Análise de condições in vitro para produção de metabólitos secundários por Streptomyces noursei CAB-C 50
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2023-03-01
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Universidade Federal de Viçosa
Resumo
A busca por novos bioinsumos que impulsionam a consolidação de uma agricultura mais sustentável é uma realidade crescente em escala global. Os insumos bioativos ou bioinsumos são produtos de origem vegetal, animal ou microbiana, que agregam benefícios ao setor agropecuário, seja no controle de pragas e doenças ou na promoção do desenvolvimento vegetal. Entre os microrganismos promissores para formulação de bioinsumos podemos destacar o gênero Streptomyces devido a sua capacidade de produzir uma ampla gama de metabólitos secundários de potencial uso biotecnológico. Este trabalho teve como objetivo investigar a produção de metabólitos secundários pela linhagem Streptomyces noursei CAB-C 50 em meios de cultivo suplementados e não suplementados com o extrato de composto orgânico (ECO) produzido pela empresa Sítio Barreiras, Bahia - Brasil. Desta forma, foram utilizados os meios de cultura: ISP-2, ISP-2+ECO, M+G+ECO, M+G, M+ECO. Para estas condições foi aferido alterações de pH, análise de unidades formadoras de colônia (UFC.mL-1 ), alterações morfológicas com auxílio do microscópio eletrônico de varredura (MEV) e produção de biomassa. Posteriormente, foi obtido o extrato orgânico utilizando acetato de etila e os metabolitos secundários foram caracterizados por cromatografia de camada delgada (CCD) e identificados por cromatografia liquida de ultra eficiência (UHPLC) acoplado ao espectrofotômetro de massas (HRMS). Realizamos atividade de biocontrole de Fusarium oxysporum f. sp. cubense (Foc) com o sobrenadante e o extrato obtido após o crescimento de S. noursei CAB-C50. Inspecionamos o genoma de S. noursei CAB-C50 para detecção de grupos gênicos codificadores de metabolitos secundários com os bancos de dados antiSMASH 7.0 e BAGEL4. Verificamos que espécie S. noursei CAB-C 50 quando cultivadas em meio ISP-2 e ISP-2+ECO o pH final foi mais alcalino, os demais meios o pH final ficou mais próximo a neutralidade. Observou-se alteração na pigmentação após quatorze dias de crescimento, não sendo influenciado pela presença ou não de ECO. Nos meios de cultura ISP-2 e ISP- 2+ECO, a produção de massa seca foi maior que nos meios M+G+ECO, M+G e M+ECO. Os meios ISP-2, ISP-2+ECO e M+G+ECO apresentaram uma população de 106 UFC.mL -1 e os meios de cultivos M+G e M+ECO uma população de 104 UFC.mL -1. Essa diferença pode estar relacionada a maior produção dos esporos. A análise de MEV revelou hifas com padrão de enovelado e produção de esporos de aspecto espiculado em todas as condições investigadas. A maior taxa de inibição de Foc foram observadas nos extratos e sobrenadantes obtidos a partir do cultivo em meio ISP-2. A atividade do sobrenadante foi maior do que o extrato. Através das análises de UHPLC- HRMS foi possível identificar a presença de duas substâncias de interesse biotecnológico produzidas por S. noursei CAB-C 50, a anisomicina e um alcaloíde bisindólico. A predição de genes envolvidos na biossíntese de metabólitos por S. noursei CAB-C 50 revelou um grande arsenal para produção de compostos bioativos caracterizados e alguns ainda não descobertos. Streptomyces noursei CAB-C 50 é uma linhagem promissora para aplicação biotecnológica, tendo a capacidade para produção de uma gama de bioativos que precisa ser explorada e investigada em condições in vivo. Palavras-chave: Actinobactérias. Bioinsumos. Fusariose. Bananicultura.
The search for new bio-inputs that drive the consolidation of more sustainable agriculture is a growing reality on a global scale. Bioactive inputs or bio-inputs are plant products, of animal or microbial origin, which add benefits to the agricultural sector, whether in the control of pests and diseases or in the promotion of plant development. Among the promising microorganisms for the formulation of bio-inputs, we can highlight the genus Streptomyces due to its ability to produce a wide range of secondary metabolites of potential biotechnological use. This work aimed to investigate the production of secondary metabolites by the strain Streptomyces noursei CAB-C50 in culture media supplemented and not supplemented with organic compound extract (ECO) produced by the company Sítio Barreiras, Bahia - Brazil. Thus, the following culture media were used: ISP-2, ISP-2+ECO, M+G+ECO, M+G, M+ECO. For these conditions, changes in pH, analysis of colony forming units (CFU.mL-1), morphological changes with the aid of a scanning electron microscope (SEM), and biomass production were measured. Subsequently, the organic extract was obtained using ethyl acetate and the secondary metabolites were characterized by thin-layer chromatography (TLC) and identified by ultra-performance liquid chromatography (UHPLC) coupled to a mass spectrophotometer (HRMS). We carried out biocontrol activities for Fusarium oxysporum f. sp. cubense (Foc) with the supernatant and the extract obtained after the growth of S. noursei CAB-C50. Was inspected the genome of S. noursei CAB-C50 for the detection of gene clusters encoding secondary metabolites with the antiSMASH 7.0 and BAGEL4 databases. We verified that the species S. noursei CAB-C50 when cultivated in ISP-2 and ISP-2+ECO medium the final pH was more alkaline, in the other media the final pH was closer to neutrality. A change in pigmentation was observed after fourteen days of growth, not being influenced by the presence or absence of ECO. The dry mass production was higher in the ISP-2 and ISP-2+ECO culture media than in the M+G+ECO, M+G, and M+ECO media. The ISP-2, ISP-2+ECO, and M+G+ECO media had a population of 106 CFU.mL-1 and the M+G and M+ECO culture media had a population of 10 4 CFU.mL-1. This difference may be related to the greater production of spores. SEM analysis revealed hyphae with a coiled pattern and the production of spiculated spores in all investigated conditions. The highest Foc inhibition rates were observed in extracts and supernatants obtained from cultivation in ISP-2 medium. The activity of the supernatant was greater than that of the extract. Through the UHPLC-HRMS analyses, it was possible to identify the presence of two substances of biotechnological interest produced by S. noursei CAB-C50, anisomycin and a bisindole alkaloid. The prediction of genes involved in the biosynthesis of metabolites by S. noursei CAB-C50 revealed a large arsenal for the production of characterized bioactive compounds and some are still undiscovered. Streptomyces noursei CAB-C50 is a promising strain for biotechnological application, having the capacity to produce a range of bioactive that needs to be explored and investigated under in vivo conditions. Keywords: Actinobacteria. Bioinsumes. Fusariosis. Banana farming
The search for new bio-inputs that drive the consolidation of more sustainable agriculture is a growing reality on a global scale. Bioactive inputs or bio-inputs are plant products, of animal or microbial origin, which add benefits to the agricultural sector, whether in the control of pests and diseases or in the promotion of plant development. Among the promising microorganisms for the formulation of bio-inputs, we can highlight the genus Streptomyces due to its ability to produce a wide range of secondary metabolites of potential biotechnological use. This work aimed to investigate the production of secondary metabolites by the strain Streptomyces noursei CAB-C50 in culture media supplemented and not supplemented with organic compound extract (ECO) produced by the company Sítio Barreiras, Bahia - Brazil. Thus, the following culture media were used: ISP-2, ISP-2+ECO, M+G+ECO, M+G, M+ECO. For these conditions, changes in pH, analysis of colony forming units (CFU.mL-1), morphological changes with the aid of a scanning electron microscope (SEM), and biomass production were measured. Subsequently, the organic extract was obtained using ethyl acetate and the secondary metabolites were characterized by thin-layer chromatography (TLC) and identified by ultra-performance liquid chromatography (UHPLC) coupled to a mass spectrophotometer (HRMS). We carried out biocontrol activities for Fusarium oxysporum f. sp. cubense (Foc) with the supernatant and the extract obtained after the growth of S. noursei CAB-C50. Was inspected the genome of S. noursei CAB-C50 for the detection of gene clusters encoding secondary metabolites with the antiSMASH 7.0 and BAGEL4 databases. We verified that the species S. noursei CAB-C50 when cultivated in ISP-2 and ISP-2+ECO medium the final pH was more alkaline, in the other media the final pH was closer to neutrality. A change in pigmentation was observed after fourteen days of growth, not being influenced by the presence or absence of ECO. The dry mass production was higher in the ISP-2 and ISP-2+ECO culture media than in the M+G+ECO, M+G, and M+ECO media. The ISP-2, ISP-2+ECO, and M+G+ECO media had a population of 106 CFU.mL-1 and the M+G and M+ECO culture media had a population of 10 4 CFU.mL-1. This difference may be related to the greater production of spores. SEM analysis revealed hyphae with a coiled pattern and the production of spiculated spores in all investigated conditions. The highest Foc inhibition rates were observed in extracts and supernatants obtained from cultivation in ISP-2 medium. The activity of the supernatant was greater than that of the extract. Through the UHPLC-HRMS analyses, it was possible to identify the presence of two substances of biotechnological interest produced by S. noursei CAB-C50, anisomycin and a bisindole alkaloid. The prediction of genes involved in the biosynthesis of metabolites by S. noursei CAB-C50 revealed a large arsenal for the production of characterized bioactive compounds and some are still undiscovered. Streptomyces noursei CAB-C50 is a promising strain for biotechnological application, having the capacity to produce a range of bioactive that needs to be explored and investigated under in vivo conditions. Keywords: Actinobacteria. Bioinsumes. Fusariosis. Banana farming
Descrição
Palavras-chave
Actinobactérias, Fusariose, Banana - Cultivo
Citação
ASSIS, Maria Eduarda Leandro. Análise de condições in vitro para produção de metabólitos secundários por Streptomyces noursei CAB-C 50. 2023. 66 f. Dissertação (Mestrado em Microbiologia Agrícola) - Universidade Federal de Viçosa, Viçosa. 2023.