Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0693-9) contains supplementary material, which is available to authorized users.

Multiplexed CRISPR-Cas9 based genome editing ofRhodosporidium toruloides

“…Combining multiple sgRNAs in an array is particularly useful for R. toruloides, as this minimizes the amount of genetic material that needs to be delivered while maximizing the number of potential gene targets. Additionally, utilizing multiple sgRNAs to target one gene at different locations allows for the possibility of removing a large DNA in between the target sites, and also increases the possibility that the target gene is successfully disrupted (20, 46, 47). Therefore, our constructs were designed to express four sgRNAs (two for each gene) such that cleavage would occur at two sites separated by ∼500bp in both genes.…”

“…However, the process of delivering and expressing fully functional CRISPR components, and the biology of each organism’s DNA repair pathways, requires organism-specific optimization to accommodate each species’ unique characteristics. The past two years have seen the publication of a wide array of studies demonstrating CRISPR-Cas9 genome engineering in fungal species for which few robust DNA editing tools existed, including Aspergillus niger (49), Cryptococcus neoformans (50), Mucor circinelloides (51), and Myceliophthora thermophila (47). The dawning of a “fungal CRISPR revolution” has occurred, empowering researchers to explore new bioproduction possibilities in obscure yet promising fungi.…”

Strategies and Challenges for the Development of Industrial Enzymes Using Fungal Cell Factories

“…7.4b) will enable faster construction of yieldoptimized strains and will fuel its use for rational design in strain construction as well as for more genome-wide searches for gene modifications that improve production of an enzyme. Genome editing in the thermophilic fungus Myceliophthora was used to increase cellulase production by 13-fold by editing up to four genes at one time (Liu et al 2017).…”

Overview of Gene Expression Using Filamentous Fungi

“…(Nødvig, Nielsen, Kogle, & Mortensen, ; Katayama et al., ), Neurospora crassa (Matsu‐ura, Baek, Kwon, & Hong, ), T. reesei (Liu, Chen, Jiang, Zhou, & Zou, ), and Myceliophtora spp. (Liu et al., ).…”