Sugar catabolism during growth on plant biomass in Aspergillus

Abstract

A growing industrial sector in which plant degrading enzymes are used is the production of alternative fuels, such as bio-ethanol, and biochemicals. Plant polysaccharides can be converted to fermentable sugars by fungal enzymes. The sugars are then fermented to ethanol and other products mainly by yeast. Aspergillus species are among the organisms of choice for enzyme production for pre-treatment of plant material, because they have high levels of protein secretion and produce a wide range of enzymes for plant polysaccharide degradation. In nature, Aspergillus degrades the polysaccharides to obtain monomeric sugars that can serve as a carbon source. Therefore, Aspergillus uses a variety of catabolic pathways to efficiently convert all the monomeric components of plant biomass. In my thesis, I focused on several of the main carbon catabolic pathways of Aspergillus niger and Aspergillus nidulans involved in converting the main monomers (D-glucose, D-xylose, L-arabinose, L-rhamnose and D-galacturonic acid) present in plant polysaccharides and their regulation. We confirmed the function of the first three putative L-rhamnose utilization genes from A. niger through gene deletion. This work showed that the inducer of RhaR is beyond L-rhamnonate dehydratase (LraC) and is likely to be the 2-keto-3-L-deoxyrhamnonate. However, we were so far not able to identify the gene encoding LraD, and can therefore not confirm that this compound is the inducer of RhaR. The same strategy has been used for the D-galacturonic acid pathway. The results indicated that 2-keto-3-deoxy-L-galactonate is the inducer of genes required for D-galacturonic acid utilization. D-xylose and L-arabinose are the most abundant monosaccharides after D-glucose in nearly all plant-derived biomass materials. We evaluated the effect of the (hemi-)cellulolytic regulator (xlnR) and xylulokinase (xkiA1) A. niger mutant strains during growth on two pentose-rich substrates, corn stover (CS) and soybean hulls (SBH), by transcriptome analysis. At a later time point, significant differences were found in the expression profiles of both mutants on CS compared to SBH. In A. niger, XlnR and the arabinanolytic transcription factor (AraR) regulate production of enzymes involved in degradation of arabinoxylan and catabolism of the released L-arabinose and D-xylose. We investigated the colonization and degradation of wheat bran by the A. niger reference strain and araR/xlnR regulatory mutants using high-resolution microscopy and exo-proteomics. This enabled us to link the reduction in colonization and degradation efficiency of the mutants to the absence of subsets of enzymes. We evaluated if it would be possible to create an A. nidulans strain that releases but does not metabolize hexoses from plant biomass. The creAΔ4 mutation in combination with preventing initial phosphorylation in glycolysis resulted in an increased expression of pentose catabolic and pentose phosphate pathway genes.This indicates that the reduced ability to use hexoses as carbon sources created a shift towards the pentose fraction of wheat bran as a major carbon source to support growth. Expression analysis also showed that CreA influences fungal physiology already at low levels of free monosaccharides, suggesting an important role for this regulator in natural habitats of fungi.