Metabolomic study of marine Streptomyces sp.: Secondary metabolites and the production of potential anticancer compounds.

Luciana Costa Furtado,Gabriel Padilla,Marcelo J Pena Ferreira,Karen Velasco-Alzate,Leticia V Costa-Lotufo,Vijai Gupta,Leandro M Garrido,Vida M B Leite,Paula C Jimenez,Norberto P Lopes,Anelize Bauermeister,Marcelo M P Tangerina

doi:10.1371/journal.pone.0244385

Luciana Costa Furtado, Gabriel Padilla + Show 10 more

Open Access

https://doi.org/10.1371/journal.pone.0244385

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Abstract

Resorting to a One Strain Many Compounds (OSMAC) approach, the marine Streptomyces sp. BRB081 strain was grown in six different media settings over 1, 2, 3 or 7 days. Extractions of mycelium and broth were conducted separately for each media and cultivation period by sonication using methanol/acetone 1:1 and agitation with ethyl acetate, respectively. All methanol/acetone and ethyl acetate crude extracts were analysed by HPLC-MS/MS and data treatment was performed through GNPS platform using MZmine 2 software. In parallel, the genome was sequenced, assembled and mined to search for biosynthetic gene clusters (BGC) of secondary metabolites using the AntiSMASH 5.0 software. Spectral library search tool allowed the annotation of desferrioxamines, fatty acid amides, diketopiperazines, xanthurenic acid and, remarkably, the cyclic octapeptides surugamides. Molecular network analysis allowed the observation of the surugamides cluster, where surugamide A and the protonated molecule corresponding to the B-E isomers, as well as two potentially new analogues, were detected. Data treatment through MZmine 2 software allowed to distinguish that the largest amount of surugamides was obtained by cultivating BRB081 in SCB medium during 7 days and extraction of culture broth. Using the same data treatment, a chemical barcode was created for easy visualization and comparison of the metabolites produced overtime in all media. By genome mining of BRB081 four regions of biosynthetic gene clusters of secondary metabolites were detected supporting the metabolic data. Cytotoxic evaluation of all crude extracts using MTT assay revealed the highest bioactivity was also observed for extracts obtained in the optimal conditions as those for surugamides production, suggesting these to be the main active compounds herein. This method allowed the identification of compounds in the crude extracts and guided the selection of best conditions for production of bioactive compounds.

Highlights

The oceans cover 70% of the surface of the Earth and harbors a large portion of the planet’s biodiversity [1], which is further connected to a great molecular diversity of natural products found in animals, algae and microorganisms [2]
Addition, this region has 7% of the gene content (ALG65317.1) related to the biosynthesis of WS9326 (MIBiG BGC0001297), which has a cinnamoyl moiety identical to that of pepticinnamin E [59]. It was found an additional 5% of the genes (ALG65334.1, ALG65335.1) described for WS9326 biosynthesis in region 3, as well as an ORF encoding cytochrome p450, reported in most diketopiperazine biosynthetic gene clusters (BGC) catalyzing peptide pathways, and an ORF encoding a protein with the tryptophan 2,3-dioxigenase activity domain, found for example in the maremycin biosynthetic pathway [60]. These results indicate that the candidate cluster for DKP biosynthesis by BRB081 may be between regions 3 and 4
Metabolomics strategies and biological activity assays guide bioprospecting towards new bioactive compounds in natural products research

Summary

Introduction

The oceans cover 70% of the surface of the Earth and harbors a large portion of the planet’s biodiversity [1], which is further connected to a great molecular diversity of natural products found in animals, algae and microorganisms [2] Such environment presents extreme conditions, e.g. high pressure, high salinity, changes in temperature, limited nutrient availability [3], among others, veering marine organisms to adapt to such conditions by developing unusual and, interesting metabolic pathways, which may provide complex chemical structures of relevance for biotechnological and pharmaceutical industries. Among all the living sea creatures, microorganisms stand out for their capacity to thrive in several marine environments, from the water surface to the lower and abyssal depths; from coastal to offshore regions; from open waters to coral reefs [4] Such a large geographical distribution throughout the oceans attests for the adaptability skills of microorganisms, merited by their genetic plasticity and rapid replication. Nearly 70% of small molecules that are utilized as medicines are derived or inspired in natural products produced by bacteria, filamentous actinobacteria [7]

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