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Characterisation of the Fucose and Rhamnose Utilising Bacterial Microcompartment system in Clostridium phytofermentans

Alrobaish, Shouaa A (2019) Characterisation of the Fucose and Rhamnose Utilising Bacterial Microcompartment system in Clostridium phytofermentans. Doctor of Philosophy (PhD) thesis, University of Kent,. (KAR id:77738)

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Abstract

Bacterial microcompartments (BMCs) are cytoplasmic polyhedral organelles that are composed of a semi-permeable proteinaceous shell made from a number of different proteins, which encapsulate a specific metabolic process. Recent studies have revealed that the basic functions of these compartments are not only to increase the metabolic efficiency of the enclosed enzymes but also to protect the cell from toxic intermediates. BMCs are found in about 20 % of all bacteria and spread widely across the kingdom. One of the bacteria that contains operons encoding for such protein compartments is Clostridium phytofermentans, a bacterium that was originally isolated from forest soil and can directly convert plant biomass into biofuels.

The bacterial genome of C. phytofermentans has three BMC-encoding loci, one of which is a 13-gene operon that houses the genetic information for a predicted fucose/rhamnose utilisation system. Within this operon, six genes encode for shell proteins, which form the BMCs outer structure, and the other seven genes encode for metabolic enzymes that are responsible for converting fucose/rhamnose into ethanol, propanol, and propionate. Based on this genome analyses, research has been undertaken to clone the various BMC-associated genes of the C. phytofermentans fucose/rhamnose system in order to generate a recombinant BMC in E. coli. This was attempted firstly by the individual cloning and recombinant expression in E. coli of the genes that encode the shell proteins. This approach also allowed the characterisation of the shell proteins and determine their ability to form functional BMCs. Secondly, engineer empty BMCs from the six shell proteins and investigate the minimum number of genes required for BMC formation.

One of the shell proteins characterised in this study, Cphy_1186 (PduT-like), was crystallised and had its structured determined by X-ray crystallography. The protein was found to have a trimeric arrangement of subunits, each of which contains a double-BMC-domain consistent with it belonging to the BMC-T class. The trimer forms a flat approximately hexagonally shaped disc with a central pore that is suitable for binding a 4Fe-4S cluster. The pore is lined with six cysteine residues, more than enough for Fe-S centre formation

The structure of the shell protein Cphy_1182 (PduA-like) was also solved. The protein contains a single-BMC-domain within each subunit and forms a hexamers, thereby belonging to the BMC-H class. Overproduction of Cphy_1182 by itself results in the formation of filaments or nanotubes in the bacterial cell.

The first attempt at engineering empty metabolosomes from the six shell proteins that are involved in the formation of the C. phytofermentans metabolosome is presented. Although ultimately this was unsuccessful the associated research has given greater insight into the structure/function relationship between the individual components.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Warren, Professor Martin
Thesis advisor: Smales, Professor Mark
Uncontrolled keywords: Bacterial microcompartment
Divisions: Divisions > Division of Natural Sciences > Biosciences
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 23 Oct 2019 09:10 UTC
Last Modified: 08 Dec 2022 16:21 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/77738 (The current URI for this page, for reference purposes)
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