Filamentous fungi have wide applications in biotechnology. The CRISPR/Cas9 system is a powerful genome-editing method that facilitates genetic alterations of genomes in a variety of organisms. However, a genome-editing approach has not been reported in filamentous fungi. Here, we demonstrated the establishment of a CRISPR/Cas9 system in the filamentous fungus Trichoderma reesei by specific codon optimization and in vitro RNA transcription. It was shown that the CRISPR/Cas9 system was controllable and conditional through inducible Cas9 expression. This system generated site-specific mutations in target genes through efficient homologous recombination, even using short homology arms. This system also provided an applicable and promising approach to targeting multiple genes simultaneously. Our results illustrate that the CRISPR/Cas9 system is a powerful genome-manipulating tool for T. reesei and most likely for other filamentous fungal species, which may accelerate studies on functional genomics and strain improvement in these filamentous fungi.

Many Penicillium species could produce extracellular enzyme systems with good lignocellulose hydrolysis performance. However, these species and their enzyme systems are still poorly understood and explored due to the lacking of genetic information. Here, we present the genomic and secretomic analyses of Penicillium decumbens that has been used in industrial production of lignocellulolytic enzymes in China for more than fifteen years. Comparative genomics analysis with the phylogenetically most similar species Penicillium chrysogenum revealed that P. decumbens has evolved with more genes involved in plant cell wall degradation, but fewer genes in cellular metabolism and regulation. Compared with the widely used cellulase producer Trichoderma reesei, P. decumbens has a lignocellulolytic enzyme system with more diverse components, particularly for cellulose binding domain-containing proteins and hemicellulases. Further, proteomic analysis of secretomes revealed that P. decumbens produced significantly more lignocellulolytic enzymes in the medium with cellulose-wheat bran as the carbon source than with glucose. The results expand our knowledge on the genetic information of lignocellulolytic enzyme systems in Penicillium species, and will facilitate rational strain improvement for the production of highly efficient enzyme systems used in lignocellulose utilization from Penicillium species.