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.

D-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.

The glucose dual-affinity transport system (low- and high-affinity) is a conserved strategy used by microorganisms to cope with natural fluctuations in nutrient availability in the environment. The glucose-sensing and uptake processes are believed to be tightly associated with cellulase expression regulation in cellulolytic fungi. However, both the identities and functions of the major molecular components of this evolutionarily conserved system in filamentous fungi remain elusive. Here, we systematically identified and characterized the components of the glucose dual-affinity transport system in the model fungus Neurospora crassa. Using RNA sequencing coupled with functional transport analyses, we assigned GLT-1 (K m = 18.42 ± 3.38 mM) and HGT-1/-2 (K m = 16.13 ± 0.95 and 98.97 ± 22.02 µM) to the low- and high-affinity glucose transport systems, respectively. The high-affinity transporters hgt-1/-2 complemented a moderate growth defect under high glucose when glt-1 was deleted. Simultaneous deletion of hgt-1/-2 led to extensive derepression of genes for plant cell wall deconstruction in cells grown on cellulose. The suppression by HGT-1/-2 was connected to both carbon catabolite repression (CCR) and the cyclic adenosine monophosphate-protein kinase A pathway. Alteration of a residue conserved across taxa in hexose transporters resulted in a loss of glucose-transporting function, whereas CCR signal transduction was retained, indicating dual functions for HGT-1/-2 as “transceptors.” In this study, GLT-1 and HGT-1/-2 were identified as the key components of the glucose dual-affinity transport system, which plays diverse roles in glucose transport and carbon metabolism. Given the wide conservation of the glucose dual-affinity transport system across fungal species, the identification of its components and their pleiotropic roles in this study shed important new light on the molecular basis of nutrient transport, signaling, and plant cell wall degradation in fungi.

Lignocellulolytic fungal cells suffer endoplasmic reticulum (ER) stress during lignocellulase synthesis; however, an understanding of this integrated process on a genome-wide scale remains poor. Here, we undertook a systematic investigation of this process in Neurospora crassa (N. crassa) using transcriptomic analysis coupled with genetic screens. A set of 766 genes was identified as the ER stress response targets (ESRTs) in N. crassa under cellulose utilization conditions. Among these, the expression of 223 and 186 genes showed dependence on IRE-1 and HAC-1, respectively. A total of 527 available mutants for ESRT genes were screened, 249 of which exhibited ER stress susceptibility, including 100 genes with unknown function. Disruption of ire-1 or hac-1 in N. crassa did not affect transcriptional induction of lignocellulase genes by cellulose but severely affected secretion of the corresponding enzymes. A global investigation of transcription factors (TFs) discovered three novel regulators (RES-1, RES-2, RRG-2) involved in lignocellulase secretion. Production of lignocellulases in Δres-1 increased by more than 30% in comparison to wild type (WT), while secretion decreased by nearly 30% in strains Δres-2 and Δrrg-2. Transcriptional profiling of the three TF mutants suggests they are deeply involved in lignocellulase secretion and ER stress response. Here, we determined the transcriptional scope of the ER stress response during lignocellulase synthesis in the model cellulolytic fungus N. crassa. Through genome-wide mutant screening and analysis, dozens of novel genes were discovered to be involved in the process. The findings of this work will be useful for strain improvement to facilitate lignocellulase and biomass-based chemical production.

Background: Cellulosic biomass is a promising resource for bioethanol production. However, various sugars in plant biomass hydrolysates including cellodextrins, cellobiose, glucose, xylose, and arabinose, are poorly fermented by microbes. The commonly used ethanol-producing microbe Saccharomyces cerevisiae can usually only utilize glucose, although metabolically engineered strains that utilize xylose have been developed. Direct fermentation of cellobiose could avoid glucose repression during biomass fermentation, but applications of an engineered cellobiose-utilizing S. cerevisiae are still limited because of its long lag phase. Bioethanol production from biomass-derived sugars by a cellulolytic filamentous fungus would have many advantages for the biorefinery industry. Results: We selected Myceliophthora thermophila, a cellulolytic thermophilic filamentous fungus for metabolic engineering to produce ethanol from glucose and cellobiose. Ethanol production was increased by 57% from glucose but not cellobiose after introduction of ScADH1 into the wild-type (WT) strain. Further overexpression of a glucose transporter GLT-1 or the cellodextrin transport system (CDT-1/CDT-2) from N. crassa increased ethanol production by 131% from glucose or by 200% from cellobiose, respectively. Transcriptomic analysis of the engineered cellobiose-utilizing strain and WT when grown on cellobiose showed that genes involved in oxidation–reduction reactions and the stress response were downregulated, whereas those involved in protein biosynthesis were upregulated in this effective ethanol production strain. Turning down the expression of pyc gene results the final engineered strain with the ethanol production was further increased by 23%, reaching up to 11.3 g/L on cellobiose. Conclusions: This is the first attempt to engineer the cellulolytic fungus M. thermophila to produce bioethanol from biomass-derived sugars such as glucose and cellobiose. The ethanol production can be improved about 4 times up to 11 grams per liter on cellobiose after a couple of genetic engineering. These results show that M. thermophila is a promising platform for bioethanol production from cellulosic materials in the future.

Background: Thermophilic filamentous fungusMyceliophthora thermophilahas great capacity for biomass degradation and is an attractive system for direct production of enzymes and chemicals from plant biomass. Its industrial importance inspired us to develop genome editing tools to speed up the genetic engineering of this fungus. First-generation CRISPR–Cas9 technology was developed in 2017 and, since then, some progress has been made in thermophilic fungi genetic engineering, but a number of limitations remain. They include the need for complex independent expression cassettes for targeting multiplex genomic loci and the limited number of available selectable marker genes.Results: In this study, we developed anAcidaminococcussp. Cas12a-based CRISPR system for efficient multiplex genome editing, using a single-array approach inM. thermophila. These CRISPR–Cas12a cassettes worked well for simultaneous multiple gene deletions/insertions. We also developed a new simple approach for marker recycling that relied on the novel cleavage activity of the CRISPR–Cas12a system to make DNA breaks in selected markers. We demonstrated its performance by targeting nine genes involved in the cellulase production pathway inM. thermophilavia three transformation rounds, using two selectable markersneoandbar. We obtained the nonuple mutant M9 in which protein productivity and lignocellulase activity were 9.0- and 18.5-fold higher than in the wild type. We conducted a parallel investigation using our transient CRISPR–Cas9 system and found the two technologies were complementary. Together we called themCRISPR–Cas-assistedmarkerrecyclingtechnology (Camr technology).Conclusions: Our study described new approaches (Camr technology) that allow easy and efficient marker recycling and iterative stacking of traits in the same thermophilic fungus strain either, using the newly established CRISPR–Cas12a system or the established CRISPR–Cas9 system. This Camr technology will be a versatile and efficient tool for engineering, theoretically, an unlimited number of genes in fungi. We expect this advance to accelerate biotechnology-oriented engineering processes in fungi.