Application of Metagenomics for Discovery of Natural Products and Virophages
Type of DegreeDissertation
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Metagenomics provided a powerful insight into an as-yet-uncultured bacterial community in different environments through construction of clone libraries and shotgun sequencing, therefore presenting potential in drug and novel microbe discovery. However, some limits such as insert size of BAC/Plasmid vector and length of sequencing reads are still big challenges for metagenomic study. In chapter II, we focus on identification of novel genomes of virophages, circular dsDNA viruses that infect giant DNA viruses, from a metagenomic DNA database of yellow stone lake. Three novel virophage genomes from Yellowstone Lake microbial assemblages were found, indicating a potentially high diversity of virophages from Yellowstone Lake in the photic zone or in Lake Floor hydrothermal vents. In chapter III, a metagenomic fosmid library constructed using DNA sample from a petroleum reservoir off the coast of Norway was screened for novel thermal-stable carbohydrate degrading enzymes. Comparison of sequencing results generated from shotgun metagenomic sequencing and fosmid library sequencing was performed as well. In the final chapter, another ~110 kb average insert sizes from soil metagenomic DNA (Cullars Rotation, Auburn, AL) achieved using a broad host range shuttle BAC vector, pSmartBAC-S was applied for discovery and expression of novel type I polyketide synthase (PKS) pathways and other secondary metabolite pathways, which is the largest soil metagenomic library so far and able to harbor large-size of biosynthetic pathway. The two approaches PCR and macroarray screening targeted in exploitation of BAC clones with type I PKS pathways. An additional approach of in silico screening combined with the next generation sequencing (NGS) was aimed to discover diverse secondary metabolite pathways. Identified BAC DNA with PKS pathways were then transformed into a heterologous expression host E.coli BTRA for further screening of produced novel polyketide, followed by multiple antibiotic assays.