Metabolismo do nitrogênio de Streptomyces clavuligerus e seus efeitos sobre a produção de compostos bioativos
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Data
2019-04-16
Autores
Baptista, Amanda Salvador
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Universidade Estadual Paulista (Unesp)
Resumo
Streptomyces clavuligerus produz uma diversidade de compostos de interesse terapêutico, com destaque para os beta-lactâmicos ácido clavulânico (um inibidor de beta-lactamases) e o antibiótico cefamicinaC, e os não beta-lactâmicos holomicina e tunicamicina. A produção destes metabólitos secundários é extremamente influenciada pela natureza e/ou concentração da(s) fonte(s) de nitrogênio do meio. Por exemplo, lisina é precursor dos antibióticos beta-lactâmicos e sua presença em culturas de S.clavuligerus estimula, principalmente, a produção de cefamicinaC. Por outro lado, lisina também é consumido no metabolismo primário, gerando duas moléculas de glutamato. O presente trabalho propôs explorar as atuações individuais de lisina e glutamato exógenos no processo em batelada de produção de compostos bioativos. Para isto, realizaram-se cultivos submersos em batelada em meios quimicamente definidos contendo maltose como principal fonte de carbono e energia e lisina ou lisina+glutamato como fonte(s) de nitrogênio. Realizaram-se cultivos preliminares em frascos agitados iniciados em pH 6,8 ou pH 7,2. Independente do pH inicial, nos meios contendo somente lisina obtiveram-se as maiores concentrações de cefamicinaC (~140 mg.L-1) e nos meios lisina+glutamato, além de cefamicina C (55 a 85 mg.L-1) também houve a produção de holomicina (15 a 35 mg.L-1). Esta última foi inesperada e inédita, pois a literatura tem afirmado que a produção de holomicina pela linhagem selvagem de S.clavuligerus é indetectável. Outra particularidade dos cultivos em lisina+glutamato foi o aumento acentuado de pH até 8,0, impedindo, assim, avaliar a atuação dos aminoácidos na produção de bioativos. Para eliminar a variação de pH e ao mesmo tempo investigar se a produção de holomicina havia sido desencadeada pela presença de glutamato e/ou justamente pelo aumento de pH, conduziram-se cultivos em biorreator convencional (5L vol. útil) com pH controlado em 6,8, valor usual em culturas submersas de S.clavuligerus, ou em pH 7,4, valor próximo do início da produção de holomicina em frascos agitados. Na condição lisina+glutamato em pH 6,8 obteve-se ca.400 mg.L-1 de cefamicinaC, um valor de gt3 a 4 vezes maior que o obtido nas demais condições. Uma concentração de cefamicinaC desta ordem de grandeza obtida em meio sintético nunca foi relatada na literatura. Este resultado também sustenta a ideia de que glutamato exógeno pode promover maior disponibilização da lisina presente no meio no sentido do aumento da produção de cefamicinaC. Holomicina foi produzida somente nos meios controlados em pH 7,4 (50 a 100 mg.L-1), independente da presença de glutamato. Concentrações desta ordem de grandeza têm sido reportadas apenas para mutantes de S.clavuligerus melhores produtores deste composto que apresentam, geralmente, danos genéticos associados à síntese de ácido clavulânico. Os dados obtidos em biorreator também foram comparados entre si por meio de estudos de modelagem e simulação, tanto do ponto de vista cinético como de distribuição de fluxos metabólicos. O modelo cinético refletiu bem o comportamento dos cultivos e as simulações do modelo metabólico indicaram que a produção de holomicina juntamente com a utilização de lisina como única fonte de nitrogênio, exigiu maior demanda energética. Os presentes resultados somam-se às evidências de que é possível configurar um ambiente artificial que simule condições necessárias e suficientes do ambiente natural do micro-organismo capazes de desencadear a produção de metabólitos secundários específicos.
Streptomyces clavuligerus produces several compounds of therapeutic interest including the beta-lactam clavulanic acid (a powerful beta-lactamase inhibitor) and the antibiotic cephamycin C as well as the not beta-lactam holomycin and tunicamycin. The production of this secondary metabolites is extremely influenced by source and/or concentration of nitrogen in the culture media. For example, lysine is a precursor of the beta-lactam antibiotics and its presence in culture S. clavuligerus stimulates especially the production of cephamycin C. On the other hand, this microorganism also consumes lysine in the primary metabolism, generating two molecules of glutamate. The present work proposed to explore the individual performances of exogenous lysine and glutamate in the batch process of bioactive compounds production. For this porpoise, submerged batch cultures were performed by using a chemically defined media containing maltose as the main source of carbon and energy coupled with lysine or lysine+glutamate as the main source(s) of N. Preliminary cultures were made in shake flasks initiated at pH 6.8 or pH 7.2. In the media containing only lysine were obtained the highest concentrations of cephamycin C (~ 140 mg.L-1). Likewise, in the media with lysine+glutamate, in which were also obtained cephamycin C (55 to 85 mg.L-1) and holomycin (15 to 35 mg.L-1). The latter was unexpected and unpublished, since the literature has stated that the production of holomycin by the wild strain of S. clavuligerus is undetectable. Another peculiarity of the glutamate cultures was the sharp increase of pH to 8,0, thus preventing the evaluation regarding the action of amino acids in the production of bioactive. This problem was eliminated by conducting the cultures in a conventional bioreactor (5L) with pH controlled at 6.8, usual value in submerged cultures of S. clavuligerus, or at pH 7.4, value close to that was observed at the start of holomycin production in shake flasks. In the lysine+glutamate condition at pH 6.8 was obtained approximately 400 mg.L-1 of cephamycin C, 3 to 4 times higher than other conditions. A concentration of cephamycin C of this order of magnitude obtained in synthetic medium has never been reported in the literature. This result also supports the idea that exogenous glutamate may promote greater availability of exogenous lysine leading to an increasing production of cephamycin C. Holomycin was produced only in the controlled media at pH 7.4 (50 to 100 mg.L-1), independent of the presence of glutamate. Concentrations of this order of magnitude have been reported only for S. clavuligerus mutants, the best holomycin producers with genetic damage associated with CA synthesis. Moreover, the data obtained in bioreactor were also compared with each other by means of modeling and simulation studies, both by kinetic point of view and distribution of metabolic fluxes. The kinetic model well reflected the behavior of the culture mediums and the simulations of the metabolic models. It also indicated that the production of holomycin combined with the presence of lysine as the only source of N, demanded the highest energy demand. The present results add to the evidence that an artificially defined environment can simulate necessary and sufficient conditions of the natural environment of the microorganism capable of triggering the production of specific secondary metabolites.
Streptomyces clavuligerus produces several compounds of therapeutic interest including the beta-lactam clavulanic acid (a powerful beta-lactamase inhibitor) and the antibiotic cephamycin C as well as the not beta-lactam holomycin and tunicamycin. The production of this secondary metabolites is extremely influenced by source and/or concentration of nitrogen in the culture media. For example, lysine is a precursor of the beta-lactam antibiotics and its presence in culture S. clavuligerus stimulates especially the production of cephamycin C. On the other hand, this microorganism also consumes lysine in the primary metabolism, generating two molecules of glutamate. The present work proposed to explore the individual performances of exogenous lysine and glutamate in the batch process of bioactive compounds production. For this porpoise, submerged batch cultures were performed by using a chemically defined media containing maltose as the main source of carbon and energy coupled with lysine or lysine+glutamate as the main source(s) of N. Preliminary cultures were made in shake flasks initiated at pH 6.8 or pH 7.2. In the media containing only lysine were obtained the highest concentrations of cephamycin C (~ 140 mg.L-1). Likewise, in the media with lysine+glutamate, in which were also obtained cephamycin C (55 to 85 mg.L-1) and holomycin (15 to 35 mg.L-1). The latter was unexpected and unpublished, since the literature has stated that the production of holomycin by the wild strain of S. clavuligerus is undetectable. Another peculiarity of the glutamate cultures was the sharp increase of pH to 8,0, thus preventing the evaluation regarding the action of amino acids in the production of bioactive. This problem was eliminated by conducting the cultures in a conventional bioreactor (5L) with pH controlled at 6.8, usual value in submerged cultures of S. clavuligerus, or at pH 7.4, value close to that was observed at the start of holomycin production in shake flasks. In the lysine+glutamate condition at pH 6.8 was obtained approximately 400 mg.L-1 of cephamycin C, 3 to 4 times higher than other conditions. A concentration of cephamycin C of this order of magnitude obtained in synthetic medium has never been reported in the literature. This result also supports the idea that exogenous glutamate may promote greater availability of exogenous lysine leading to an increasing production of cephamycin C. Holomycin was produced only in the controlled media at pH 7.4 (50 to 100 mg.L-1), independent of the presence of glutamate. Concentrations of this order of magnitude have been reported only for S. clavuligerus mutants, the best holomycin producers with genetic damage associated with CA synthesis. Moreover, the data obtained in bioreactor were also compared with each other by means of modeling and simulation studies, both by kinetic point of view and distribution of metabolic fluxes. The kinetic model well reflected the behavior of the culture mediums and the simulations of the metabolic models. It also indicated that the production of holomycin combined with the presence of lysine as the only source of N, demanded the highest energy demand. The present results add to the evidence that an artificially defined environment can simulate necessary and sufficient conditions of the natural environment of the microorganism capable of triggering the production of specific secondary metabolites.
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Palavras-chave
Streptomyces clavuligerus, Bioprocesso, Metabólito secundário, Modelagem matemática e simulação, Fluxos metabólicos