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.

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: 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.

The thermophilic filamentous fungus Myceliophthora thermophila has many suitable characteristics for industrial biotechnology and could be a promising new chassis system for synthetic biology, particularly the ATCC 42464 strain, whose genome was sequenced in 2011. However, metabolic engineering of this strain using genetic approaches has not been reported owing to a lack of genetic tools for this organism. In the present study, we developed a high efficiency Agrobacterium tumefaciens mediated transformation system for M. thermophila, including an approach for targeted gene deletion using green fluorescence protein (GFP) as a marker for selection. Up to 145 transformants per 105 conidia were obtained in one transformation plate. Moreover, a ku70 deletion mutant was constructed in the ATCC 42464 background using the tools developed in present study and subsequently characterized. The ku70 deletion construct was designed using resistance to phosphinothricin as the selection marker. Additionally, a GFP-encoding cassette was incorporated that allowed for the selection of site-specific (no fluorescence) or ectopic (fluorescence) integration of the ku70 construct. Transformants with ectopically integrated ku70 deletion constructs were therefore identified using the fluorescent signal of GFP. PCR and Southern blotting analyses of non-fluorescent putative ku70 deletion transformants revealed all 11 tested transformants to be correct deletions. The deletion frequency in a pool of 116 transformants analyzed was 58 %. Moreover, the homologous rate improved about 3 folds under ku70 mutant using the pyrG as a test gene to disrupt in M. thermophila. We successfully developed an efficient transformation and target gene disruption approach for M. thermophila ATCC 42464 mediated by A. tumefaciens. The tools and the ku70 deletion strain developed here should advance the development of M. thermophila as an industrial host through metabolic engineering and accelerate the elucidation of the mechanism of rapid cellulose degradation in this thermophilic fungus.

Background: Consolidated bioprocessing (CBP) technique is a promising strategy for biorefinery construction, producing bulk chemicals directly from plant biomass without extra hydrolysis steps. Fixing and channeling CO₂ into carbon metabolism for increased carbon efficiency in producing value-added compounds is another strategy for cost-effective bio-manufacturing. It has not been reported whether these two strategies can be combined in one microbial platform. Results: In this study, using the cellulolytic thermophilic fungus Myceliophthora thermophila, we designed and constructed a novel biorefinery system DMCC (Direct microbial conversion of biomass with CO₂ fixation) through incorporating two CO₂ fixation modules, PYC module and Calvin–Benson–Bassham (CBB) pathway. Harboring the both modules, the average rate of fixing and channeling ¹³CO₂ into malic acid in strain CP51 achieved 44.4, 90.7, and 80.7 mg/L/h, on xylose, glucose, and cellulose, respectively. The corresponding titers of malic acid were up to 42.1, 70.4, and 70.1 g/L, respectively, representing the increases of 40%, 10%, and 7%, respectively, compared to the parental strain possessing only PYC module. The DMCC system was further improved by enhancing the pentose uptake ability. Using raw plant biomass as the feedstock, yield of malic acid produced by the DMCC system was up to 0.53 g/g, with ¹³C content of 0.44 mol/mol malic acid, suggesting DMCC system can produce 1 t of malic acid from 1.89 t of biomass and fix 0.14 t CO₂ accordingly. Conclusions: This study designed and constructed a novel biorefinery system named DMCC, which can convert raw plant biomass and CO₂ into organic acid efficiently, presenting a promising strategy for cost-effective production of value-added compounds in biorefinery. The DMCC system is one of great options for realization of carbon neutral economy.

The cellulolytic fungus Neurospora crassa is considered a potential host for enzyme and bioethanol production. However, large scale applications are hindered by its filamentous growth. Although previous investigations have shown that mycelial morphology in submerged culture can be controlled by altering physical factors, there is little knowledge available about the potential for morphology control by genetic modification. In this study, we screened morphological mutants in the filamentous fungus N. crassa. Of the 90 morphological mutants screened, 14 mutants exhibited considerably higher viscosity compared with that of the wild type strain, and only two mutants showed low-viscosity morphologies in submerged culture. We observed that disruption of gul-1 (NCU01197), which encodes an mRNA binding protein involved in cell wall remodeling, caused pellet formation as the fermentation progressed, and resulted in the most significant decrease in viscosity of culture broth. Moreover, over-expression of gul-1 caused dramatically increased viscosity, suggesting that the gul-1 had an important function in mycelial morphology during submerged cultivation. Transcriptional profiling showed that expression of genes encoding eight GPI-anchored cell wall proteins was lowered in Δgul-1 while expression of genes associated with two non-anchored cell wall proteins was elevated. Meanwhile, the expression levels of two hydrophobin genes were also significantly altered. These results suggested that GUL-1 affected the transcription of cell wall-related genes, thereby influencing cell wall structure and mycelial morphology. Additionally, the deletion of gul-1 caused increased protein secretion, probably due to a defect in cell wall integrity, suggesting this as an alternative strategy of strain improvement for enzyme production. To confirm practical applications, deletion of gul-1 in the hyper-cellulase producing strain (∆ncw-1∆Ncap3m) significantly reduced the viscosity of culture broth. Using the model filamentous fungus N. crassa, genes that affect mycelial morphology in submerged culture were explored through systematic screening of morphological mutants. Disrupting several candidate genes altered viscosities in submerged culture. This work provides an example for controlling fungal morphology in submerged fermentation by genetic engineering, and will be beneficial for industrial fungal strain improvement.