Uncovering factors allowing high carbohydrate active enzyme production in the thermophilic filamentous fungus Thermoascus aurantiacus

Fungal carbohydrate active enzymes (CAZy) are vital for various industrial applications such as the conversion of plant biomass to biofuels and bio-based chemicals. The thermophilic filamentous fungus Thermoascus aurantiacus is an intriguing host for cellulase production and produces considerable quantities of cellulases and xylanases which retain high activity above 65 °C. The goal of this thesis was to uncover (1) CAZy induction and (2) CAZy gene regulation in T. aurantiacus and (3) learn from classical fungal CAZy hypersecreting Neurospora crassa mutants how to improve fungal enzyme secretion to design improved genetic- and bioprocess engineering strategies. This work involved extensive methods development and established vital knowledge for improving cellulase production with T. aurantiacus. Xylan lead to secretion of high amounts of cellulases and xylanases, while simple C5 and C6 sugars and cellobiose failed to significantly induce CAZy expression in batch cultures, presumably due to carbon catabolite repression (CCR). The development of a continuous feed setup allowed to circumvent CCR while feeding low amounts of potential inducers into 50 mL shake flask cultures. One major obstacle that prevents employing new fungal strains is the lack of genetic transformation systems. Here we report on successful Agrobacterium tumefaciens mediated transformation of the thermophilic fungus T. aurantiacus, a producer of highly heat-stable plant cell wall degrading enzymes. We optimized the transformation efficiency of the newly developed protocol and overexpressed the transcriptional xylanase regulator xlnR, clr1 and clr2, which led to increase in xylanase activity of the mutant strain. Finally, we sought to learn from classical fungal CAZy hypersecreting mutants how to increase enzyme secretion in T. aurantiacus. The mutation causing CAZy hypersecretion in the long-known N. crassa exo-1 mutant was identified through genome resequencing and genetic tests: a premature stop in the F-box protein-encoding gene exo-1. As known for the exo-1 mutant, high invertase, amylase and pectinase activity was found in the corresponding deletion strain upon sugar depletion. While the exo-1 gene homolog is missing in T. aurantiacus, the enzyme hypersecretion phenotype could be successfully reverse-engineered through collaborators into the industrially employed thermophilic fungus Myceliophthora thermophila through CRISPR/Cas9, leading to high amylase secretion.