Discovery and characterisation of carbohydrate-active enzyme associated domains of unknown function via metagenomics

Jackson, Mingaile (2024) Discovery and characterisation of carbohydrate-active enzyme associated domains of unknown function via metagenomics. Doctoral thesis, Northumbria University.

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Abstract

Facing the issue of climate change countries and companies are motivated and innovating to fight against it. A crucial step in this fight are Net Zero commitments which aim to fully stop carbon dioxide release in commercial processes. One innovation to achieve this is the use of biomass, which can be degraded by Carbohydrate Active Enzymes (CAZymes) to produce products to be used as chemical catalysts, food additives or for biofuel production.

CAZymes are multi modular enzymes, where each module has its own distinct structure and function. To fully understand the process involving these enzymes, it is important to figure out the function of the Domains of Unknown Function (DUFs), which make up over 20% of all protein domains currently. DUFs can play important roles in the biorefinery process as they may be carbohydrate binding modules and assists in the degradation process or may be undiscovered enzymes that degrade biomass efficiently. It is therefore important to discover DUF activities to improve the biorefinery process.

Using metagenomic mining and bioinformatics tools forty-one target DUFs were identified, via architectural analysis, to be associated with degradative CAZymes in order to assist in or carry out degradative activities. They were then cloned from metagenomes, heterologously expressed and analysed via microarrays and biochemical assays in an attempt to functionally characterise the target DUFs.

In summary, the DUF bioinformatics screening methodology allowed for targeted DUFs to be selected for the study. This resulted in some DUFs being functionally characterised and some having functional predictions that needs to be further investigated. Three DUFs families (DUF1080 clone 5f1, DUF1573 clone d76 and DUF4979 clone 5da) were discovered to be novel alginate lyase type enzymes, other DUF clones (DUF1080 clones 5f1, bc9, 27f and DUF4980 clone 040) were identified to have potential PGA, pectin, amylopectin and inulin degradation, DUF5011 clone 3a3 showed potential chitin binding activity. The discovery of DUF functions allows for their production and use in appropriate commercial sectors such as biofuel production, the pharmaceutical and chemical industries and in the gut microbiome.

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