Habilidades Intrapersonales

One of the main issues with antibiotic discovery expeditions based on natural products is the probability of rediscovering known metabolites. This requires introducing one or more elements of novelty into the screening endeavour, which in turn requires an understanding of the screening approaches employed in the past. Most of the past screening efforts involved isolating, among bacteria, strains belonging to the genus Streptomyces from a variety of sources, growing them in nutrient-rich media, and testing the resulting cultures for the ability to inhibit growth of one or more pathogens relevant at that time. The compounds responsible for bioactivity were then purified by bioassay-based fractionation, characterized for their antibacterial properties and structurally elucidated. This approach introduced a number of biases, namely: i) the type of microorganisms isolated and screened; ii) the conditions under which those strains were grown; iii) the type of pathogens that were screened against; and iv) the requirement for growth inhibition. Overall, this approach favoured the discovery of compounds that were potent and/or were produced in high amounts. Therefore, an element of novelty could address any of the above biases. For example, it could involve the use of previously unexplored bacterial group; or it could involve growing known strains under conditions that trigger the expression of the several biosynthetic gene clusters (BGCs) present in their genomes and not expressed under routine cultivation conditions; or it could involve screening against pathogens that are of medical relevance today but not considered important few decades ago (e.g., Acinetobacter baumanii or C. difficile); or it could employ assays more sensitive than growth inhibition of a standard strain.

An alternative, paradigm-shifting approach is the so called genome mining, in which genomic information is used as a starting point to identify metabolites predicted from the organization of the corresponding BCGs. If the strain does not normally produce the predicted metabolite, it can be engineered to do so by manipulating its regulatory circuits or the cluster can be moved and expressed in a heterologous host. While the genomic mining approach simultaneously addresses previous screening biases and it represents an enormous opportunity to unveil novel chemistry, awakening silent clusters is at the moment an ineffective trial and

error procedure. For example, the most studied organism among actinomycetes, Streptomyces

coelicolor is slowly delivering new chemical structures after the identification of the corresponding BGCs in its genome and thorough analyses of the metabolite profiles by different laboratories under a variety of conditions.

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In the pursuit of my PhD program, it was decided to introduce an element of novelty by exploring different strains, namely a taxonomic group that had not witnessed extensive analyses of its secondary metabolites. Taxonomic diversity can be seen as a surrogate for chemical diversity (Jaspars and Challis, 2014; Kim et al., 2005). The main idea is that organisms that have been subjected to different evolutionary pressures have developed unique biology to survive and secondary metabolites are also part of their biology. While horizontal gene transfer can blur this picture and lead to the transfer of BGCs between unrelated bacterial taxa, as it does occasionally with housekeeping genes, one would expect that, on average, the BGCs present in a given taxon would be more related to each other than to BGCs present in an unrelated taxon. Thus, there is a higher probability of discovering novel BGCs, and hence novel chemistry, by exploring novel taxa.

Though we are aware of the universe of uncharacterized bacteria awaiting to be studied, we have also observed that production of secondary metabolites is not distributed equally amongst all species, with some groups – not surprisingly, those with large genomes – more prolific than others. Therefore, when endeavouring in a new expedition for bioactive natural products from new taxonomical groups it is important to select a taxon with a high potential to produce such compounds in order not to increase the probability of finding the desired compounds with a reasonable effort. A good example of a taxonomic group well known for the

production of secondary metabolites is the phylum Actinobacteria, which stands out as being

responsible for almost 9,000 molecules out of the 17,000 produced by microorganisms (Bull, 2004; Goodfellow and Fiedler, 2010). Among them is the genus Streptomyces which is the most studied and the one that has contributed with the largest amount of compounds (Bull, 2004; Goodfellow and Fiedler, 2010; Lazzarini et al., 2000; Pozzi et al., 2011; Stackebrandt et al., 1997; Tamura et al., 2009; Ventura et al., 2007; Watve et al., 2001). Previous reports have stressed the use of underexplored group of actinomycetes as an effective strategy to discover novel chemistry, an approach that has been recently reinvigorated by the results obtained from exploring unknown marine actinomycetes (Subramani and Aalbersberg, 2012). Moreover, drug discovery expeditions with new taxonomic groups have more than the obvious role of finding new drug candidates. The work goes beyond the screening itself, since it is also a characterization of the producer strains. For example, a significant amount of information is generated by simply recording which known metabolites are produced by the screened strains. This information will contribute to the better understanding of the chosen group, which can become an important factor for the success of posterior screening campaigns. In particular, this

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work was designed to assess the metabolic potential of the genus Actinoallomurus while enriching the knowledge on this recently described group.