Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering
Top Cited Papers
Open Access
- 3 January 2017
- journal article
- research article
- Published by Springer Science and Business Media LLC in Biotechnology for Biofuels
- Vol. 10 (1), 1-14
- https://doi.org/10.1186/s13068-016-0693-9
Abstract
Over 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. In 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. A 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.Keywords
Funding Information
- Chinese Academy of Sciences (ZDRW-ZS-2016-3)
- National Natural Science Foundation of China (CN) (31471186, 31640049)
This publication has 60 references indexed in Scilit:
- One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome EngineeringCell, 2013
- Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systemsNucleic Acids Research, 2013
- Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene ExpressionCell, 2013
- Multiplex Genome Engineering Using CRISPR/Cas SystemsScience, 2013
- A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial ImmunityScience, 2012
- Conserved and essential transcription factors for cellulase gene expression in ascomycete fungiProceedings of the National Academy of Sciences of the United States of America, 2012
- Induction of lignocellulose-degrading enzymes in Neurospora crassa by cellodextrinsProceedings of the National Academy of Sciences of the United States of America, 2012
- Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestrisNature Biotechnology, 2011
- Genetic Modification of Carbon Catabolite Repression in Trichoderma reesei for Improved Protein ProductionApplied and Environmental Microbiology, 2009
- Rapid preparation of DNA from filamentous fungiLetters in Applied Microbiology, 1985