Abstract
This thesis aims to understand the regulation of plant biomass degradation in promising cell factory candidates such as Aspergillus vadensis and Penicillium subrubescens and capitalize on this knowledge to develop them into efficient fungal biofactories for diverse biotechnological applications.
In Chapter 1, the background and the objectives of this thesis are
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described. In Chapter 2, several well-used fungal cell factories for heterologous protein production are described, and approaches employed to improve heterologous protein production in filamentous fungi are reviewed. A. vadensis, a close relative of A. niger, has been suggested as a more favorable alternative for recombinant protein production as it does not acidify the culture medium and produces very low levels of extracellular proteases. P. subrubescens is a promising candidate for industrial applications as its plant cell wall-degrading enzyme production levels and saccharification abilities are similar to that of the well-established industrial species A. niger. Understanding the molecular mechanisms and regulatory interactions governing protein/enzyme production in A. vadensis and P. subrubescens is imperative for their potential application at the industrial level.
Results shown in Chapter 3, revealed that the non-amylolytic nature of A. vadensis is due to the non-function of amyR promoter. However, the non-proteolytic nature of A. vadensis is still not clear. It seems not only due to the non-function of prtT, as its promoter showed no functional role in A. vadensis, but also the non-function of other putative proteolytic TF. Regarding the understanding of the regulatory processes involved in plant biomass degradation in this species, in Chapter 4 we showed the influence of the (hemi-)cellulolytic regulator XlnR and the arabinanolytic regulator AraR on the degradation of agro-industrial by-products soybean hulls (SBH), sugar beet pulp (SBP) and wheat bran (WB) in A. vadensis. Our results showed that the XlnR is the major TF affecting WB degradation, while AraR plays a larger role in SBH and SBP degradation. However, the deletion of xlnR resulted in strongly reduced growth on solid SBH but did not significantly reduce the overall amounts of extracellular proteins in liquid SBH. In contrast, the deletion of araR showed the opposite effect, indicating that the regulatory system of these two TFs appears to be influenced by cultural conditions.
In Chapter 5, we generated P. subrubescens ∆xlnR, ∆araR and ∆xlnR∆araR mutants and analyzed the transcriptional response of these strains to D-xylose and L-arabinose, and wheat arabinoxylan to identify the genes and metabolic pathways regulated by these TFs in P. subrubescens. The transcriptome analysis showed that deleting araR in P. subrubescens had a more significant impact than deleting xlnR, affecting the expression of hemicellulolytic genes, as well as genes involved in the pentose catabolic pathway and pentose phosphate pathway. These findings suggest that AraR plays a larger role in plant biomass degradation in P. subrubescens compared to XlnR.
In conclusion, A. vadensis may be a preferable host for recombinant protein production, whereas P. subrubescens appears more suitable for producing large quantities of enzyme cocktails.
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