CORNISH, KATY,ALEXANDRIA,SOPHIE (2022) Searching for novel enzymes from microbial dark matter. Doctoral thesis, Durham University.
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Author-imposed embargo until 18 November 2023.
The vast majority of inhabited environments on Earth are dominated by uncultivated microbes, described as ‘microbial dark matter’. Metagenomic analyses of various ecological niches have unearthed an abundance of genomes from unculturable organisms that have evaded polymerase chain reaction (PCR)-based and cultivation-dependent isolation efforts. Many such organisms fall within a recently uncovered expanse of the bacterial domain, known as the Candidate Phyla Radiation (CPR), which is thought to harbour a third of all biodiversity within the tree of life. The genomes present within this mysterious microbial world present a major source of uncharted genetic diversity and are untapped sources of molecular tools for biological research, encoding swathes of novel proteins with innovation potential.
The Virus-X consortium endeavoured to probe the sequence diversity of extreme environments through metagenomics and identify commercially valuable enzymes. From the pool of over 50 million genes discovered during the Virus-X project, 18 from the CPR and five from a newly discovered giant phage dubbed the Ubervirus were selected as targets of particular interest. From 23 cloned targets, 18 proteins were successfully expressed in trials, leading to large-scale soluble expression of 12 and successful purification of 10 proteins. 9 of these targets were characterised through analytical size exclusion chromatography, mass spectrometry, and thermal shift analysis. Promising crystallisation conditions have been identified for four targets, leading to novel X-ray crystal structures for two CPR enzymes.
The structure of a DNA processing enzyme, CPR-DprA, is reported to a resolution of 2.10 Å (R/Rfree 0.20/0.23), showing a core domain formed from an extended Rossmann fold. Also presented are three crystal structures of a hypothetical protein, CPR-C4, to a maximum resolution of 2.25 Å (R/Rfree 0.18/0.23). Through remarkable structural homology to human vasohibin proteins, CPR-C4 was characterised as a cysteine protease utilising a noncanonical cysteine-histidine-leucine(carbonyl) catalytic triad, with protease activity confirmed using fluorescence-based assays. The structural and functional similarities between CPR-C4 and the human vasohibins point to an evolutionary relationship undetectable at the sequence level, which is addressed through phylogenetic analysis. The production and characterisation of a DnaK/ClpB bi-chaperone system from the CPR, with tangible biotechnology applications, is also reported, including preliminary cryo-electron microscopy analysis of the hexameric CPR-ClpB disaggregase.
Along with laying the groundwork for future commercialisation, the investigation of these CPR proteins takes strides towards addressing the substantial gaps in our knowledge of this little understood, yet pervasive, branch of the tree of life.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Faculty and Department:||Faculty of Science > Chemistry, Department of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||22 Nov 2022 13:26|