Construction of an enzyme-constrained metabolic network model for Myceliophthora thermophila using machine learning-based kcat data.

BACKGROUND: Genome-scale metabolic models (GEMs) serve as effective tools for understanding cellular phenotypes and predicting engineering targets in the development of industrial strain. Enzyme-constrained genome-scale metabolic models (ecGEMs) have emerged as a valuable advancement, providing more accurate predictions and unveiling new engineering targets compared to models lacking enzyme constraints. In 2022, a stoichiometric GEM, iDL1450, was reconstructed for the industrially significant fungus Myceliophthora thermophila. To enhance the GEM's performance, an ecGEM was developed for M. thermophila in this study. RESULTS: Initially, the model iDL1450 underwent refinement and updates, resulting in a new version named iYW1475. These updates included adjustments to biomass components, correction of gene-protein-reaction (GPR) rules, and a consensus on metabolites. Subsequently, the first ecGEM for M. thermophila was constructed using machine learning-based kcat data predicted by TurNuP within the ECMpy framework. During the construction, three versions of ecGEMs were developed based on three distinct kcat collection methods, namely AutoPACMEN, DLKcat and TurNuP. After comparison, the ecGEM constructed using TurNuP-predicted kcat values performed better in several aspects and was selected as the definitive version of ecGEM for M. thermophila (ecMTM). Comparing ecMTM to iYW1475, the solution space was reduced and the growth simulation results more closely resembled realistic cellular phenotypes. Metabolic adjustment simulated by ecMTM revealed a trade-off between biomass yield and enzyme usage efficiency at varying glucose uptake rates. Notably, hierarchical utilization of five carbon sources derived from plant biomass hydrolysis was accurately captured and explained by ecMTM. Furthermore, based on enzyme cost considerations, ecMTM successfully predicted reported targets for metabolic engineering modification and introduced some new potential targets for chemicals produced in M. thermophila. CONCLUSIONS: In this study, the incorporation of enzyme constraint to iYW1475 not only improved prediction accuracy but also broadened the model's applicability. This research demonstrates the effectiveness of integrating of machine learning-based kcat data in the construction of ecGEMs especially in situations where there is limited measured enzyme kinetic parameters for a specific organism.

The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi.

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein-encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain Δcre-1, while glucose repression was still mostly functional in Δexo-1 Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in Δexo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in Δexo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

The transcriptional factor Clr-5 is involved in cellulose degradation through regulation of amino acid metabolism in Neurospora crassa.

BACKGROUND: Filamentous fungi are efficient degraders of plant biomass and the primary producers of commercial cellulolytic enzymes. While the transcriptional regulation mechanisms of cellulases have been continuously explored in lignocellulolytic fungi, the induction of cellulase production remains a complex multifactorial system, with several aspects still largely elusive. RESULTS: In this study, we identified a Zn2Cys6 transcription factor, designated as Clr-5, which regulates the expression of cellulase genes by influencing amino acid metabolism in Neurospora crassa during growth on cellulose. The deletion of clr-5 caused a significant decrease in secreted protein and cellulolytic enzyme activity of N. crassa, which was partially alleviated by supplementing with yeast extract. Transcriptomic profiling revealed downregulation of not only the genes encoding main cellulases but also those related to nitrogen metabolism after disruption of Clr-5 under Avicel condition. Clr-5 played a crucial role in the utilization of multiple amino acids, especially leucine and histidine. When using leucine or histidine as the sole nitrogen source, the Δclr-5 mutant showed significant growth defects on both glucose and Avicel media. Comparative transcriptomic analysis revealed that the transcript levels of most genes encoding carbohydrate-active enzymes and those involved in the catabolism and uptake of histidine, branched-chain amino acids, and aromatic amino acids, were remarkably reduced in strain Δclr-5, compared with the wild-type N. crassa when grown in Avicel medium with leucine or histidine as the sole nitrogen source. These findings underscore the important role of amino acid metabolism in the regulation of cellulase production in N. crassa. Furthermore, the function of Clr-5 in regulating cellulose degradation is conserved among ascomycete fungi. CONCLUSIONS: These findings regarding the novel transcription factor Clr-5 enhance our comprehension of the regulatory connections between amino acid metabolism and cellulase production, offering fresh prospects for the development of fungal cell factories dedicated to cellulolytic enzyme production in bio-refineries.

Consolidated bioprocessing for bioethanol production by metabolically engineered cellulolytic fungus Myceliophthora thermophila.

Using cellulosic ethanol as fuel is one way to help achieve the world's decarbonization goals. However, the economics of the present technology are unfavorable, especially the cost of cellulose degradation. Here, we reprogram the thermophilic cellulosic fungus Myceliophthora thermophila to directly ferment cellulose into ethanol by mimicking the aerobic ethanol fermentation of yeast (the Crabtree effect), including optimizing the synthetic pathway, enhancing the glycolytic rate, inhibiting mitochondrial NADH shuttles, and knocking out ethanol consumption pathway. The final engineered strain produced 52.8 g/L ethanol directly from cellulose, and 39.8 g/L from corncob, without the need for any added cellulase, while the starting strain produced almost no ethanol. We also demonstrate that as the ethanol fermentation by engineered M. thermophila increases, the composition and expression of cellulases that facilitate the degradation of cellulose, especially cellobiohydrolases, changes. The simplified production process and significantly increased ethanol yield indicate that the fungal consolidated bioprocessing technology that we develop here (one-step, one-strain ethanol production) is promising for fueling sustainable carbon-neutral biomanufacturing in the future.

Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants.

BACKGROUND: Glucoamylase is an important enzyme for starch saccharification in the food and biofuel industries and mainly produced from mesophilic fungi such as Aspergillus and Rhizopus species. Enzymes produced from thermophilic fungi can save the fermentation energy and reduce costs as compared to the fermentation system using mesophiles. Thermophilic fungus Myceliophthora thermophila is industrially deployed fungus to produce enzymes and biobased chemicals from biomass during optimal growth at 45 °C. This study aimed to construct the M. thermophila platform for glucoamylase hyper-production by broadening genomic targeting range of the AsCas12a variants, identifying key candidate genes and strain engineering. RESULTS: In this study, to increase the genome targeting range, we upgraded the CRISPR-Cas12a-mediated technique by engineering two AsCas12a variants carrying the mutations S542R/K607R and S542R/K548V/N552R. Using the engineered AsCas12a variants, we deleted identified key factors involved in the glucoamylase expression and secretion in M. thermophila, including Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2. Deletion of four targets led to more than 1.87- and 1.85-fold higher levels of secretion and glucoamylases activity compared to wild-type strain MtWT. Transcript level of the major amylolytic genes showed significantly increased in deletion mutants. The glucoamylase hyper-production strain MtGM12 was generated from our previously strain MtYM6 via genetically engineering these targets Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2 and overexpressing Mtamy1 and Mtpga3. Total secreted protein and activities of amylolytic enzymes in the MtGM12 were about 35.6-fold and 51.9‒55.5-fold higher than in MtWT. Transcriptional profiling analyses revealed that the amylolytic gene expression levels were significantly up-regulated in the MtGM12 than in MtWT. More interestingly, the MtGM12 showed predominantly short and highly bulging hyphae with proliferation of rough ER and abundant mitochondria, secretion vesicles and vacuoles when culturing on starch. CONCLUSIONS: Our results showed that these AsCas12a variants worked well for gene deletions in M. thermophila. We successfully constructed the glucoamylase hyper-production strain of M. thermophila by the rational redesigning and engineering the transcriptional regulatory and secretion pathway. This targeted engineering strategy will be very helpful to improve industrial fungal strains and promote the morphology engineering for enhanced enzyme production.

Fungal carboxylate transporters: recent manipulations and applications.

Carboxylic acids containing acidic groups with additional keto/hydroxyl-groups or unsaturated bond have displayed great applicability in the food, agricultural, cosmetic, textile, and pharmaceutical industries. The traditional approach for carboxylate production through chemical synthesis is based on petroleum derivatives, resulting in concerns for the environmental complication and energy crisis, and increasing attention has been attracted to the eco-friendly and renewable bio-based synthesis for carboxylate production. The efficient and specific export of target carboxylic acids through the microbial membrane is essential for high productivity, yield, and titer of bio-based carboxylates. Therefore, understanding the characteristics, regulations, and efflux mechanisms of carboxylate transporters will efficiently increase industrial biotechnological production of carboxylic acids. Several transporters from fungi have been reported and used for improved synthesis of target products. The transport activity and substrate specificity are two key issues that need further improvement in the application of carboxylate transporters. This review presents developments in the structural and functional diversity of carboxylate transporters, focusing on the modification and regulation of carboxylate transporters to alter the transport activity and substrate specificity, providing new strategy for transporter engineering in constructing microbial cell factory for carboxylate production. KEY POINTS: • Structures of multiple carboxylate transporters have been predicted. • Carboxylate transporters can efficiently improve production. • Modification engineering of carboxylate transporters will be more popular in the future.

Dissecting key residues of a C4-dicarboxylic acid transporter to accelerate malate export in Myceliophthora.

Efficient transporters are necessary for high concentration and purity of desired products during industrial production. In this study, we explored the mechanism of substrate transport and preference of the C4-dicarboxylic acid transporter AoMAE in the fungus Myceliophthora thermophila, and investigated the roles of 18 critical amino acid residues within this process. Among them, the residue Arg78, forming a hydrogen bond network with Arg23, Phe25, Thr74, Leu81, His82, and Glu94 to stabilize the protein conformation, is irreplaceable for the export function of AoMAE. Furthermore, varying the residue at position 100 resulted in changes to the size and shape of the substrate binding pocket, leading to alterations in transport efficiencies of both malic acid and succinic acid. We found that the mutation T100S increased malate production by 68%. Using these insights, we successfully generated an AoMAE variant with mutation T100S and deubiquitination, exhibiting an 81% increase in the selective export activity of malic acid. Simply introducing this version of AoMAE into M. thermophila wild-type strain increased production of malic acid from 1.22 to 54.88 g/L. These findings increase our understanding of the structure-function relationships of organic acid transporters and may accelerate the process of engineering dicarboxylic acid transporters with high efficiency. KEY POINTS: • This is the first systematical analysis of key residues of a malate transporter in fungi. • Protein engineering of AoMAE led to 81% increase of malate export activity. • Arg78 was essential for the normal function of AoMAE in M. thermophila. • Substitution of Thr100 affected export efficiency and substrate selectivity of AoMAE.

MtTRC-1, a Novel Transcription Factor, Regulates Cellulase Production via Directly Modulating the Genes Expression of the Mthac-1 and Mtcbh-1 in Myceliophthora thermophila.

The thermophilic fungus Myceliophthora thermophila has been used to produce industrial enzymes and biobased chemicals. In saprotrophic fungi, the mechanisms regulating cellulase production have been studied, which revealed the involvement of multiple transcription factors. However, in M. thermophila, the transcription factors influencing cellulase gene expression and secretion remain largely unknown. In this study, we identified and characterized a novel cellulase regulator (MtTRC-1) in M. thermophila through a combination of functional genomics and genetic analyses. Deletion of Mttrc-1 resulted in significantly decreased cellulase production and activities. Transcriptome analysis revealed downregulation of not only the encoding genes of main cellulases but also the transcriptional regulator MtHAC-1 of UPR pathway after disruption of MtTRC-1 under cellulolytic induction conditions. Herein, we also characterized the ortholog of the yeast HAC1p in M. thermophila. We show that Mthac-1 mRNA undergoes an endoplasmic reticulum (ER) stress-induced splicing by removing a 23-nucleotide (nt) intron. Notably, the protein secretion on cellulose was dramatically impaired by the deletion of MtHAC-1. Moreover, the colonial growth on various carbon sources was defective in the absence of MtHAC-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays verified MtTRC-1 regulates the transcription of Mthac-1 and the major cellulase gene Mtcbh-1 by binding directly to the promoters in vitro and in vivo. Furthermore, DNase I footprinting assays identified the putative consensus binding site (5'-GNG/C-3'). These results revealed the importance of MtTRC-1 for positively regulating cellulase production. This finding has clarified the complex regulatory pathways involved in cellulolytic enzyme production. IMPORTANCE In the present study, we characterized a novel regulator MtTRC-1 in M. thermophila, which regulated cellulase production through direct transcriptional regulation of the Mthac-1 and Mtcbh-1 genes. Our data demonstrated that MtHAC-1 is a key factor for the cellulase secretion capacity of M. thermophila. Our data indicate that this thermophilic fungus regulates cellulase production through a multilevels network, in which the protein secretory pathway is modulated by MtHAC-1-dependent UPR pathway and the cellulase gene expression is directly regulated in parallel by transcription factors. The conservation of Mttrc1 in filamentous fungi suggests this mechanism may be exploited to engineer filamentous fungal cell factories capable of producing proteins on an industrial scale.

Reconstruction and analysis of genome-scale metabolic model for thermophilic fungus Myceliophthora thermophila.

Myceliophthora thermophila, a thermophilic fungus that can degrade and utilize all major polysaccharides in plant biomass, has great potential in biotechnological industries. Here, the first manually curated genome-scale metabolic model iDL1450 for M. thermophila was reconstructed using an autogenerating pipeline with thorough manual curation. The model contains 1450 genes, 2592 reactions, and 1784 unique metabolites. High accuracy was shown in predictions related to carbon and nitrogen source utilization based on data obtained from Biolog experiments. Besides, metabolism profiles were analyzed using iDL1450 integrated with transcriptomics data of M. thermophila at various growth temperatures. The refined model provides new insights into thermophilic fungi metabolism and sheds light on model-driven strain design to improve biotechnological applications of this thermophilic lignocellulosic fungus.

Development of a flow cytometry-based plating-free system for strain engineering in industrial fungi.

Recent technical advances regarding filamentous fungi have accelerated the engineering of fungal-based production and benefited basic science. However, challenges still remain and limit the speed of fungal applications. For example, high-throughput technologies tailored to filamentous fungi are not yet commonly available for genetic modification. The currently used fungal genetic manipulations are time-consuming and laborious. Here, we developed a flow cytometry-based plating-free system to directly screen and isolate the transformed protoplasts in industrial fungi Myceliophthora thermophila and Aspergillus niger. This system combines genetic engineering via the 2A peptide and the CRISPR-Cas9 system, strain screening by flow cytometry, and direct sorting of colonies for deep-well-plate incubation and phenotypic analysis while avoiding culturing transformed protoplasts in plates, colony picking, conidiation, and cultivation. As a proof of concept, we successfully applied this system to generate the glucoamylase-hyperproducing strains MtYM6 and AnLM3 in M. thermophila and A. niger, respectively. Notably, the protein secretion level and enzyme activities in MtYM6 were 17.3- and 25.1-fold higher than in the host strain. Overall, these findings suggest that the flow cytometry-based plating-free system can be a convenient and efficient tool for strain engineering in fungal biotechnology. We expect this system to facilitate improvements of filamentous fungal strains for industrial applications. KEY POINTS: • Development of a flow cytometry-based plating-free (FCPF) system is presented. • Application of FCPF system in M. thermophila and A. niger for glucoamylase platform. • Hyper-produced strains MtYM6 and AnLM3 for glucoamylase production are generated.

STK-12 acts as a transcriptional brake to control the expression of cellulase-encoding genes in Neurospora crassa.

Cellulolytic fungi have evolved a complex regulatory network to maintain the precise balance of nutrients required for growth and hydrolytic enzyme production. When fungi are exposed to cellulose, the transcript levels of cellulase genes rapidly increase and then decline. However, the mechanisms underlying this bell-shaped expression pattern are unclear. We systematically screened a protein kinase deletion set in the filamentous fungus Neurospora crassa to search for mutants exhibiting aberrant expression patterns of cellulase genes. We observed that the loss of stk-12 (NCU07378) caused a dramatic increase in cellulase production and an extended period of high transcript abundance of major cellulase genes. These results suggested that stk-12 plays a critical role as a brake to turn down the transcription of cellulase genes to repress the overexpression of hydrolytic enzymes and prevent energy wastage. Transcriptional profiling analyses revealed that cellulase gene expression levels were maintained at high levels for 56 h in the Δstk-12 mutant, compared to only 8 h in the wild-type (WT) strain. After growth on cellulose for 3 days, the transcript levels of cellulase genes in the Δstk-12 mutant were 3.3-fold over WT, and clr-2 (encoding a transcriptional activator) was up-regulated in Δstk-12 while res-1 and rca-1 (encoding two cellulase repressors) were down-regulated. Consequently, total cellulase production in the Δstk-12 mutant was 7-fold higher than in the WT. These results strongly suggest that stk-12 deletion results in dysregulation of the cellulase expression machinery. Further analyses showed that STK-12 directly targets IGO-1 to regulate cellulase production. The TORC1 pathway promoted cellulase production, at least partly, by inhibiting STK-12 function, and STK-12 and CRE-1 functioned in parallel pathways to repress cellulase gene expression. Our results clarify how cellulase genes are repressed at the transcriptional level during cellulose induction, and highlight a new strategy to improve industrial fungal strains.

CLR-4, a novel conserved transcription factor for cellulase gene expression in ascomycete fungi.

Fungal degradation of lignocellulosic biomass requires various (hemi-)cellulases and is an important part of the natural carbon cycle. Although induction of cellulases has been described for some saprobic filamentous fungi, the regulation of cellulase transcription is complex and many aspects are still poorly understood. Here, we identified and characterized the novel cellulase regulation factor NcCLR-4 in Neurospora crassa and its ortholog MtCLR-4 in Myceliophthora thermophila. Deletion of CLR-4 resulted in similarly defective cellulolytic enzyme production and activities. Transcriptome analyses of ΔNcclr-4/ΔMtclr-4 revealed the down-regulation of genes encoding (hemi-)cellulases and pivotal regulators (clr-1, clr-2 and xyr-1) and key genes in the cAMP signaling pathway such as adenylate cyclase Nccr-1. Intracellular cAMP levels were markedly lower in ΔNcclr-4/ΔMtclr-4 than in wild-type during cellulose utilization. In electrophoretic mobility shift (EMSA) and DNase I footprinting assays, NcCLR-4/MtCLR-4 can directly bound to the promoters of Nccr-1/Mtcr-1 (encoding adenylyl cyclase). EMSAs also demonstrated that NcCLR-4/MtCLR-4 could directly bound to clr-1 (encoding a key cellulase regulator), Mtclr-2 and Mtxyr-1 (encoding biomass deconstruction regulators). These findings about the novel cellulase expression regulators NcCLR-4 and MtCLR-4 enrich our understanding of how cellulose degradation is regulated and provide new targets for engineering fungi to deconstruct plant biomass in biorefineries.

Direct production of commodity chemicals from lignocellulose using Myceliophthora thermophila.

The production of fuels and chemicals from renewable plant biomass has been proposed as a feasible strategy for global sustainable development. However, the economic efficiency of biorefineries is low. Here, through metabolic engineering, Myceliophthora thermophila, a cellulolytic thermophilic fungus, was constructed into a platform that can efficiently convert lignocellulose into important bulk chemicals-four carbon 1, 4-diacids (malic and succinic acid), building blocks for biopolymers-without the need for extra hydrolytic enzymes. Titers of >200 g/L from crystalline cellulose and 110 g/L from plant biomass (corncob) were achieved during fed-batch fermentation. Our study represents a milestone in consolidated bioprocessing technology and offers a new and promising system for the cost-effective production of chemicals and fuels from biomass.

Improving cellulases production by Myceliophthora thermophila through disruption of protease genes.

OBJECTIVE: To identify main protease genes for the proteolytic degradation of cellulases in M. thermophila and generate a lower-proteases fungal host that can be used for further metabolic engineering to increase cellulase production and heterologous protein expression. RESULTS: Systematic transcriptomic analysis were conducted on the expression of proteases genes in M. thermophila genome and five highly expressed genes encoding extracellular proteases were selected for mutation analyses. A series of single- and multi-gene mutants of these five selected genes was constructed using the CRISPR-Cas9 technique. Compared with WT, the ΔMtalp1 and the quintuple mutant showed significantly lower protease activity (decreased 52.7% and 58.4%, respectively) and at least double enhanced cellulase production. CONCLUSIONS: The results indicated that Mtalp1 is a critical protease gene in cellulase degradation in M. thermophila and disruption of protease genes showed significantly decreased protease activity and obviously enhanced cellulase production in the fermentation broth of ΔMtalp1 and the quintuple mutant.

Construction of a new thermophilic fungus Myceliophthora thermophila platform for enzyme production using a versatile 2A peptide strategy combined with efficient CRISPR-Cas9 system.

OBJECTIVE: To construct a new thermophilic platform for glucoamylase production through 2A peptide strategy combined with CRISPR-Cas9 system using Myceliophthora thermophila as host, thermophilic filamentous fungus with industrial attractiveness to produce enzymes and chemicals from biomass. RESULTS: We adapted the viral 2A peptide approach for M. thermophila and constructed a bicistronic vector for co-expressing two heterologous genes MhglaA and egfp. We obtained positive transformants OE-MhglaA-gfp overexpressing MhGlaA-9 ×His-2A-eGFP through convenient fluorescence screening, western blotting and RT-qPCR. We purified and characterized the recombinant MhGlaA, which exhibited stability in a broader pH range of 3.0-9.0 and thermostable stability at 65 °C, suggesting its potential industrial application. Furthermore, to improve glucoamylase secretion, we genetically engineered the obtained strain OE-MhglaA-gfp through our efficient CRISPR/Cas9 system and generated the quintuple mutant OE-MhglaA-gfpOE-amyRΔalp-1Δres-1Δcre-1, in which protein productivity and amylase activity were increased by approximately 12.0- and 8.2-fold compared with WT. CONCLUSIONS: The 2A peptide approach worked well in M. thermophila and can be used to heterologously co-express two different proteins, and thus in combination with efficient CRISPR-Cas system will accelerate establishing hyper-secretion platforms for biotechnological applications.

Disruption of gul-1 decreased the culture viscosity and improved protein secretion in the filamentous fungus Neurospora crassa.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Disruption of non-anchored cell wall protein NCW-1 promotes cellulase production by increasing cellobiose uptake in Neurospora crassa.

OBJECTIVES: To elucidate the mechanism of cellulase signal transduction in filamentous fungi including the components of the cellulase induction pathway. RESULTS: Neurospora crassa ncw-1 encodes a non-anchored cell wall protein. The absence of ncw-1 increased cellulase gene expression and this is not due to relieving carbon catabolite repression mediated by the cre-1 pathway. A mutant lacking genes encoding both three major ß-glucosidase enzymes and NCW-1 (Δ3ßGΔncw-1) was constructed. Transcriptome analysis of the quadruple mutant demonstrated enhanced expression of cellodextrin transporters after ncw-1 deletion, indicating that ncw-1 affects cellulase expression and production by inhibiting the uptake of the cellodextrin. CONCLUSIONS: NCW-1 is a novel component that plays a critical role in the cellulase induction signaling pathway.

The putative cellodextrin transporter-like protein CLP1 is involved in cellulase induction in Neurospora crassa.

Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3ßG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3ßG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators.

Heterologous expression of a GH3 ß-glucosidase from Neurospora crassa in Pichia pastoris with high purity and its application in the hydrolysis of soybean isoflavone glycosides.

Previous studies have shown isoflavone aglycones to have more biological effects than their counterparts, isoflavone glycones. Some ß-glucosidases can hydrolyze isoflavone glucosides to release aglycones, and discovery of these has attracted great interest. A glycoside hydrolase (GH) family 3 ß-glucosidase (bgl2) gene from Neurospora crassa was heterologously expressed in Pichia pastoris with high purity. The recombinant BGL2 enzyme displayed its highest activity at pH 5.0 and 60 °C, and had its maximum activity against p-nitrophenyl-ß-d-glucopyranoside (pNPG) (143.27 ± 4.79 U/mg), followed by cellobiose (74.99 ± 0.78 U/mg), gentiobiose (47.55 ± 0.15 U/mg), p-nitrophenyl-ß-d-cellobioside (pNPC) (40.07 ± 0.87 U/mg), cellotriose (12.31 ± 0.36 U/mg) and cellotetraose (9.04 ± 0.14 U/mg). The kinetic parameters of Km and Vmax were 0.21 ± 0.01 mM and 147.93 ± 2.77 µM/mg/min for pNPG. The purified enzyme showed a heightened ability to convert the major soybean isoflavone glycosides (daidzin, genistin and glycitin) into their corresponding aglycone forms (daidzien, genistein and glycitein). With this activity against soybean isoflavone glycosides, BGL2 shows great potential for applications in the food, animal feed, and pharmaceutical industries.

Unveiling equal importance of two 14-3-3 proteins for morphogenesis, conidiation, stress tolerance and virulence of an insect pathogen.

Two conserved 14-3-3 proteins orthologous to Saccharomyces cerevisiae Bmh1/2 are poorly understood in filamentous fungi. Here we show that Bmh1 and Bmh2 contribute equally to the fundamental biology and physiology of Beauveria bassiana by targeting many sets of proteins/enzymes. Single Bmh deletion caused similar upregulation of another. Excellent knockdown (∼91%) expressions of Bmh1 in ΔBmh2 and Bmh2 in ΔBmh1 resulted in equally more severe multiphenotypic defects than the single deletions, including G2 /M transition, blastospore size, carbon/nitrogen utilization, conidiation, germination and conidial tolerances to high osmolarity, oxidation, cell wall stress, high temperature and UV-B irradiation. All the deletion and deletion/knockdown mutants showed similar defects in blastospore yield and density, hyphal septation and cell size, hyphal responses to most chemical stresses and virulence. All the defects were evident with altered transcripts of phenotype-related genes and well restored by each Bmh complementation. Our Bmh1- and Bmh2-specific transcriptomes generated under osmotic and oxidative stresses revealed up to 6% genes differentially expressed by at least twofold in the fungal genome. Many of those were greatly depressed or co-depressed in ΔBmh1 and ΔBmh2. Our findings provide a thorough insight into the functions and complementary effects of the two 14-3-3 proteins in the filamentous entomopathogen.