CRISPR/Cas9 mediated targeted knock-in of eglA gene to improve endoglucanase activity of Aspergillus fumigatus LMB-35Aa

Abstract

Abstract Bioeconomy goals for using biomass feedstock for biofuels and bio-based production has arisen the demand for fungal strains and enzymes for biomass processing. Despite well-known Trichoderma and Aspergillus commercial strains, continuous bioprospecting has revealed the fungal biodiversity potential for production of biomass degrading enzymes. The strain Aspergillus fumigatus LMB-35Aa has revealed a great potential as source of lignocellulose-degrading enzymes. Nevertheless, genetic improvement should be considered to increase its biotechnological potential. Molecular manipulation based on homologous direct recombination (HDR) in filamentous fungi poses a challenge since its low recombination rate. Currently, CRISPR/Cas9-mediated mutagenesis can enable precise and efficient editing of filamentous fungi genomes. In this study, a CRISPR/Cas9-mediated gene editing strategy for improving endoglucanase activity of A. fumigatus LMB-35Aa strain was successfully used, which constitutes the first report of heterologous cellulase production in filamentous fungi using this technology. For this, egl A gene from A. niger ATCC 10,864 was integrated into conidial melanin pks P gene locus, which facilitated the selection of edited events discerned by the emergence of albino colonies. Heterologous production of the EglA enzyme in a biofilm fermentation system resulted in a 40% improvement in endoglucanase activity of the mutant strain compared to the wild type.