AbstractEliminating carbon catabolite repression (CCR) from microbial cell factories is desirable for industrial fermentation of sugars derived from mixed-carbohydrate feedstocks such as lignocellulosic biomass. Co-utilisation of different sugars in the substrate would be beneficial for process simplification, facilitating complete utilization of all available sugars and potentially improving product yield. The work in this thesis has furthered our understanding on how CCR is regulated in Parageobacillus thermoglucosidasius DSM 2542, a thermophilic strain of industrial interest. Despite suggestions that thermophiles may not exhibit CCR, preliminary studies showed that glucose is a repressor of xylose utilisation by P. thermoglucosidasius DSM 2542 under both aerobic and anaerobic conditions. However, unlike in some organisms, repression was only partial. Physiological and molecular studies under fermentative conditions revealed a loosely controlled CCR pattern in DSM 2542. Furthermore, bioinformatic analysis showed that DSM 2542 encoded all the components of a typical firmicute CCR system. Based on a bioinformatic analysis it was proposed that CCR of xylose utilisation in P. thermoglucosidasius DSM 2542 may occur primarily through regulation of expression of the pentose transporters.
Subsequently, based on the evidence for the operation of a classical firmicute CCR system, various strategies were used to remove CCR from P. thermoglucosidasius DSM 2542. The first approach was site-directed mutagenesis of the HPr and Crh proteins at their Ser46 regulatory phosphorylation sites, which had been reported to alleviate CCR in B. subtilis. However, despite DSM 2542 having genes encoding for all of the key components of a typical CCR regulatory system in low-GC Gram-positive bacteria, the HPr-S46A and Crh-S46A mutations have yielded some unexpected results and failed to produce the desired phenotype. While the Crh-S46A mutation had no obvious fitness effect in DSM 2542, the HPr-S46A mutation had a negative impact on cell growth and sugar utilisation under fermentative conditions. Intriguingly, it was not possible to generate a double mutant containing both mutations.
The second attempt involved an adaptive evolution approach using the non-metabolizable glucose analogue, 2-deoxy-D-glucose (2-DG). Cells cannot grow on 2-DG, and the presence of 2-DG inhibited the metabolism of xylose, presumably by activation of CCR. This was effective as a selection pressure to remove CCR from DSM 2542. Two selection strategies were applied to optimise the phenotypes of evolved strains. Single-nucleotide polymorphisms (SNPs) identified by genome sequencing followed by complementation analysis revealed key mutations affecting the ribose operon repressor (RbsR) and PTS components EI and EIIBGlu. These results suggest that the CCR in P. thermoglucosidasius DSM 2542, or possibly a wider community of Gram-positive bacteria and possibly other similar bacteria might be more complex than the picture currently presented in the literature.
Detailed bioinformatic analysis revealed that, despite being able to grow on xylose as a sole carbon source, P. thermoglucosidasius DSM 2542 did not encode a dedicated xylose transporter. Through a combination of bioinformatics and experimentation, alternative genes, alternative genes which might contribute to xylose transport have been identified. It has been suggested that pentose transporters in DSM 2542 or even other microbes have a wide substrate-specificity.
|Date of Award||17 Jan 2022|
|Supervisor||David Leak (Supervisor), Albert Bolhuis (Supervisor) & Richard van Kranenburg (Supervisor)|
- catabolite repression
- Parageobacillus thermoglucosidasius
- metabolic engineering