Chemical Elicitation Strategies Combined with Mass Spectrometry Approaches for the Discovery of Microbial Metabolites

vol. 1, 2019 - 117633
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Resumo

Natural products from microorganisms have a crucial role in modern drug discovery. Genome sequencing of several lineages of microorganisms reveal that the number of biosynthetic genes in bacteria and fungi clearly outnumber the already identified secondary metabolites of these organisms, indicating that there are still many novel natural products to be discovered from these sources1. One possible reason for this observation is that only some biosynthetic pathway genes are expressed in standard laboratory culture conditions, and that other genes are silenced or barely expressed without a particular stimulus, such as specific nutrients, environmental factors, and signaling compounds2. Therefore, to access a wider reservoir of potentially bioactive metabolites, the biosynthesis of these substances needs to be induced through the manipulation of nutritional or environmental factors. One method of triggering secondary metabolite biosynthesis in bacteria is through the influence of stress conditions by chemical elicitation, adding defense metabolites of microorganisms (antibiotics in subinhibitory concentrations) to microbial cultures3. For this experiment, endophytic lineages of the actinobacteria Streptomyces sp. were isolated from the plant Tithonia diversifolia. The isolated lineages were cultivated in a DS solid culture incorporated with the antibiotics kanamycin A or apramycin in a minimum inhibitory concentration (MIC). After a 10-day inoculation period, microextraction with methanol was performed and the metabolic profile of the antibiotic incorporated cultures were analyzed using high performance liquid chromatography (HPLC) and compared to the profiles of the bacterial cultures without chemical elicitation. The HPLC chromatogram of lineages Rtd5 and Rtd8 incorporated with apramycin and Rtd2 incorporated with kanamycin showed new and more intense peaks compared to the chromatograms of the same lineages without antibiotic treatment. These lineages were selected for further analysis by mass spectrometry (MS). The great quantity of MS data was organized in a molecular network, and MS/MS spectra libraries were used to identify known substances in the samples. Statistical analysis of the replicates and the molecular network results (Figure 1) both showed a clear separation of metabolite clusters present in the bacteria cultures with antibiotic treatment from those without. This study demonstrates that chemical elicitation using antibiotics can stimulate the biosynthesis of microorganisms leading to the production of substances either not observed or produced in much lesser quantities when elicitation is not introduced. Furthermore, MS-based molecular networking is a powerful tool to identify known molecules and aid in the discovery of novel natural products.

References:
1. Walsh, C. T.; Fischbach, M. A., Natural products version 2.0: connecting genes to molecules. Journal of the American Chemical Society 2010, 132 (8), 2469-2493.
2. Challis, G. L. Mining microbial products biosynthesis in microorganisms. Organic & Biomolecular Chemistry 2009, 7(9), 1753-1760.
3. Pettit, R. K. Small-molecule elicitation of microbial secondary metabolites. Microbial Biotechnology 2011, 4 (4), 471-478.

Instituições
  • 1 Universidade Federal do Rio de Janeiro
  • 2 FCFRP (Faculdade de Ciências Farmacêuticas de Ribeirão Preto) / Universidade de São Paulo
Palavras-chave
chemical ecology
Streptomyces
Molecular Networking
Mass Spectrometry
Chemical Elicitation