RESUMO
In an earlier work on lovastatin production by Aspergillus terreus, we found that reactive oxygen species (ROS) concentration increased to high levels precisely at the start of the production phase (idiophase) and that these levels were sustained during all idiophase. Moreover, it was shown that ROS regulate lovastatin biosynthesis. ROS regulation has also been reported for aflatoxins. It has been suggested that, due to their antioxidant activity, aflatoxins are regulated and synthesized like a second line of defense against oxidative stress. To study the possible ROS regulation of other industrially important secondary metabolites, we analyzed the relationship between ROS and penicillin biosynthesis by Penicillium chrysogenum and cephalosporin biosynthesis by Acremonium chrysogenum. Results revealed a similar ROS accumulation in idiophase in penicillin and cephalosporin fermentations. Moreover, when intracellular ROS concentrations were decreased by the addition of antioxidants to the cultures, penicillin and cephalosporin production were drastically reduced. When intracellular ROS were increased by the addition of exogenous ROS (H2O2) to the cultures, proportional increments in penicillin and cephalosporin biosyntheses were obtained. It was also shown that lovastatin, penicillin, and cephalosporin are not antioxidants. Taken together, our results provide evidence that ROS regulation is a general mechanism controlling secondary metabolism in fungi.
Assuntos
Acremonium/metabolismo , Cefalosporinas/biossíntese , Penicilinas/biossíntese , Penicillium chrysogenum/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acremonium/efeitos dos fármacos , Vias Biossintéticas , Fermentação , Regulação Fúngica da Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Penicillium chrysogenum/efeitos dos fármacos , Espécies Reativas de Oxigênio/farmacologia , Metabolismo SecundárioRESUMO
Three different transformation strategies were tested and compared in an attempt to facilitate and improve the genetic transformation of Acremonium chrysogenum, the exclusive producer of the pharmaceutically relevant ß-lactam antibiotic cephalosporin C. We investigated the use of high-voltage electric pulse to transform germinated conidia and young mycelium and compared these procedures with traditional PEG-mediated protoplast transformation, using phleomycin resistance as selection marker in all cases. The effect of the field strength and capacitance on transformation frequency and cell viability was evaluated. The electroporation of germinated conidia and young mycelium was found to be appropriate for transforming A. chrysogenum with higher transformation efficiencies than those obtained with the conventional protoplast-based transformation procedures. The developed electroporation strategy is fast, simple to perform, and highly reproducible and avoids the use of chemicals toxic to cells. Electroporation of young mycelium represents an alternative method for transformation of fungal strains with reduced or no sporulation, as often occurs in laboratory-developed strains in the search for high-yielding mutants for industrial bioprocesses.
Assuntos
Acremonium/genética , Eletroporação/métodos , Transformação Genética , Acremonium/efeitos dos fármacos , Acremonium/metabolismo , Cefalosporinas/biossíntese , Farmacorresistência Bacteriana , Viabilidade Microbiana , Micélio/efeitos dos fármacos , Micélio/genética , Micélio/metabolismo , Fleomicinas/farmacologia , Protoplastos/fisiologia , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismoRESUMO
Esfenvalerate biodegradation by marine-derived fungi is reported here. Esfenvalerate (S,S-fenvalerate) and its main metabolites [3-phenoxybenzaldehyde (PBAld), 3-phenoxybenzoic acid (PBAc), 3-phenoxybenzyl alcohol (PBAlc), and 2-(4-chlorophenyl)-3-methylbutyric acid (CLAc)] were quantitatively analyzed by a validated method in triplicate experiments. All the strains (Penicillium raistrickii CBMAI 931, Aspergillus sydowii CBMAI 935, Cladosporium sp. CBMAI 1237, Microsphaeropsis sp. CBMAI 1675, Acremonium sp. CBMAI 1676, Westerdykella sp. CBMAI 1679, and Cladosporium sp. CBMAI 1678) were able to degrade esfenvalerate, however, with different efficiencies. Initially, 100 mg L(-1) esfenvalerate (Sumidan 150SC) was added to each culture in 3 % malt liquid medium. Residual esfenvalerate (64.8-95.2 mg L(-1)) and the concentrations of PBAc (0.5-7.4 mg L(-1)), ClAc (0.1-7.5 mg L(-1)), and PBAlc (0.2 mg L(-1)) were determined after 14 days. In experiments after 7, 14, 21, and 28 days of biodegradation with the three most efficient strains, increasing concentrations of the toxic compounds PBAc (2.7-16.6 mg L(-1), after 28 days) and CLAc (6.6-13.4 mg L(-1), after 28 days) were observed. A biodegradation pathway was proposed, based on HPLC-ToF results. The biodegradation pathway includes PBAld, PBAc, PBAlc, ClAc, 2-hydroxy-2-(3-phenoxyphenyl)acetonitrile, 3-(hydroxyphenoxy)benzoic acid, and methyl 3-phenoxy benzoate. Marine-derived fungi were able to biodegrade esfenvalerate in a commercial formulation and showed their potential for future bioremediation studies in contaminated soils and water bodies.
Assuntos
Acremonium/metabolismo , Aspergillus/metabolismo , Cladosporium/metabolismo , Nitrilas/metabolismo , Penicillium/metabolismo , Praguicidas/metabolismo , Piretrinas/metabolismo , Poluentes Químicos da Água/metabolismo , Benzaldeídos/metabolismo , Benzoatos/metabolismo , Álcoois Benzílicos/metabolismo , Biodegradação Ambiental , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Poluentes do Solo/metabolismoRESUMO
Production of ethanol with two corn endophytic fungi, Fusarium verticillioides and Acremonium zeae, was studied. The yield of ethanol from glucose, xylose and a mixture of both sugars were 0.47, 0.46 and 0.50g/g ethanol/sugar for F. verticillioides and 0.37, 0.39 and 0.48g/g ethanol/sugar for A. zeae. Both fungi were able to co-ferment glucose and xylose. Ethanol production from 40g/L of pre-treated sugarcane bagasse was 4.6 and 3.9g/L for F. verticillioides and A. zeae, respectively, yielding 0.31g/g of ethanol per consumed sugar. Both fungi studied were capable of co-fermenting glucose and xylose at high yields. Moreover, they were able to produce ethanol directly from lignocellulosic biomass, demonstrating to be suitable microorganisms for consolidated bioprocessing.
Assuntos
Acremonium/metabolismo , Celulose/metabolismo , Etanol/metabolismo , Fusarium/metabolismo , Glucose/metabolismo , Saccharum/química , Xilose/metabolismo , Zea mays/microbiologia , Microbiologia IndustrialRESUMO
In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2(Ë)) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.
Assuntos
Acremonium/metabolismo , Fragaria/microbiologia , Proteínas Fúngicas/metabolismo , Doenças das Plantas/microbiologia , Subtilisina/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Sequência de Bases , Bioensaio , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Complementar/metabolismo , Resistência à Doença , Eletroforese em Gel de Poliacrilamida , Fragaria/imunologia , Espectrometria de Massas , Dados de Sequência Molecular , Imunidade Vegetal , Espécies Reativas de Oxigênio , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Subtilisinas/metabolismo , Tripsina , UltrafiltraçãoRESUMO
Uranium(VI) sorption by two highly uranium-resistant air-borne fungi in a low-pH medium was measured by means of gamma spectrometry. Growth kinetics and stoichiometry of the two fungal species were also studied. Results show acceptable growth rates in synthetic medium with glucose and ammonia as carbon and nitrogen sources, respectively. Typical oxidative metabolism of carbohydrates was found. In vivo uptake of the radionuclide was negligible, but biosorption dry biomass presented a remarkable performance. The fungal strains showed potential for use in bioremediation.
Assuntos
Biodegradação Ambiental , Fungos Mitospóricos/metabolismo , Desintoxicação por Sorção/métodos , Espectrometria gama/métodos , Urânio/metabolismo , Poluentes Radioativos da Água/metabolismo , Acremonium/metabolismo , Biomassa , Concentração de Íons de Hidrogênio , Cinética , Especificidade da Espécie , Bactérias Redutoras de Enxofre/metabolismoRESUMO
Acremonium chrysogenum NCIM 1069 was used for the biosynthesis of cephalosporin-C (CPC) in batch mode of cultivation. The effect of different medium constituents for better yield of CPC was thoroughly investigated. From the results of the fermentation, it was found that ammonium sulphate as inorganic nitrogen source and methionine at the concentration of 0.4 percent are most suitable for higher yield of antibiotic. The variation in the C/N ratio on the biosynthesis of CPC showed that a C/N ratio of 8.0 is most suitable for maximum production of CPC
Assuntos
Acremonium/metabolismo , Cefalosporinas/biossíntese , Reatores Biológicos , Meios de Cultura , Fermentação , Metionina/metabolismoRESUMO
In a screening for antimicrobial metabolites, amides of D-allo- and L-isoleucine derivatives were isolated from the culture of a marine strain of Acremonium furcatum. Structural elucidation of these compounds was performed by analysis of spectroscopic data and confirmed by synthesis. All of the compounds, natural and synthetic intermediates, were bioassayed against bacteria and phytopathogenic fungi, with many showing remarkable antifungal activities.
Assuntos
Acremonium/química , Acremonium/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Bactérias/efeitos dos fármacos , Estrutura MolecularRESUMO
The industrial production of antibiotics with filamentous fungi is usually carried out in conventional aerated and agitated tank fermentors. Highly viscous non-Newtonian broths are produced and a compromise must be found between convenient shear stress and adequate oxygen transfer. In this work, cephalosporin C production by bioparticles of immobilized cells of Cephalosporium acremonium ATCC 48272 was studied in a repeated batch tower bioreactor as an alternative to the conventional process. Also, gas-liquid oxygen transfer volumetric coefficients, k(L)a, were determined at various air flow-rates and alumina contents in the bioparticle. The bioparticles were composed of calcium alginate (2.0% w/w), alumina ( < 44 micra), cells, and water. A model describing the cell growth, cephalosporin C production, oxygen, glucose, and sucrose consumption was proposed. To describe the radial variation of oxygen concentration within the pellet, the reaction-diffusion model forecasting a dead core bioparticle was adopted. The k(L)a measurements with gel beads prepared with 0.0, 1.0, 1.5, and 2.0% alumina showed that a higher k(L)a value is attained with 1.5 and 2.0%. An expression relating this coefficient to particle density, liquid density, and air velocity was obtained and further utilized in the simulation of the proposed model. Batch, followed by repeated batch experiments, were accomplished by draining the spent medium, washing with saline solution, and pouring fresh medium into the bioreactor. Results showed that glucose is consumed very quickly, within 24 h, followed by sucrose consumption and cephalosporin C production. Higher productivities were attained during the second batch, as cell concentration was already high, resulting in rapid glucose consumption and an early derepression of cephalosporin C synthesizing enzymes. The model incorporated this improvement predicting higher cephalosporin C productivity.
Assuntos
Acremonium/crescimento & desenvolvimento , Acremonium/metabolismo , Reatores Biológicos/microbiologia , Cefalosporinas/biossíntese , Glucose/metabolismo , Modelos Biológicos , Consumo de Oxigênio/fisiologia , Sacarose/metabolismo , Acremonium/citologia , Divisão Celular/fisiologia , Células Imobilizadas/fisiologia , Simulação por ComputadorRESUMO
One serious difficulty in modeling a fermentative process is the forecasting of the duration of the lag phase. The usual approach to model biochemical reactors relies on first-principles, unstructured mathematical models. These models are not able to take into account changes in the process response caused by different incubation times or by repeated fedbatches. To overcome this problem, we have proposed a hybrid neural network algorithm. Feedforward neural networks were used to estimate rates of cell growth, substrate consumption, and product formation from on-line measurements during cephalosporin C production. These rates were included in the mass balance equations to estimate key process variables: concentrations of cells, substrate, and product. Data from fed-batch fermentation runs in a stirred aerated bioreactor employing the microorganism Cephalosporium acremonium ATCC 48272 were used. On-line measurements strongly related to the mass and activity of the cells used. They include carbon dioxide and oxygen concentrations in the exhausted gas. Good results were obtained using this approach.