| Popis: |
Natural products with antimicrobial activity have played an important role in the treatment of infection since their discovery. The increasing emergence of pathogens resistant to multiple antibiotics has raised awareness of the urgent need for novel antibiotics. Soil microorganisms are the major source of antibiotics and Actinobacteria in particular have an impressive capacity for production of diverse bioactive secondary metabolites. However, culture-independent studies have shown a greater microbial diversity present in soil with potential for novel chemical structures and these can be explored further using metagenomic approaches capturing genes without the need to cultivate the host. Different metagenomic tools were used to study and explore microbial secondary metabolite diversity in soil. In particular, amplicon sequencing of 16S rRNA gene, NRPS and PKS biosynthetic genes allowed the identification of novel potential phylogenetic drivers of secondary metabolite diversity in the less characterized phyla Verrucomicrobia and Bacteroidetes and potential geographic hotspots harbouring unique biosynthetic diversity such as Antarctica and Cuba. The exploitation of these hotspots presented some bottlenecks in the form of DNA extraction efficiency, library creation, screening and heterologous expression. These were overcome by comparative analysis of different eDNA extraction methods to optimise fragment size and purity combined with development of new cloning tools for both DNA capture and expression. Modification of the microbial community through the amendment of the soil with chitin, highlighted the beneficial effect of microbial enrichment allowing a higher recovery of eDNA and higher detection of the biosynthetic gene of interest related to secondary metabolite production. Further additions were made to the metagenomic molecular toolbox in the form of BAC vectors (pBCaBAC and pBCkBAC) which were tested with suitable heterologous host systems (Streptomyces sp. and the engineered Pseudomonas putida species) potentially facilitating heterologous expression. In conclusion this is the first study to identify the drivers of microbial secondary metabolite diversity in situ and provided a comparative analysis of a range of diverse soil types. This approach paired with new developments in metagenomic technologies will make a substantial contribution to improving the likelihood for discovery and exploitation of new drugs for treating multi-resistant pathogenic bacteria. |
