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.

Siliceous mesostructured cellular foams (MCF) impregnated with polyethylenimine (PEI) of various molecular weights and structures were evaluated as CO 2 adsorbents. The MCF solid support consisted of a well-defined interconnected three-dimensional mesoporous structure with large cell diameter of 30.3 nm and large window diameter of 11.3 nm, filled with polyethylenimine up to 70 weight percent or about 22.3% nitrogen atom by weight of the adsorbents. While other mesoporous solid supports lost their porosity after PEI impregnation, our MCF solid support maintained its pore volume over the range of 1.12 to 1.64 cm 3 g 21 . The importance of the porosity of PEI-impregnated MCF adsorbents for high capacity CO 2 adsorbents was demonstrated. The highest CO 2 sorption capacity (180.6 mg-CO 2 /g-adsorbent or 393.6 mg-CO 2 /g-PEI at 75 uC) was obtained for silica supports loaded with 50 weight percent branched PEI with average molecular weight of 600 g mol 21 . Under dry atmospheric CO 2 gas, this adsorbent reached the theoretical CO 2 capacity of 0.50 mole-CO 2 per mole-nitrogen within less than about 8 min, making this adsorbent one of the most effective CO 2 adsorbents reported. Repeated multiple sorption cycles demonstrated good stability of this adsorbent for CO 2 capture. The initial sorption kinetics determined the overall CO 2 sorption capacity, which was limited by the formation of a carbamate layer as a result of the CO 2 -PEI complexation that due to inhibition of CO 2 diffusion; the kinetics of ''ionic'' gelation of the impregnated PEI by CO 2 controlled the overall performance of the CO 2 adsorbents. At 75 uC, the operating temperature favored the molecular mobility of PEI and unrestricted diffusion of CO 2 to allow the theoretical CO 2 capacity of the PEI to be attained. Lower temperatures limited the mobilities of PEI and CO 2 and the kinetics of ''ionic'' gel formation dominated, causing a lowered overall performance of the CO 2 adsorbents. Overall, this study points to the importance of interconnected porous channel networks to optimize the performance of PEI-impregnated mesoporous silica particles.

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