Abstract
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.
Highlights
Over the past 3 years, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized the field of genome engineering
The Cas9OE strains showed an identical dry weight when grown on sucrose and Avicel (Additional file 1: Fig. S1F). All of these results indicated that constitutive expression of Cas9 nuclease did not alter the growth, sporulation, or cellulolytic production of the ATCC 42464 strain of M. thermophila
In this study, an efficient CRISPR/Cas9 system for genome editing was successfully developed in the thermophilic species M. thermophila and M. heterothallica
Summary
Over the past 3 years, the CRISPR/Cas system has revolutionized the field of genome engineering. Lignocellulosic biomass is an abundant renewable, conventional energy source for many industrial applications [1, 2]. Through their secretion of large amounts of. A high-efficiency Agrobacterium tumefaciensmediated transformation system was developed by our group for the sequenced M. thermophila strain ATCC 42464 [12]. Using this technique, a ku deletion strain was constructed. The lack of a powerful and versatile genome-editing tool for M. thermophila impedes wider biotechnological exploitation of this fungus
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