Arabinose catabolism in Trichoderma reesei

Fungal strains of the genus Trichoderma are not only the best cellulase producers, but also very efficient producers of various types of polysaccharide hydrolases, including a whole array of hemicellulolytic enzymes. These enzymes hydrolyze the non-cellulosic plant polysaccharides which have been named "hemicelluloses" and which are found in plant cell walls in close association with cellulose, and which are composed of two or more monosaccharides such as D-xylose, L-arabinose, D-mannose, D-glucose, D-galactose and 4-O-methyl-D-glucuronic acid. The most abundant hemicelluloses of cereals and grasses are arabinoxylans, which are also abundant in maize. Because of their great industrial potential, the enzyme systems that are produced by Trichoderma spp. to hydrolyze a variety of these xylans (e.g. xylanases, arabinofuranosidases and others) have been extensively investigated. Despite the comprehensive knowledge on the enzyme systems degrading hemicelluloses to their monomers, little is however known about the further metabolism of the hemicellulose monomers in Trichoderma, and how this metabolism regulates the synthesis of the respective hemicellulases. As will be explained in this proposal, such a knowledge would be important both for the production of cellulases and hemicellulases, as well as from a general biochemical point of view (the pathway has not yet been studied azt the molecular genetic level). In this proposal, we therefore plan to clone the genes involved in the initial, specific stages of L-arabinose metabolism in Trichoderma reesei, and plan to study their transcriptional regulation under conditions relevant to hemicellulose degradation. We furthermore plan to construct mutants in the various steps to investigate how they influence the triggering of hemicellulase gene transcription in this fungus.

Plant hemicelluloses constitute 20 - 35 % of the annually renewable biomass, and therefore are an important substrates for microbial fermentations. In order to provide a basis for the construction of tailor-made strains for such fermentations, we have decided to clone the genes involved in the pathway of catabolism of L-arabinose and D-xylose in the industrially important fungus Hypocrea jecorina (Trichoderma reesei). L-arabinose utilization involves an NADPH-linked aldose reductase, which forms L-arabinitol. This is converted to L-xylulose by an L- arabinitol-4-dehydrogenase, followed by an NADPH-linked L-xylulose reductase, which forms xylitol from L- xylulose. Finally, xylitol is catabolized to D-xylulose (which can be phosphorylated and chanelled into the pentose phosphate pathway) by xylitol dehydrogenase. In the course of this project, we cloned the genes encoding one of the three aldose reductases (alr1), L-arabinitol dehydrogenase (lad1), and xylitol dehydrogenase (xdh1), and produced single and double knockout mutants in various combinations. The respective results show that the aldose reductase step is shared between multiple enzymes, and that lad1 and xdh1 also compensate for a loss of each other. Overexpression, purification and enzymological characterization of the encoded enzymes yielded data consistent with the results from gene deletions. The knock out mutants had altered profiles of induction of hemicellulases, thus offering a promising tool for the use of the alr1, lad1 and xdh1 genes for strain improvement.