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1.
J Agric Food Chem ; 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32054270

RESUMO

D-xylose is the most abundant hemicellulosic monomer on earth, but wild-type Saccharomyces cerevisiae has very limited D-xylose uptake capacity. We conducted bioprospecting for new sugar transporters from the D-xylose-consuming filamentous fungus Trichoderma reesei and identified three candidates belonging to the major facilitator superfamily. When they were expressed in yeast and assayed for D-xylose uptake, one of them, Xltr1p, had D-xylose transport activity that was more efficient than that of Gal2p, an endogenous yeast transporter. Site-directed mutagenesis was used to examine the functional contributions of 13 amino acid residues for the uptake of D-xylose, and these experiments identified particular amino acids that function distinctly in D-xylose vs. glucose transport (e.g., F300). Excitingly, the yeast strain expressing the N326FXltr1p variant was able to carry a 'high efficiency' transporter for D-xylose but was completely unable to utilize glucose; in contrast, the strain with the F300AXltr1p variant grew on glucose but lost D-xylose transport activity.

2.
Biotechnol Lett ; 42(2): 219-229, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31802333

RESUMO

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.

3.
PLoS Genet ; 15(11): e1008510, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31765390

RESUMO

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.

4.
Front Microbiol ; 10: 2317, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31736884

RESUMO

Fungal plant cell wall degradation processes are governed by complex regulatory mechanisms, allowing the organisms to adapt their metabolic program with high specificity to the available substrates. While the uptake of representative plant cell wall mono- and disaccharides is known to induce specific transcriptional and translational responses, the processes related to early signal reception and transduction remain largely unknown. A fast and reversible way of signal transmission are post-translational protein modifications, such as phosphorylations, which could initiate rapid adaptations of the fungal metabolism to a new condition. To elucidate how changes in the initial substrate recognition phase of Neurospora crassa affect the global phosphorylation pattern, phospho-proteomics was performed after a short (2 min) induction period with several plant cell wall-related mono- and disaccharides. The MS/MS-based peptide analysis revealed large-scale substrate-specific protein phosphorylation and de-phosphorylations. Using the proteins identified by MS/MS, a protein-protein-interaction (PPI) network was constructed. The variance in phosphorylation of a large number of kinases, phosphatases and transcription factors indicate the participation of many known signaling pathways, including circadian responses, two-component regulatory systems, MAP kinases as well as the cAMP-dependent and heterotrimeric G-protein pathways. Adenylate cyclase, a key component of the cAMP pathway, was identified as a potential hub for carbon source-specific differential protein interactions. In addition, four phosphorylated F-Box proteins were identified, two of which, Fbx-19 and Fbx-22, were found to be involved in carbon catabolite repression responses. Overall, these results provide unprecedented and detailed insights into a so far less well known stage of the fungal response to environmental cues and allow to better elucidate the molecular mechanisms of sensory perception and signal transduction during plant cell wall degradation.

5.
Artigo em Inglês | MEDLINE | ID: mdl-31754437

RESUMO

Background: Lignocellulosic biomass has long been recognized as a potential sustainable source for industrial applications. The costs associated with conversion of plant biomass to fermentable sugar represent a significant barrier to the production of cost-competitive biochemicals. Consolidated bioprocessing (CBP) is considered a potential breakthrough for achieving cost-efficient production of biomass-based fuels and commodity chemicals. During the degradation of cellulose, cellobiose (major end-product of cellulase activity) is catabolized by hydrolytic and phosphorolytic pathways in cellulolytic organisms. However, the details of the two intracellular cellobiose metabolism pathways in cellulolytic fungi remain to be uncovered. Results: Using the engineered malic acid production fungal strain JG207, we demonstrated that the hydrolytic pathway by ß-glucosidase and the phosphorolytic pathway by phosphorylase are both used for intracellular cellobiose metabolism in Myceliophthora thermophila, and the yield of malic acid can benefit from the energy advantages of phosphorolytic cleavage. There were obvious differences in regulation of the two cellobiose catabolic pathways depending on whether M. thermophila JG207 was grown on cellobiose or Avicel. Disruption of Mtcpp in strain JG207 led to decreased production of malic acid under cellobiose conditions, while expression levels of all three intracellular ß-glucosidase genes were significantly up-regulated to rescue the impairment of the phosphorolytic pathway under Avicel conditions. When the flux of the hydrolytic pathway was reduced, we found that ß-glucosidase encoded by bgl1 was the dominant enzyme in the hydrolytic pathway and deletion of bgl1 resulted in significant enhancement of protein secretion but reduction of malate production. Combining comprehensive manipulation of both cellobiose utilization pathways and enhancement of cellobiose uptake by overexpression of a cellobiose transporter, the final strain JG412Δbgl2Δbgl3 produced up to 101.2 g/L and 77.4 g/L malic acid from cellobiose and Avicel, respectively, which corresponded to respective yields of 1.35 g/g and 1.03 g/g, representing significant improvement over the starting strain JG207. Conclusions: This is the first report of detailed investigation of intracellular cellobiose catabolism in cellulolytic fungus M. thermophila. These results provide insights that can be applied to industrial fungi for production of biofuels and biochemicals from cellobiose and cellulose.

6.
mBio ; 10(4)2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266859

RESUMO

It is essential for microbes to acquire information about their environment. Fungi use soluble degradation products of plant cell wall components to understand the substrate composition they grow on. Individual perception pathways have been well described. However, the interconnections between pathways remain poorly understood. In the present work, we provide evidence of crosstalk between the perception pathways for cellulose and the hemicellulose mannan being conserved in several filamentous fungi and leading to the inhibition of cellulase expression. We used the functional genomics tools available for Neurospora crassa to investigate this overlap at the molecular level. Crosstalk and competitive inhibition could be identified both during uptake by cellodextrin transporters and intracellularly. Importantly, the overlap is independent of CRE-1-mediated catabolite repression. These results provide novel insights into the regulatory networks of lignocellulolytic fungi and will contribute to the rational optimization of fungal enzyme production for efficient plant biomass depolymerization and utilization.IMPORTANCE In fungi, the production of enzymes for polysaccharide degradation is controlled by complex signaling networks. Previously, these networks were studied in response to simple sugars or single polysaccharides. Here, we tackled for the first time the molecular interplay between two seemingly unrelated perception pathways: those for cellulose and the hemicellulose (gluco)mannan. We identified a so far unknown competitive inhibition between the respective degradation products acting as signaling molecules. Competition was detected both at the level of the uptake and intracellularly, upstream of the main transcriptional regulator CLR-2. Our findings provide novel insights into the molecular communication between perception pathways. Also, they present possible targets for the improvement of industrial strains for higher cellulase production through the engineering of mannan insensitivity.

7.
Metab Eng ; 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31078793

RESUMO

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.

8.
Nat Commun ; 10(1): 1378, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30914637

RESUMO

Acetyl-CoA is a fundamental metabolite for all life on Earth, and is also a key starting point for the biosynthesis of a variety of industrial chemicals and natural products. Here we design and construct a Synthetic Acetyl-CoA (SACA) pathway by repurposing glycolaldehyde synthase and acetyl-phosphate synthase. First, we design and engineer glycolaldehyde synthase to improve catalytic activity more than 70-fold, to condense two molecules of formaldehyde into one glycolaldehyde. Second, we repurpose a phosphoketolase to convert glycolaldehyde into acetyl-phosphate. We demonstrated the feasibility of the SACA pathway in vitro, achieving a carbon yield ~50%, and confirmed the SACA pathway by 13C-labeled metabolites. Finally, the SACA pathway was verified by cell growth using glycolaldehyde, formaldehyde and methanol as supplemental carbon source. The SACA pathway is proved to be the shortest, ATP-independent, carbon-conserving and oxygen-insensitive pathway for acetyl-CoA biosynthesis, opening possibilities for producing acetyl-CoA-derived chemicals from one-carbon resources in the future.


Assuntos
Acetaldeído/análogos & derivados , Acetilcoenzima A/biossíntese , Aldeído Liases/metabolismo , Carbono/metabolismo , Escherichia coli/metabolismo , Formaldeído/metabolismo , Organofosfatos/metabolismo , Acetaldeído/metabolismo , Aldeído Liases/genética , Escherichia coli/genética , Engenharia Metabólica , Plasmídeos
9.
Mol Microbiol ; 111(2): 373-394, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30474279

RESUMO

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.


Assuntos
Celulase/biossíntese , Regulação Fúngica da Expressão Gênica , Sordariales/enzimologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Sordariales/genética , Fatores de Transcrição/deficiência
10.
Biotechnol Biofuels ; 11: 323, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30534201

RESUMO

Background: Fumaric acid is widely used in food and pharmaceutical industries and is recognized as a versatile industrial chemical feedstock. Increasing concerns about energy and environmental problems have resulted in a focus on fumaric acid production by microbial fermentation via bioconversion of renewable feedstocks. Filamentous fungi are the predominant microorganisms used to produce organic acids, including fumaric acid, and most studies to date have focused on Rhizopus species. Thermophilic filamentous fungi have many advantages for the production of compounds by industrial fermentation. However, no previous studies have focused on fumaric acid production by thermophilic fungi. Results: We explored the feasibility of producing fumarate by metabolically engineering Myceliophthora thermophila using the CRISPR/Cas9 system. Screening of fumarases suggested that the fumarase from Candida krusei was the most suitable for efficient production of fumaric acid in M. thermophila. Introducing the C. krusei fumarase into M. thermophila increased the titer of fumaric acid by threefold. To further increase fumarate production, the intracellular fumarate digestion pathway was disrupted. After deletion of the two fumarate reductase and the mitochondrial fumarase genes of M. thermophila, the resulting strain exhibited a 2.33-fold increase in fumarate titer. Increasing the pool size of malate, the precursor of fumaric acid, significantly increased the final fumaric acid titer. Finally, disruption of the malate-aspartate shuttle increased the intracellular malate content by 2.16-fold and extracellular fumaric acid titer by 42%, compared with that of the parental strain. The strategic metabolic engineering of multiple genes resulted in a final strain that could produce up to 17 g/L fumaric acid from glucose in a fed-batch fermentation process. Conclusions: This is the first metabolic engineering study on the production of fumaric acid by the thermophilic filamentous fungus M. thermophila. This cellulolytic fungal platform provides a promising method for the sustainable and efficient-cost production of fumaric acid from lignocellulose-derived carbon sources in the future.

11.
Int J Biol Macromol ; 118(Pt A): 1035-1044, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-29964118

RESUMO

The lignocellulosic biomass usually need pre-treatment with acid at high temperature or solved in ionic liquid (IL) before cellulases hydrolysis. Thus, thermoacidophilic, thermostable and IL-tolerant cellulases are greatly desired, but rarely reported. In this study, a novel endo-ß-1,4-glucanase gene nmGH45 was directly cloned from saline-alkaline lake soil microbial metagenomic DNA and expressed in Pichia pastoris. Recombinant NMgh45 was active and stable at pH 3.0-9.0, with maximum activity at pH 4.5 and >80% residual activities at pH 3.0-11.0. It exhibited maximal activity at 60-70 °C and remaining >90% and 68% residual activities at 80 °C for 2 h and 90 °C for 1 h. Besides, NMgh45 retained >88% relative activities in 4 M NaCl, and was stable for 24 h. Significantly, it was highly IL-tolerant, remaining 92.8% and 43.8% residual activities in 10% and 20% 1-butyl-3-methylimidazolium chloride. Compared with other non-halo-tolerant counterparts, NMgh45 was enriched with acidic amino acids on the protein surface, and the conformation of NMgh45 was stable at high salinity condition based on molecular dynamic simulation. Finally, the productivity of recombinant NMgh45 was optimized at multi-levels. The thermoacidphilic and acid-alkaline-halo-tolerant properties make NMgh45 a promising enzyme for basic research and industrial applications.


Assuntos
Celulase/química , DNA , Lagos/microbiologia , Metagenoma , Microbiologia do Solo , Microbiologia da Água , Celulase/genética , Celulase/metabolismo , Líquidos Iônicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
Microb Cell Fact ; 17(1): 96, 2018 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-29908565

RESUMO

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.


Assuntos
Proteínas Fúngicas/genética , Neurospora crassa/genética , Viscosidade
13.
Bioresour Technol ; 265: 558-562, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29843921

RESUMO

Thermophilic fungus Myceliophthora thermophila has great capacity for biomass degradation and is an attractive option for use as cell factory to produce chemicals directly from renewable polysaccharides, such as starch, rather than monomer glucose. To date, there has been no transcriptomic analysis of this thermophilic fungus on starch. This study determined the transcriptomic profile of M. thermophila responding to soluble starch and a 342-gene set was identified as a "starch regulon", including the major amylolytic enzyme (Mycth_72393). Its overexpression led to increased amylase activities on starch by 35%. Furthermore, overexpressing the key amylolytic enzyme regulator AmyR in M. thermophila significantly increased amylase activity by 30%. Deletion of amyR by the CRISPR/Cas9 system led to the relief of carbon catabolite repression and 3-fold increased lignocellulase activities on cellulose. This study will accelerate rational fungal strain engineering for biochemical production from biomass substrates such as raw corn starch and even crop straw.


Assuntos
Perfilação da Expressão Gênica , Sordariales , Amido/metabolismo , Amilases , Repressão Catabólica , Sordariales/enzimologia , Sordariales/genética
14.
Free Radic Biol Med ; 117: 218-227, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29421311

RESUMO

Catalase is an important enzyme found in nearly all aerobic organisms and plays an essential role in protecting cells from oxidative damage by catalyzing the degradation of hydrogen peroxide into water and oxygen. In filamentous fungus Neurospora crassa, the expression levels of catalases are rigorously regulated by morphogenetic transition during growth and development in cells. Our study revealed that catalase-3 transcription is positively regulated by histone acetyltransferase GCN5 and the cross-pathway control gene cpc-1, as the cat-3 expression level is significantly decreased in gcn5KO and cpc-1 (j-5) mutants. Moreover, gcn5KO and cpc-1 (j-5) mutants could not respond to H2O2 treatment due to the inadequate cat-3 transcription, while wild-type strains showed high expression levels of catalase upon H2O2 treatment. The global H3 acetylation and the acetylation of H3 at cat-3 locus dramatically decreased in gcn5KO under normal or oxidative stress conditions. Meanwhile, the expression of CAT-3 is reduced in gcn5E146Q, the catalytically dead mutant, suggesting that the catalytic activity of GCN5 functions in regulation of cat-3 transcription. In addition, GCN5 cannot acetylate histone H3 efficiently at cat-3 locus in cpc-1 (j-5) mutant strains under normal or oxidative stress conditions. Furthermore, ChIP assays data revealed that the CPC1/GCN4 can directly target the cat-3 promoter region, which may recruit GCN5 to modify the histone acetylation of this region. These results disclosed a distinctive function of CPC1/GCN4 in the regulatory pathway of cat-3 transcription, which is mediated by GCN5-dependent acetylation.


Assuntos
Catalase/biossíntese , Proteínas Fúngicas/metabolismo , Genes Fúngicos/fisiologia , Neurospora crassa/fisiologia , Estresse Oxidativo/fisiologia , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/metabolismo
15.
Sheng Wu Gong Cheng Xue Bao ; 33(1): 79-89, 2017 Jan 25.
Artigo em Chinês | MEDLINE | ID: mdl-28959865

RESUMO

The lignocellulolytic filamentous fungus Neurospora crassa is able to assimilate various mono- and oligo-saccharides. However, more than half of predicted sugar transporters in the genome are still waiting for functional elucidation. In this study, system analysis of substrate spectra of predicted sugar transporters in N. crassa was performed at genome-wide level. NCU01868 and NCU08152 have the capability of uptaking various hexose, which are named as NcHXT-1 and NcHXT-2 respectively. Their transport activities for glucose were further confirmed by fluorescence resonance energy transfer analysis. Over-expression of either NcHXT-1 or NcHXT-2 in the null-hexose-transporter yeast EBY.VW4000 restored the growth and ethanol fermentation under submerged fermentation with glucose, galactose, or mannose as the sole carbon source. NcHXT-1/-2 homologues were found in a variety of cellulolytic fungi. Functional identification of two filamentous fungal-conserved hexose transporters NcHXT-1/-2 via genome scanning would represent novel targets for ongoing efforts in engineering cellulolytic fungi and hexose fermentation in yeast.


Assuntos
Fermentação , Neurospora crassa/metabolismo , Saccharomyces cerevisiae/metabolismo , Açúcares/metabolismo , Transporte Biológico , Carboidratos , Galactose , Glucose , Hexoses , Proteínas de Transporte de Monossacarídeos
16.
Biotechnol Lett ; 39(4): 545-551, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28039555

RESUMO

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.


Assuntos
Celobiose/metabolismo , Celulase/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Neurospora crassa/enzimologia , Transdução de Sinais , Parede Celular/metabolismo , Celulase/genética , Celulose/análogos & derivados , Celulose/metabolismo , Dextrinas/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Neurospora crassa/genética , beta-Glucosidase/metabolismo
17.
Biotechnol Biofuels ; 10: 1, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28053662

RESUMO

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

18.
Biotechnol Biofuels ; 10: 17, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28115989

RESUMO

BACKGROUND: 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. RESULTS: Using RNA sequencing coupled with functional transport analyses, we assigned GLT-1 (Km = 18.42 ± 3.38 mM) and HGT-1/-2 (Km = 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." CONCLUSIONS: 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.

19.
Protein Expr Purif ; 119: 75-84, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26596358

RESUMO

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.


Assuntos
Proteínas Fúngicas/biossíntese , Glicosídeos/química , Isoflavonas/química , beta-Glucosidase/biossíntese , Sequência de Aminoácidos , Celobiose/química , Cromatografia de Afinidade , Sequência Conservada , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Expressão Gênica , Glucose/química , Glicosilação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Neurospora crassa/enzimologia , Pichia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Soja/química , Especificidade por Substrato , beta-Glucosidase/química , beta-Glucosidase/isolamento & purificação
20.
Biotechnol Biofuels ; 8: 124, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26300971

RESUMO

BACKGROUND: Lignocellulase hypersecretion has been achieved in industrial fungal workhorses such as Trichoderma reesei, but the underlying mechanism associated with this process is not well understood. Although previous comparative genomic studies have revealed that the mutagenic T. reesei strain RUT-C30 harbors hundreds of mutations compared with its parental strain QM6a, how these mutations actually contribute to the hypersecretion phenotype remains to be elucidated. RESULTS: In this study, we systematically screened gene knockout (KO) mutants in the cellulolytic fungus Neurospora crassa, which contains orthologs of potentially defective T. reesei RUT-C30 mutated genes. Of the 86 deletion mutants screened in N. crassa, 12 exhibited lignocellulase production more than 25% higher than in the wild-type (WT) strain and 4 showed nearly 25% lower secretion. We observed that the deletion of Ncap3m (NCU03998), which encodes the µ subunit of the adaptor protein 3 (AP-3) complex in N. crassa, led to the most significant increase in lignocellulase secretion under both Avicel and xylan culture conditions. Moreover, strains lacking the ß subunit of the AP-3 complex, encoded by Ncap3b (NCU06569), had a similar phenotype to ΔNcap3m, suggesting that the AP-3 complex is involved in lignocellulase secretion in N. crassa. We also found that the transcriptional abundance of major lignocellulase genes in ΔNcap3m was maintained at a relatively higher level during the late stage of fermentation compared with the WT, which might add to the hypersecretion phenotype. Finally, we found that importation of the T. reesei ap3m ortholog Trap3m into ΔNcap3m can genetically restore secretion of lignocellulases to normal levels, which suggests that the effect of the AP-3 complex on lignocellulase secretion is conserved in cellulolytic ascomycetes. CONCLUSIONS: Using the model cellulolytic fungus N. crassa, we explored potential hypersecretion-related mutations in T. reesei strain RUT-C30. Through systematic genetic screening of 86 corresponding orthologous KO mutants in N. crassa, we identified several genes, particularly those encoding the AP-3 complex that contribute to lignocellulase secretion. These findings will be useful for strain improvement in future lignocellulase and biomass-based chemical production.

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