BackgroundOver the past 3 years, the CRISPR/Cas9 system has revolutionized the field of genome engineering. However, its application has not yet been validated in thermophilic fungi. Myceliophthora thermophila, an important thermophilic biomass-degrading fungus, has attracted industrial interest for the production of efficient thermostable enzymes. Genetic manipulation of Myceliophthora is crucial for metabolic engineering and to unravel the mechanism of lignocellulose deconstruction. The lack of a powerful, versatile genome-editing tool has impeded the broader exploitation of M. thermophila in biotechnology.ResultsIn this study, a CRISPR/Cas9 system for efficient multiplexed genome engineering was successfully developed in the thermophilic species M. thermophila and M. heterothallica. This CRISPR/Cas9 system could efficiently mutate the imported amdS gene in the genome via NHEJ-mediated events. As a proof of principle, the genes of the cellulase production pathway, including cre-1, res-1, gh1-1, and alp-1, were chosen as editing targets. Simultaneous multigene disruptions of up to four of these different loci were accomplished with neomycin selection marker integration via a single transformation using the CRISPR/Cas9 system. Using this genome-engineering tool, multiple strains exhibiting pronounced hyper-cellulase production were generated, in which the extracellular secreted protein and lignocellulase activities were significantly increased (up to 5- and 13-fold, respectively) compared with the parental strain.ConclusionsA genome-wide engineering system for thermophilic fungi was established based on CRISPR/Cas9. Successful expansion of this system without modification to M. heterothallica indicates it has wide adaptability and flexibility for use in other Myceliophthora species. This system could greatly accelerate strain engineering of thermophilic fungi for production of industrial enzymes, such as cellulases as shown in this study and possibly bio-based fuels and chemicals in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0693-9) contains supplementary material, which is available to authorized users.

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BackgroundD-glucose, D-xylose and L-arabinose are the three major monosaccharides in plant cell walls. Complete utilization of all three sugars is still a bottleneck for second-generation cellulolytic bioethanol production, especially for L-arabinose. However, little is known about gene expression profiles during L-arabinose utilization in fungi and a comparison of the genome-wide fungal response to these three major monosaccharides has not yet been reported.ResultsUsing next-generation sequencing technology, we have analyzed the transcriptome of N. crassa grown on L-arabinose versus D-xylose, with D-glucose as the reference. We found that the gene expression profiles on L-arabinose were dramatically different from those on D-xylose. It appears that L-arabinose can rewire the fungal cell metabolic pathway widely and provoke the expression of many kinds of sugar transporters, hemicellulase genes and transcription factors. In contrast, many fewer genes, mainly related to the pentose metabolic pathway, were upregulated on D-xylose. The rewired metabolic response to L-arabinose was significantly different and wider than that under no carbon conditions, although the carbon starvation response was initiated on L-arabinose. Three novel sugar transporters were identified and characterized for their substrates here, including one glucose transporter GLT-1 (NCU01633) and two novel pentose transporters, XAT-1 (NCU01132), XYT-1 (NCU05627). One transcription factor associated with the regulation of hemicellulase genes, HCR-1 (NCU05064) was also characterized in the present study.ConclusionsWe conducted the first transcriptome analysis of Neurospora crassa grown on L-arabinose and performed a comparative analysis with cells grown on D-xylose and D-glucose, which deepens the understanding of the utilization of L-arabinose and D-xylose in filamentous fungi. The dataset generated by this research will be useful for mining target genes for D-xylose and L-arabinose utilization engineering and the novel sugar transportes identified are good targets for pentose untilization and biofuels production. Moreover, hemicellulase production by fungi could be improved by modifying the hemicellulase regulator discovered here.