities, and the relevance of investing more efforts in understanding the ecology of halo/osmotolerant fungi.
Asunto(s)
Ascomicetos/metabolismo , Endófitos/metabolismo , Phaeophyceae/microbiología , Antibacterianos/farmacología , Antifúngicos/farmacología , Ascomicetos/enzimología , Ascomicetos/aislamiento & purificación , Bioprospección/métodos , Ecosistema , Endófitos/enzimología , Hongos/aislamiento & purificación , Hongos/metabolismo , Hongos no Clasificados/aislamiento & purificación , Hongos no Clasificados/metabolismo , Pruebas de Sensibilidad Microbiana , Portugal , Algas Marinas/microbiologíaRESUMEN
This study was undertaken to explore alternative applications of the widely known entomopathogenic/endophytic fungus, Beauveria bassiana, besides its sole use as a biocontrol agent. B. bassiana SAN01, was investigated for the production of two glycoside hydrolases, xylanase and endoglucanase under submerged conditions. Among the different biomass tested, wheat bran provided the best results for both xylanase and endoglucanase, and their production levels were further enhanced using response surface methodology. Under optimised conditions, heightened yields of 1061 U/ml and 23.03 U/ml were observed for xylanase and endoglucanase, respectively, which were 3.44 and 1.35 folds higher than their initial yields. These are the highest ever production levels reported for xylanase and endoglucanase from any B. bassiana strain or any known entomopathogenic fungi. Furthermore, the efficacy of xylanase/endoglucanase cocktail in the saccharification of sugarcane bagasse was evaluated. The highest amount of reducing sugar released from the pretreated biomass by the action of the crude Beauveria enzyme cocktail was recorded at 30°C after 8 h incubation. The significant activities of the hydrolytic enzymes recorded with B. bassiana in this study thus present promising avenues for the use of the entomopathogen as a new source of industrial enzymes and by extension, other biotechnological applications.
Asunto(s)
Beauveria , Celulasa , Xilosidasas , Beauveria/enzimología , Celulasa/metabolismo , Endófitos/enzimología , Saccharum/microbiología , Xilosidasas/metabolismoRESUMEN
Salt stress negatively affects plant growth, and the fungal endophyte Epichloëgansuensis increases the tolerance of its host grass species, Achnatherum inebrians, to abiotic stresses. In this work, we first evaluated the effects of E. gansuensis on glucose-6-phosphate dehydrogenase (G6PDH) and plasma membrane (PM) H+-ATPase activity of Achnatherum inebrians plants under varying NaCl concentrations. Our results showed that the presence of E. gansuensis increased G6PDH, PM H+-ATPase, superoxide dismutase and catalase activity to decrease O2â¢-, H2O2 and Na+ contents in A. inebrians under NaCl stress, resulting in enhanced salt tolerance. In addition, the PM NADPH oxidase activity and NADPH/NADP+ ratios were all lower in A. inebrians with E. ganusensis plants than A. inebrians plants without this endophyte under NaCl stress. In conclusion, E. gansuensis has a positive role in improving host grass yield under NaCl stress by enhancing the activity of G6PDH and PM H+-ATPase to decrease ROS content. This provides a new way for the selection of stress-resistant and high-quality forage varieties by the use of systemic fungal endophytes.
Asunto(s)
Endófitos/enzimología , Epichloe/enzimología , Glucosafosfato Deshidrogenasa/metabolismo , Poaceae/enzimología , ATPasas de Translocación de Protón/metabolismo , Cloruro de Sodio/metabolismo , Membrana CelularRESUMEN
Endophytic microbes are known to live asymptomatically inside their host throughout different stages of their life cycle and play crucial roles in the growth, development, fitness, and diversification of plants. The plant-endophyte association ranges from mutualism to pathogenicity. These microbes help the host to combat a diverse array of biotic and abiotic stressful conditions. Endophytic microbes play a major role in the growth promotion of their host by solubilizing of macronutrients such as phosphorous, potassium, and zinc; fixing of atmospheric nitrogen, synthesizing of phytohormones, siderophores, hydrogen cyanide, ammonia, and act as a biocontrol agent against wide array of phytopathogens. Endophytic microbes are beneficial to plants by directly promoting their growth or indirectly by inhibiting the growth of phytopathogens. Over a long period of co-evolution, endophytic microbes have attained the mechanism of synthesis of various hydrolytic enzymes such as pectinase, xylanases, cellulase, and proteinase which help in the penetration of endophytic microbes into tissues of plants. The effective usage of endophytic microbes in the form of bioinoculants reduce the usage of chemical fertilizers. Endophytic microbes belong to different phyla such as Actinobacteria, Acidobacteria, Bacteroidetes, Deinococcus-thermus, Firmicutes, Proteobacteria, and Verrucomicrobia. The most predominant and studied endophytic bacteria belonged to Proteobacteria followed by Firmicutes and then by Actinobacteria. The most dominant among reported genera in most of the leguminous and non-leguminous plants are Bacillus, Pseudomonas, Fusarium, Burkholderia, Rhizobium, and Klebsiella. In future, endophytic microbes have a wide range of potential for maintaining health of plant as well as environmental conditions for agricultural sustainability. The present review is focused on endophytic microbes, their diversity in leguminous as well as non-leguminous crops, biotechnological applications, and ability to promote the growth of plant for agro-environmental sustainability.
Asunto(s)
Bacterias/clasificación , Biodiversidad , Productos Agrícolas/microbiología , Endófitos/clasificación , Endófitos/fisiología , Desarrollo de la Planta , Agricultura/métodos , Endófitos/enzimología , Fijación del Nitrógeno , Reguladores del Crecimiento de las Plantas , Raíces de Plantas/microbiología , SimbiosisRESUMEN
Grammothele lineata strain SDL-CO-2015-1, jute (Corchorus olitorius) endophyte has been reported to produce anti-cancer drug paclitaxel in culture condition. Here we investigated the genome using different bioinformatic tools to find its association with the production of commercially important compounds including taxol. Carbohydrate-active enzymes, proteases, and secretory proteins were annotated revealing a complex endophytic relationship with its plant host. The presences of a diverse range of CAZymes including numerous lignocellulolytic enzymes support its potentiality in biomass degradation. Genome annotation led to the identification of 28 clusters for secondary metabolite biosynthesis. Several biosynthesis gene clusters were identified for terpene biosynthesis from antiSMASH analysis but none could be specifically pinned to taxol synthesis. This study will direct us to understand the genomic organization of endophytic basidiomycetes with a potential for producing numerous commercially important enzymes and secondary metabolites taking G. lineata as a model.
Asunto(s)
Genoma Fúngico , Polyporaceae/genética , Polyporaceae/metabolismo , Metabolismo de los Hidratos de Carbono/genética , Sistema Enzimático del Citocromo P-450/genética , Endófitos/enzimología , Endófitos/genética , Endófitos/metabolismo , Proteínas Fúngicas/genética , Ontología de Genes , Lignina/metabolismo , Proteínas de Transporte de Membrana/genética , Anotación de Secuencia Molecular , Péptido Hidrolasas/genética , Filogenia , Polyporaceae/clasificación , Polyporaceae/enzimología , Metabolismo Secundario/genéticaRESUMEN
Abstract Endophytic fungi belonging to the genus Muscodor now transferred to Induratia are known producers of bioactive volatile organic compounds (VOCs) with many industrial applications. However, the members of this genus have rarely been reported to produce non-volatile metabolites including enzyme. Enzymes of the endophytes are degraders of the polysaccharides available in the host plants and the knowledge of enzyme production by Induratia spp. may provide insights into their possible biotechnological applications. The aim of this study was to evaluate the activity of amylase, cellulase, lipase, pectinase, phytase, protease, endo β-1,4 glucanase and exo β-1,4 glucanase enzymes produced by fungi of the species Induratia coffeana, Induratia yucatanensis and Induratia sp. isolated from organic coffee plants. All Induratia spp. were able to produce the extracellular enzymes cellulase, pectinase, protease, and phytase. Eight fungi were able to produce lipase and four produced amylase. The specific activity of endo β-1, 4 glucanase and exo β-1,4 glucanase enzymes were detected for 9 and 8 endophytic fungi, respectively. This work demonstrated for the first time, the array of enzymes produced by Induratia spp. isolated from Coffea arabica in organic systems in Brazil.
Asunto(s)
Coffea/microbiología , Activación Enzimática , Compuestos Orgánicos Volátiles/metabolismo , Endófitos/enzimología , BrasilRESUMEN
The fungal strain Alternaria alternata JS0515 was isolated from Vitex rotundifolia (beach vitex). Twelve secondary metabolites, including one new altenusin derivative (1), were isolated. The isolated metabolites included seven known altenusin derivatives (2-8), two isochromanones (9, 10), one perylenequinone (11), and one benzocycloalkanone (12). Their structures were determined via 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), and computational electronic circular dichroism (ECD) analysis. Compounds 3 and 11 increased pyruvate dehydrogenase (PDH) activity in AD-293 human embryonic kidney cells and significantly inhibited PDH phosphorylation. The IC50 values of 3 and 11 were 32.58 and 27.82 µM, respectively.
Asunto(s)
Alternaria/aislamiento & purificación , Alternaria/metabolismo , Endófitos/aislamiento & purificación , Endófitos/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Metabolismo Secundario , Vitex/microbiología , Alternaria/enzimología , Bioensayo , Espectroscopía de Resonancia Magnética con Carbono-13 , Endófitos/enzimología , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
Endophytic fungi provide benefits to host plants by producing a diverse class of secondary metabolites (natural products). Arrays of polyketide natural products are synthesized by specific classes of polyketide synthases (PKS I, II and III) in host organisms. In the present study, we attempt to screen and identify type III PKSs in culturable fungal endophytes isolated from the ethno medicinal plants including Arbus precatorius, Bacopa monnieri,Citrus aurantifolia and Datura metel to detect the genetic potential of endophytic fungi in producing bioactive compounds. A total of seventeen endophytic fungal strains belonging to eight genera were identified using fungal morphology and rDNA-ITS phylogenetic analyses. A CODEHOP-PCR based strategy was followed to design degenerate primers for the screening of type III PKS genes from fungal endophytes. We had successfully amplified partial PKS genes from eight endophytes. The amplified PKS sequences showed 60-99% identity to already characterized/putative PKS genes. From the partial sequence of FiPKS from Fusarium incarnatum BMER1, a full-length gene was amplified, cloned and characterized. FiPKScDNA was cloned and expressed in E. coli Lemo21 (DE3) and the purified protein was shown to produce pyrones and resorcinols using acyl-CoA thioesters as substrates. FiPKS showed the highest catalytic efficiency of 7.6 × 104 s-1 M-1 with stearoyl CoA as a starter unit. This study reports the identification and characterization of type III PKS from endophytes of medicinal plants by CODEHOP PCR.
Asunto(s)
Aciltransferasas/genética , Endófitos/enzimología , Hongos/enzimología , Plantas Medicinales/microbiología , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Hongos/clasificación , Hongos/genética , Hongos/aislamiento & purificación , Expresión Génica , Cinética , Técnicas Microbiológicas , Filogenia , Pironas/metabolismo , Resorcinoles/metabolismo , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Verticillium wilt, caused by Verticillium dahliae, results in a dramatic loss of cotton yields in China. There is great potential for biocontrol to manage this destructive crop disease. In this study, we obtained the endophytic bacterium Bacillus halotolerans Y6 from Verticillium wilt-resistant cotton Gossypium barbadense Xinhai15; this bacterium possesses strong antagonistic abilities that inhibit V. dahliae spore germination and mycelial growth. The results of the enzyme activity assay, heterologous expression, and gene knockdown showed that the key virulence factor of Y6 for antagonizing V. dahliae was ß -glucanase Bgy6. To facilitate field tests of biological control, we constructed the homologous Bgy6-overexpression strain OY6. Compared with the wild-type Y6 strain, the ß-glucanase activity of OY6 was increased by 91.79%, and the inhibition rate of OY6 against V. dahliae V991 exceeded 96.7%. Moreover, the spores of V. dahliae V991 treated with OY6 showed more mucus and larger holes on the surface, as observed by scanning electron microscopy. Potting test results illustrated that both OY6 and Y6 could improve the resistance of upland cotton to Verticillium wilt. With the inoculation of V. dahliae V991 for 45 days, the disease index of G. hirsutum TM-1 treated with OY6 was only 8.33, which was significantly lower than that in plants treated with the wild-type strain Y6 (17.86) or the controls without bacteria (35.94). Our research provides a new idea for the control of Verticillium wilt in upland cotton via transforming endophytic bacteria of Verticillium wilt-resistant cotton and proposes a new solution to prevent and control Verticillium wilt.
Asunto(s)
Bacillus/enzimología , Proteínas Bacterianas/genética , Endo-1,3(4)-beta-Glucanasa/genética , Endófitos/enzimología , Gossypium/microbiología , Enfermedades de las Plantas/inmunología , Verticillium/fisiología , Factores de Virulencia/genética , Antibiosis , Bacillus/genética , Bacillus/aislamiento & purificación , Bacillus/fisiología , Proteínas Bacterianas/metabolismo , Resistencia a la Enfermedad , Endo-1,3(4)-beta-Glucanasa/metabolismo , Endófitos/genética , Endófitos/aislamiento & purificación , Endófitos/fisiología , Expresión Génica , Regulación de la Expresión Génica de las Plantas , Gossypium/inmunología , Enfermedades de las Plantas/microbiología , Factores de Virulencia/inmunologíaRESUMEN
10-deacetylbaccatin III-10-ß-O-acetyltransferase (DBAT) is a key rate-limiting enzyme of the Taxol biosynthetic pathway, which is uncharacterized in Taxol-producing endophytic fungi. Here, an open reading frame of DBAT was cloned from the Taxol-producing endophytic fungus Lasiodiplodia theobromae (LtDBAT). The LtDBAT enzyme was heterologously expressed and purified by the affinity and gel filtration chromatography methods. The molecular weight of the purified protein was 49 kDa and its identity was confirmed by western blot. The purified LtDBAT enzyme was capable of catalyzing 10-deacetylbaccatin III into baccatin III, as shown by liquid chromatography-mass spectroscopy. The mass spectra of baccatin III were identical to the authentic baccatin III. The LtDBAT enzyme was characterized and the kinetic parameters of catalysis were determined. In addition, localization of LtDBAT was performed by using confocal microscopy and the result showed that the enzyme was localized in lipid droplets. Together, this study provides biochemical insights into the fungal recombinant DBAT enzyme that is involved in the Taxol biosynthetic pathway. In the near future, engineering of the LtDBAT enzyme and the Taxol biosynthetic pathway in endophytic fungi could be an eco-friendly and economically feasible alternative source for production of Taxol and its precursors.
Asunto(s)
Acetiltransferasas/química , Acetiltransferasas/metabolismo , Ascomicetos/enzimología , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Paclitaxel/biosíntesis , Acetiltransferasas/genética , Alcaloides/metabolismo , Ascomicetos/química , Ascomicetos/genética , Ascomicetos/metabolismo , Biocatálisis , Clonación Molecular , Endófitos/enzimología , Endófitos/genética , Endófitos/metabolismo , Estabilidad de Enzimas , Proteínas Fúngicas/genética , Concentración de Iones de Hidrógeno , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Taxoides/metabolismo , TemperaturaRESUMEN
The filamentous Bipolaris and Curvularia genera consist of species known to cause severe diseases in plants and animals amounting to an estimated annual loss of USD $10 billion worldwide. Despite the harmful effect of Bipolaris and Curvularia species, scarce attention is paid on beneficial areas where the fungi are used in industrial processes to generate biotechnological products. Catalytic potential of Bipolaris and Curvularia species in the production of biodiesel, bioflucculant, biosorbent, and mycoherbicide are promising for the bioeconomy. It is herein demonstrated that knowledge-based application of some endophytic Bipolaris and Curvularia species are indispensable vectors of sustainable economic development. In the twenty-first century, India, China, and the USA have taken progress in the biotechnological application of these fungi to generate wealth. As such, some Bipolaris and Curvularia species significantly impact on global crop improvement, act as catalyst in batch-reactors for biosynthesis of industrial enzymes and medicines, bioengineer of green-nanoparticle, agent of biofertilizer, bioremediation and bio-hydrometallurgy. For the first time, this study discusses the current advances in biotechnological application of Bipolaris and Curvularia species and provide new insights into the prospects of optimizing their bioengineering potential for developing bioeconomy.
Asunto(s)
Ascomicetos , Bioingeniería , Biotecnología , Endófitos , Ascomicetos/clasificación , Ascomicetos/enzimología , Ascomicetos/metabolismo , Biodegradación Ambiental , Biocombustibles , Agentes de Control Biológico , Biotransformación , Endófitos/clasificación , Endófitos/enzimología , Endófitos/metabolismo , Fertilizantes , Floculación , Virus Fúngicos , Herbicidas , Metalurgia , Nanopartículas , Suelo/química , Simbiosis , Termotolerancia , UranioRESUMEN
The huge applications of cellulosic and lignocellulosic materials in the various fields of life lead to accumulation of its wastes that became one of the major sources of environmental pollution. In this study, a Gram-positive cellulose-decomposing endophytic bacterium (Chi-04) was isolated from medicinal plant Chiliadenus montanus which inhabitant Saint Catherine (Sinai) region in Egypt. The bacterial strain was identified based on the sequence analysis of 16S rRNA genes as Lysinibacillus xylanilyticus. This isolate was capable of degrading 58% of cellulosic filter paper (100 g/l) within 15 days of incubation. The soluble and reduced sugars were spectrophotometrically determined as cellulose decomposition metabolites. The bacterial isolate exhibited an obvious activity toward cellulase enzyme production. The maximum cellulase activity (0.18 U/min) was detected after 12 days of incubation while the maximum release of soluble sugars (11.85 mg/ml) was detected after 15 days of incubation. CaCl2 nanoparticles (100 nm) were chemically prepared to enhance the activity of the enzyme. The optimum concentration of CaCl2 nanoparticles that showed the highest activity of cellulase (0.3 mg/ml reduced sugar) was 0.6%. The bacterial isolates showed potential convert of cellulose into reducing sugars which could be used in several applications.