BIOPROSPECTING PLANCTOMYCETOTA FOR CARBOHYDRATE HYDROLYSIS POTENTIAL THROUGH CULTURE-DEPENDENT AND CULTURE-INDEPENDENT METHODS

The Planctomycetota phylum has been identified as a group of bacteria with significant carbohydrate hydrolytic potential, making them one of key candidates for applications in diverse biotechnology sectors. However, comprehensive studies on their diversity, ecology, and enzymatic capacities remain limited. This PhD thesis aims to characterise the carbohydrolytic potential of Planctomycetota through an integrated approach that combines phylogenetic, ecological, and functional analyses. To achieve a more robust understanding, both culture-dependent and culture-independent approaches were employed in a complementary manner.
Initially, a comparative genomic analysis was conducted to examine the encoded carbohydrate-active enzyme (CAZyme) families across planctomycetotal genomes from different phylogenetic lineages, which were reconstructed from genomic and metagenomic studies spanning a wide range of habitats. This characterisation revealed that Planctomycetota encompass a wide variety of the currently known CAZyme diversity assigned to glycoside hydrolase families and that many members encode a versatile enzymatic machinery towards complex carbohydrate degradation, including lignocellulose. As a result, three planctomycetotal orders were identified as high priority targets for future bioprospecting efforts. Subsequently, diversity of Planctomycetota across various environmental niches with high loads of organic matter (so-called biomass-rich environments) was examined using 16S rRNA gene sequencing, revealing the presence of diverse planctomycetotal communities represented by distinct phylogenetic clusters, especially in anaerobic digester and termite gut communities.
In parallel, isolation studies were conducted to obtain representative strains of Planctomycetota from the selected biomass-rich environments e.g., marine sediment and anaerobic digestion reactor. A robust culture-based approach allowed for the successful isolation of several strains, which were subsequently phenotypically and genotypically characterised using a suite of molecular and standard microbiology techniques. Novel species from digesters were identified as mixotrophs, highlighting their potential capacity for glycoprotein scavenging. The next assessment of enzymatic capacities centred on evaluating the marine isolated strains, with a particular focus on their ability to degrade complex carbohydrates. Microbial growth assays were performed, and enzyme crude extracts were obtained to evaluate the utilisation of predicted carbohydrates based on the CAZyme gene content. As a result, the non-canonical utilisation of terrestrial plant polysaccharides highlights the potential of marine strains for future discovery of new enzymatic mechanisms.
Overall, this thesis characterises Planctomycetota carbohydrolytic potential, linking their diversity and metabolic capabilities, enhancing understanding of their roles in biomass-rich environments and potential industrial applications.