Assessment of the biosynthesis potential of microbial dark matter by cultivation independent techniques

Aileen Ute Geers*

*Corresponding author for this work

Research output: Book/ReportPh.D. thesis

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The number of reported infections with antibiotic resistant pathogens and their associated deaths have increased dramatically over the last two decades. Limiting the use of anti- infective agents will not be sufficient to combat this looming crisis, and we are therefore in dire need to discover and extract novel bioactive compounds. Secondary metabolites produced by isolated microorganisms have shown to have a high potential to act as antibiotics and other modern day medicines. While rediscovery rates of known compounds from cultivated microorganisms steadily increased, high-throughput sequencing of environmental microbiomes has revealed that the majority of environmental strains remain uncultured. Moreover, with the use of culture-independent techniques it has become increasingly clear that these strains, the ‘microbial dark matter’, harbour an immense genetic potential to synthesise chemical novelty. Yet tapping into this potential is not straightforward and little is still known on how this potential is distributed in the environment.
In this PhD project, we set out to assess the biosynthesis potential of the microbial dark matter, reviewing published literature, and performing our own culture-independent cross- system study. Furthermore, we evaluated different methods for the characterisation and exploitation of this potential.
In all studies investigating the biosynthetic potential of environmental microbiomes, the common denominator is a very high percentage of around 80 to 99 % of functional domains or biosynthetic gene cluster (BGC) sequences being novel. We found the overall biosynthetic domain diversity to be affected by a range of different habitat specific environmental factors, such as pH in soil and salinity in aquatic biomes. Across ecosystems, microbiomes with a high bacterial diversity had the highest biosynthetic richness and diversity. Interestingly, each ecosystem had a unique composition of biosynthetic genes, while at the same time being highly affected by dispersal limitation. Therefore, it is vital to sample as many different ecosystems as possible. Accordingly, we investigated the secondary metabolite biosynthesis genes of permafrost microbiomes and found that even these extreme environments harbour a large biosynthetic diversity, which decreased and shifted with the transition from active layer soil to frozen permafrost soil.

While these culture-independent methods have greatly expanded our knowledge on the biosynthesis of SM, they are not without complications and biases. We found the majority of primers for targeted amplicon sequencing have a very low sensitivity for detecting functional domains in silico. This observation was further confirmed by assessing the sensitivity and precision of the most commonly used primer pair for detecting functional domains in the genomic DNA of two proficient secondary metabolite producer microorganisms. Moreover, differences in the bioinformatics processing for the generation of operational biosynthetic units (OBUs) can lead to notably different total richness estimates. Still, targeted amplicon sequencing remains useful for the comparison of a large number of samples. In contrast, shotgun metagenomic sequencing can be used to give a broader picture of the present BGCs in selected samples. Challenges with the assembly of repeat sequences and large BGCs, can be overcome by using long read sequencing, further increasing the potential to directly or indirectly use environmental DNA for the discovery and expression of novel BGCs. Alternatively, innovative cultivation techniques, as for example the iChip, hold the promise of bringing the microbial dark matter into the laboratory for the discovery of novel compounds. In conclusion, this thesis will guide future research efforts selecting environments and methods for the discovery of novel bioactive compounds.
Original languageEnglish
Place of PublicationKgs. Lyngby, Denmark
PublisherDTU Bioengineering
Number of pages157
Publication statusPublished - 2022


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