Susan Mampusta Madrid

Regulation of pentose utilisation by AraR, but not XlnR, differs in Aspergillus nidulans and Aspergillus niger.

Filamentous fungi are important producers of plant polysaccharide degrading enzymes that are used in many industrial applications. These enzymes are produced by the fungus to liberate monomeric sugars that are used as carbon source. Two of the main components of plant polysaccharides are l-arabinose and d-xylose, which are metabolized through the pentose catabolic pathway (PCP) in these fungi. In Aspergillus niger, the regulation of pentose release from polysaccharides and the PCP involves the transcriptional activators AraR and XlnR, which are also present in other Aspergilli such as Aspergillus nidulans. The comparative analysis revealed that the regulation of the PCP by AraR differs in A. nidulans and A. niger, whereas the regulation of the PCP by XlnR was similar in both species. This was demonstrated by the growth differences on l-arabinose between disruptant strains for araR and xlnR in A. nidulans and A. niger. In addition, the expression profiles of genes encoding l-arabinose reductase (larA), l-arabitol dehydrogenase (ladA) and xylitol dehydrogenase (xdhA) differed in these strains. This data suggests evolutionary changes in these two species that affect pentose utilisation. This study also implies that manipulating regulatory systems to improve the production of polysaccharide degrading enzymes, may give different results in different industrial fungi.

Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo‐xylanase or ferulic acid esterase in the endosperm

Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm-specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu) KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and threefold relative to wild type. The grains were shrivelled and had a 25%-33% decrease in mass. Extensive analysis of the cell walls showed a 10%-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water-extractable arabinoxylan, and a shift in the MW of the water-extractable arabinoxylan from being mainly larger than 85 kD to being between 2 and 85 kD. Ferulic acid esterase-expressing grains were also shrivelled, and the seed weight was decreased by 20%-50%. No ferulic acid esterase activity could be detected in wild-type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15%-40% increase in water-unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13% and 34%. In all the plants, the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.

Unique Regulatory Mechanism for d-Galactose Utilization in Aspergillus nidulans

ABSTRACT This study describes two novel regulators, GalX and GalR, that control d-galactose utilization in Aspergillus nidulans. This system is unique for A. nidulans since no GalR homologs were found in other ascomycetes. GalR shares significant sequence identity with the arabinanolytic and xylanolytic regulators AraR and XlnR, but GalX is more distantly related.

Disruption of the L-arabitol dehydrogenase encoding gene in Aspergillus tubingensis results in increased xylanase production.

Fungal xylanases are of major importance to many industrial sectors, such as food and feed, paper and pulp, and biofuels. Improving their production is therefore highly relevant. We determined the molecular basis of an improved xylanase-producing strain of Aspergillus tubingensis that was generated by UV mutagenesis in an industrial strain improvement program. Using enzyme assays, gene expression, sequencing of the ladA locus in the parent and mutant, and complementation of the mutation, we were able to show that improved xylanase production was mainly caused by a chromosomal translocation that occurred between a subtilisin-like protease pepD gene and the L-arabitol dehydrogenase encoding gene (ladA), which is part of the L-arabinose catabolic pathway. This genomic rearrangement resulted in disruption of both genes and, as a consequence, the inability of the mutant to use L-arabinose as a carbon source, while growth on D-xylose was unaffected. Complementation with constitutively expressed ladA confirmed that the xylanase overproducing phenotype was mainly caused by loss of ladA function, while a knockout of xlnR in the UV mutant demonstrated that improved xylanase production was mediated by XlnR. This study demonstrates the potential of metabolic manipulation for increased production of fungal enzymes.