No abstract

The urgency for reducing the dependence on fossil-based materials is increasing the interest in the utilization of renewable feedstocks. Lignocellulosic residual biomass can be used as feedstock to produce chemicals and energy without generating food security problems. Wheat straw (WS) has a clear potential for developing sustainable processes in a circular bioeconomy context. However, the development of processes requires a strategy for utilizing the hemicellulosic, cellulosic, and lignin fractions. This work covers the utilization of the hemicellulosic fraction as the first stage of a wheat straw biorefinery. The aim was to evaluate the hydrolysis of WS by using liquid hot water (LHW) treatment, the detoxification of the produced wheat straw hydrolysate (WSH), and the cultivation of Trichoderma reesei using it as the only carbon source as proof of detoxification. LHW treatment was performed at 160 °C and 90 min and yielded a WSH rich in monomeric and oligomeric saccharides (~ 14 g/L) and containing degradation products in low concentration (furfural, HMF, and acetic acid). As part of the development of the extraction and detoxification strategy, we determined the specific inhibition thresholds for T. reesei for the mentioned degradation products. Detoxification was carried out by evaporation by modifying the % of volume evaporated and the pH of the solution. Approximately 55.9% of acetic acid and 100% of furfural were removed from the WSH. The fungal biomass obtained in the medium containing WSH was equivalent to 98% of the biomass obtained in the control medium.

Aureobasidium pullulans is a ubiquitous, polyextremotolerant, yeast-like ascomycete used for the industrial production of pullulan and other products and as biocontrol agent in the agriculture. Its application potential and its wide-spread occurrence make A. pullulans an interesting study object. The availability of a fast and efficient genome editing method is an obvious advantage for future basic and applied research on A. pullulans. In this study, we describe the development of a CRISPR/Cas9-based genome editing method using ribonucleoproteins (RNPs). We demonstrate that this method can be used for single and multiplex genome editing using only RNPs by targeting ura3 (encoding for orotidine-5'-phosphate decarboxylase), praics (encoding for phosphoribosyl aminoimidazole-succinocarboxamide synthase) and asl (encoding for arginine succinate lyase). We demonstrate the applicability of Trichoderma reesei pyr4 and Aspergillus fumigatus pyrG to complement the ura3 deficiency. Further, we show that the usage of RNPs can boost the homologous recombination rate up to nearly 100%, even when using only 20bp long homologous flanks. Therefore, the repair cassettes can be constructed by a single PCR, abolishing the need for laborious and time-consuming cloning. The here presented method allows fast and efficient genome editing for gene deletions, modifications, and insertions in A. pullulans.