RESUMO
Electroporation is a method for the introduction of molecules (usually nucleic acids) into a cell, consisting of submitting the cells to high-voltage and short electric pulses in the presence of the exogenous DNA/molecule. It is a versatile method, adaptable to different types of cells, from bacteria to cultured cells to higher eukaryotes, and thus has applications in many diverse fields, such as environmental biology, biotechnology, genetic engineering, and medicine. Electroporation has some advantages over other genetic transformation strategies, including the simplicity of the method, a wide range of adjustable parameters (possibility of optimization), high reproducibility and avoidance of the use of chemicals toxic to cells. Here we describe an optimized electroporation procedure for the industrially important fungus Acremonium chrysogenum, using germinated conidia and fragmented young mycelium. In both cases, the transformation efficiency was higher compared to the conventional polyethylene glycol (PEG)-mediated transformation of protoplasts.
Assuntos
Eletroporação/métodos , Fungos/genética , Acremonium/genética , Biotecnologia/métodos , Engenharia Genética/métodos , Micélio/genética , Polietilenoglicóis/química , Protoplastos , Reprodutibilidade dos Testes , Transformação Genética/genéticaRESUMO
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
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