Quantitative Proteome Profiling Reveals Cellobiose-Dependent Protein Processing and Export Pathways for the Lignocellulolytic Response in Neurospora crassa

ABSTRACT Filamentous fungi are intensively used for producing industrial enzymes, including lignocellulases. Employing insoluble cellulose to induce the production of lignocellulases causes some drawbacks, e.g., a complex fermentation operation, which can be overcome by using soluble inducers such as cellobiose. Here, a triple β-glucosidase mutant of Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to profile the proteome responses to cellobiose and cellulose (Avicel). Our results revealed a shared proteomic response to cellobiose and Avicel, whose elements included lignocellulases and cellulolytic product transporters. While the cellulolytic proteins showed a correlated increase in protein and mRNA levels, only a moderate correlation was observed on a proteomic scale between protein and mRNA levels (R2 = 0.31). Ribosome biogenesis and rRNA processing were significantly overrepresented in the protein set with increased protein but unchanged mRNA abundances in response to Avicel. Ribosome biogenesis, as well as protein processing and protein export, was also enriched in the protein set that showed increased abundance in response to cellobiose. NCU05895, a homolog of yeast CWH43, is potentially involved in transferring a glycosylphosphatidylinositol (GPI) anchor to nascent proteins. This protein showed increased abundance but no significant change in mRNA levels. Disruption of CWH43 resulted in a significant decrease in cellulase activities and secreted protein levels in cultures grown on Avicel, suggesting a positive regulatory role for CWH43 in cellulase production. The findings should have an impact on a systems engineering approach for strain improvement for the production of lignocellulases. IMPORTANCE Lignocellulases are important industrial enzymes for sustainable production of biofuels and bio-products. Insoluble cellulose has been commonly used to induce the production of lignocellulases in filamentous fungi, which causes a difficult fermentation operation and enzyme loss due to adsorption to cellulose. The disadvantages can be overcome by using soluble inducers, such as the disaccharide cellobiose. Quantitative proteome profiling of the model filamentous fungus Neurospora crassa revealed cellobiose-dependent pathways for cellulase production, including protein processing and export. A protein (CWH43) potentially involved in protein processing was found to be a positive regulator of lignocellulase production. The cellobiose-dependent mechanisms provide new opportunities to improve the production of lignocellulases in filamentous fungi.

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Deciphering Transcriptional Regulatory Mechanisms Associated with Hemicellulose Degradation in Neurospora crassa

Eukaryotic Cell ◽

10.1128/ec.05327-11 ◽

2012 ◽

Vol 11 (4) ◽

pp. 482-493 ◽

Cited By ~ 111

Author(s):

Jianping Sun ◽

Chaoguang Tian ◽

Spencer Diamond ◽

N. Louise Glass

Keyword(s):

Neurospora Crassa ◽

Filamentous Fungi ◽

Transcriptome Analysis ◽

Plant Cell Wall ◽

Value Added ◽

Regulatory Mechanisms ◽

Transcriptional Level ◽

Cellulolytic Activity ◽

Content Type ◽

Hemicellulose Degradation

ABSTRACTHemicellulose, the second most abundant plant biomass fraction after cellulose, is widely viewed as a potential substrate for the production of liquid fuels and other value-added materials. Degradation of hemicellulose by filamentous fungi requires production of many different enzymes, which are induced by biopolymers or its derivatives and regulated mainly at the transcriptional level through transcription factors (TFs).Neurospora crassa, a model filamentous fungus, expresses and secretes enzymes required for plant cell wall deconstruction. To better understand genes specifically associated with degradation of hemicellulose, we applied secretome and transcriptome analysis toN. crassagrown on beechwood xylan. We identified 34 secreted proteins and 353 genes with elevated transcription on xylan. The xylanolytic phenotype of strains with deletions in genes identified from the secretome and transcriptome analysis of the wild type was assessed, revealing functions for known and unknown proteins associated with hemicellulose degradation. By evaluating phenotypes of strains containing deletions of predicted TF genes inN. crassa, we identified a TF (XLR-1;xylan degradationregulator1) essential for hemicellulose degradation that is an ortholog to XlnR/XYR1 inAspergillusandTrichodermaspecies, respectively, a major transcriptional regulator of genes encoding both cellulases and hemicellulases. Deletion ofxlr-1inN. crassaabolished growth on xylan and xylose, but growth on cellulose and cellulolytic activity were only slightly affected. To determine the regulatory mechanisms for hemicellulose degradation, we explored the transcriptional regulon of XLR-1 under xylose, xylanolytic, and cellulolytic conditions. XLR-1 regulated only some predicted hemicellulase genes inN. crassaand was required for a full induction of several cellulase genes. Hemicellulase gene expression was induced by a combination of release from carbon catabolite repression (CCR) and induction. This systematic analysis illustrates the similarities and differences in regulation of hemicellulose degradation among filamentous fungi.

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Crosstalk of Cellulose and Mannan Perception Pathways Leads to Inhibition of Cellulase Production in Several Filamentous Fungi

mBio ◽

10.1128/mbio.00277-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 8

Author(s):

Lara Hassan ◽

Liangcai Lin ◽

Hagit Sorek ◽

Laura E. Sperl ◽

Thomas Goudoulas ◽

...

Keyword(s):

Filamentous Fungi ◽

Regulatory Networks ◽

Plant Cell Wall ◽

Competitive Inhibition ◽

Plant Biomass ◽

Degradation Products ◽

Cellulase Production ◽

Content Type ◽

Industrial Strains ◽

Genomics Tools

ABSTRACTIt 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 forNeurospora crassato 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.IMPORTANCEIn 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.

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The Oxygenase CAO-1 of Neurospora crassa Is a Resveratrol Cleavage Enzyme

Eukaryotic Cell ◽

10.1128/ec.00084-13 ◽

2013 ◽

Vol 12 (9) ◽

pp. 1305-1314 ◽

Cited By ~ 19

Author(s):

Violeta Díaz-Sánchez ◽

Alejandro F. Estrada ◽

M. Carmen Limón ◽

Salim Al-Babili ◽

Javier Avalos

Keyword(s):

Neurospora Crassa ◽

Sequence Similarity ◽

Hydroxyl Groups ◽

Mrna Levels ◽

Pronounced Increase ◽

Content Type ◽

Carotenoid Metabolism ◽

Cleavage Enzyme ◽

Β Carotene

ABSTRACT The genome of the ascomycete Neurospora crassa encodes CAO-1 and CAO-2, two members of the carotenoid cleavage oxygenase family that target double bonds in different substrates. Previous studies demonstrated the role of CAO-2 in cleaving the C 40 carotene torulene, a key step in the synthesis of the C 35 apocarotenoid pigment neurosporaxanthin. In this work, we investigated the activity of CAO-1, assuming that it may provide retinal, the chromophore of the NOP-1 rhodopsin, by cleaving β-carotene. For this purpose, we tested CAO-1 activity with carotenoid substrates that were, however, not converted. In contrast and consistent with its sequence similarity to family members that act on stilbenes, CAO-1 cleaved the interphenyl Cα-Cβ double bond of resveratrol and its derivative piceatannol. CAO-1 did not convert five other similar stilbenes, indicating a requirement for a minimal number of unmodified hydroxyl groups in the stilbene background. Confirming its biological function in converting stilbenes, adding resveratrol led to a pronounced increase in cao-1 mRNA levels, while light, a key regulator of carotenoid metabolism, did not alter them. Targeted Δ cao-1 mutants were not impaired by the presence of resveratrol, a phytoalexin active against different fungi, which did not significantly affect the growth and development of wild-type Neurospora . However, under partial sorbose toxicity, the Δ cao-1 colonies exhibited faster radial growth than control strains in the presence of resveratrol, suggesting a moderate toxic effect of resveratrol cleavage products.

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Engineering Neurospora crassa for Improved Cellobiose and Cellobionate Production

Applied and Environmental Microbiology ◽

10.1128/aem.02885-14 ◽

2014 ◽

Vol 81 (2) ◽

pp. 597-603 ◽

Cited By ~ 22

Author(s):

Amanda Hildebrand ◽

Edyta Szewczyk ◽

Hui Lin ◽

Takao Kasuga ◽

Zhiliang Fan

Keyword(s):

Neurospora Crassa ◽

Catabolite Repression ◽

Cellulase Production ◽

Cellobiose Dehydrogenase ◽

Hydrolysis Rate ◽

Wild Type ◽

Content Type ◽

Product Consumption ◽

Cellulase Expression ◽

Engineered Strain

ABSTRACTWe report engineeringNeurospora crassato improve the yield of cellobiose and cellobionate from cellulose. A previously engineered strain ofN. crassa(F5) with six of seven β-glucosidase (bgl) genes knocked out was shown to produce cellobiose and cellobionate directly from cellulose without the addition of exogenous cellulases. In this study, the F5 strain was further modified to improve the yield of cellobiose and cellobionate from cellulose by increasing cellulase production and decreasing product consumption. The effects of two catabolite repression genes,cre-1andace-1, on cellulase production were investigated. The F5 Δace-1mutant showed no improvement over the wild type. The F5 Δcre-1and F5 Δace-1Δcre-1strains showed improved cellobiose dehydrogenase and exoglucanase expression. However, this improvement in cellulase expression did not lead to an improvement in cellobiose or cellobionate production. The cellobionate phosphorylase gene (ndvB) was deleted from the genome of F5 Δace-1Δcre-1to prevent the consumption of cellobiose and cellobionate. Despite a slightly reduced hydrolysis rate, the F5 Δace-1Δcre-1ΔndvBstrain converted 75% of the cellulose consumed to the desired products, cellobiose and cellobionate, compared to 18% converted by the strain F5 Δace-1Δcre-1.

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Strain Improvement of Trichoderma spp. Through Two Steps Protoplast Fusion for Cellulase Production Enhancement

Canadian Journal of Microbiology ◽

10.1139/cjm-2020-0438 ◽

2020 ◽

Author(s):

Zahra Papzan ◽

Mojegan Kowsari ◽

Mohammad Javan-Nikkhah ◽

Amir Mirzadi Gohari ◽

M. Carmen Limón

Keyword(s):

Protoplast Fusion ◽

Strain Improvement ◽

Cellulase Activity ◽

Cellulase Production ◽

Mrna Levels ◽

Hybrid Strain ◽

Trichoderma Spp ◽

Wide Range ◽

Natural Isolates ◽

Rut C30

Fungal protoplast fusion is an approach to introduce novel characteristics into industrially important strains. Cellulases, essential enzymes with a wide range of biotechnological applications, are produced by many species of the filamentous fungi Trichoderma. In this study, a collection of 60 natural isolates have been screened for Avicel and CMC degradation, and two cellulase producers of Trichoderma virens and Trichoderma harzianum were used for protoplast fusion. One of resulting hybrids with an improvement in cellulase activity, C1-3, was fused with the hyper producer Trichoderma reesei Rut-C30. A new selected hybrid, F7, increased its cellulase activity 1.8 and 5 times in comparison with Rut-C30 and C1-3, respectively. The increases in enzyme activity correlated with an upregulation of cellulolytic genes cbh1, cbh2, egl3, and bgl1 in the parents. Amount of mRNA of cbh1 and cbh2 in F7 resembled Rut-C30 while the bgl1 mRNA levels were similar to C1-3. AFLP fingerprinting and GC-MS analysis represented variations in parental strains and fusants. In conclusion, results demonstrate that a 3-interspecific hybrid strain has been isolated with improved characteristics for cellulase degradation showing genetic polymorphisms and differences in the volatile profile which suggests reorganizations at genetic level.

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The Predicted Mannosyltransferase GT69-2 Antagonizes RFW-1 To Regulate Cell Fusion in Neurospora crassa

mBio ◽

10.1128/mbio.00307-21 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Yang Li ◽

Jens Heller ◽

A. Pedro Gonçalves ◽

N. Louise Glass

Keyword(s):

Cell Wall ◽

Neurospora Crassa ◽

Somatic Cell ◽

Filamentous Fungi ◽

Cell Fusion ◽

Wild Type ◽

Content Type ◽

Asexual Sporulation ◽

Somatic Cell Fusion ◽

Cell Wall Remodeling

ABSTRACT Filamentous fungi undergo somatic cell fusion to create a syncytial, interconnected hyphal network which confers a fitness benefit during colony establishment. However, barriers to somatic cell fusion between genetically different cells have evolved that reduce invasion by parasites or exploitation by maladapted genetic entities (cheaters). Here, we identified a predicted mannosyltransferase, glycosyltransferase family 69 protein (GT69-2) that was required for somatic cell fusion in Neurospora crassa. Cells lacking GT69-2 prematurely ceased chemotropic signaling and failed to complete cell wall dissolution and membrane merger in pairings with wild-type cells or between Δgt69-2 cells (self fusion). However, loss-of-function mutations in the linked regulator of cell fusion and cell wall remodeling-1 (rfw-1) locus suppressed the self-cell-fusion defects of Δgt69-2 cells, although Δgt69-2 Δrfw-1 double mutants still failed to undergo fusion with wild-type cells. Both GT69-2 and RFW-1 localized to the Golgi apparatus. Genetic analyses indicated that RFW-1 negatively regulates cell wall remodeling-dependent processes, including cell wall dissolution during cell fusion, separation of conidia during asexual sporulation, and conidial germination. GT69-2 acts as an antagonizer to relieve or prevent negative functions on cell fusion by RFW-1. In Neurospora species and N. crassa populations, alleles of gt69-2 were highly polymorphic and fell into two discrete haplogroups. In all isolates within haplogroup I, rfw-1 was conserved and linked to gt69-2. All isolates within haplogroup II lacked rfw-1. These data indicated that gt69-2/rfw-1 are under balancing selection and provide new mechanisms regulating cell wall remodeling during cell fusion and conidial separation. IMPORTANCE Cell wall remodeling is a dynamic process that balances cell wall integrity versus cell wall dissolution. In filamentous fungi, cell wall dissolution is required for somatic cell fusion and conidial separation during asexual sporulation. In the filamentous fungus Neurospora crassa, allorecognition checkpoints regulate the cell fusion process between genetically different cells. Our study revealed two linked loci with transspecies polymorphisms and under coevolution, rfw-1 and gt69-2, which form a coordinated system to regulate cell wall remodeling during somatic cell fusion, conidial separation, and asexual spore germination. RFW-1 acts as a negative regulator of these three processes, while GT69-2 functions antagonistically to RFW-1. Our findings provide new insight into the mechanisms involved in regulation of fungal cell wall remodeling during growth and development.

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Aspects of the Neurospora crassa Sulfur Starvation Response Are Revealed by Transcriptional Profiling and DNA Affinity Purification Sequencing

mSphere ◽

10.1128/msphere.00564-21 ◽

2021 ◽

Author(s):

Lori B. Huberman ◽

Vincent W. Wu ◽

Juna Lee ◽

Chris Daum ◽

Ronan C. O’Malley ◽

...

Keyword(s):

Neurospora Crassa ◽

Filamentous Fungi ◽

Affinity Purification ◽

Transcriptional Profiling ◽

Nutrient Source ◽

Starvation Response ◽

Content Type ◽

Sulfur Starvation ◽

Electron Carriers

Identification of nutrients present in the environment is a challenge common to all organisms. Sulfur is an important nutrient source found in proteins, lipids, and electron carriers that are required for the survival of filamentous fungi such as Neurospora crassa .

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Intron Retention Coupled with Nonsense-Mediated Decay is Involved in Cellulase Biosynthesis in Cellulolytic Fungi

10.21203/rs.3.rs-1214111/v1 ◽

2022 ◽

Author(s):

Yichen Gao ◽

Ai-Ping Pang ◽

Leyao Ma ◽

Haiyan Wang ◽

Samran Durrani ◽

...

Keyword(s):

Gene Expression ◽

Filamentous Fungi ◽

Regulatory Networks ◽

Intron Retention ◽

Low Cost ◽

Cellulase Production ◽

Mrna Levels ◽

Nonsense Mediated Decay ◽

Tor Pathway ◽

Regulatory Systems

Abstract Background Knowledge on regulatory networks associated with cellulase biosynthesis is prerequisite for exploitation of such regulatory systems in ehancing cellulase production with low cost. The biological functions of intron retention (IR) and nonsense-mediated mRNA decay (NMD) in filamentous fungi is lack of study, let alone their roles in cellulase biosynthesis. Result We found that major cellulase genes (cel7a, cel7b, and cel3a) exhibited concomitant decrease in IR rates and increase in their gene expression in T. reesei under cellulase-producing condition (cellulose and lactose) that was accompanied with a more active NMD pathway, as compared to non cellulase-producing condition (glucose). In the presence of the NMD pathway inhibitor that successfully repressed the NMD pathway, the mRNA levels of cellulase genes were sharply down-regulated, but the rates of IR in these genes were significantly up-regulated. Consistently, the cellulase activities were severely inhibited. In addition, the NMD pathway inhibitor caused the downregulated mRNA levels of two important genes of the target of rapamycin (TOR) pathway, trfkbp12 and trTOR1. The absence of gene trfkbp12 made the cellulase production in T. reesei more sensitive to the NMD pathway inhibitor. Conclusion All these findings suggest that the IR of cellulase genes regulates their own gene expression by coupling with the NMD pathway, which might involve the TOR pathway. Our results provide better understanding on intron retention, the NMD pathway, and cellulase production mechanism in filamentous fungi.

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Reprogramming mRNA Expression in Response to Defect in RNA Polymerase III Assembly in the Yeast Saccharomyces cerevisiae

International Journal of Molecular Sciences ◽

10.3390/ijms22147298 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7298

Author(s):

Izabela Rudzińska ◽

Małgorzata Cieśla ◽

Tomasz W. Turowski ◽

Alicja Armatowska ◽

Ewa Leśniewska ◽

...

Keyword(s):

Mrna Expression ◽

Ribosome Biogenesis ◽

Rna Polymerase Iii ◽

Mrna Levels ◽

Rna Seq ◽

Yeast Saccharomyces Cerevisiae ◽

Protein Coding ◽

General Transcription Factor ◽

Pol I ◽

Pol Iii

The coordinated transcription of the genome is the fundamental mechanism in molecular biology. Transcription in eukaryotes is carried out by three main RNA polymerases: Pol I, II, and III. One basic problem is how a decrease in tRNA levels, by downregulating Pol III efficiency, influences the expression pattern of protein-coding genes. The purpose of this study was to determine the mRNA levels in the yeast mutant rpc128-1007 and its overdose suppressors, RBS1 and PRT1. The rpc128-1007 mutant prevents assembly of the Pol III complex and functionally mimics similar mutations in human Pol III, which cause hypomyelinating leukodystrophies. We applied RNAseq followed by the hierarchical clustering of our complete RNA-seq transcriptome and functional analysis of genes from the clusters. mRNA upregulation in rpc128-1007 cells was generally stronger than downregulation. The observed induction of mRNA expression was mostly indirect and resulted from the derepression of general transcription factor Gcn4, differently modulated by suppressor genes. rpc128-1007 mutation, regardless of the presence of suppressors, also resulted in a weak increase in the expression of ribosome biogenesis genes. mRNA genes that were downregulated by the reduction of Pol III assembly comprise the proteasome complex. In summary, our results provide the regulatory links affected by Pol III assembly that contribute differently to cellular fitness.

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Production and optimization of cellulase from agricultural waste and its application in bioethanol production by simultaneous saccharification and fermentation

Management of Environmental Quality An International Journal ◽

10.1108/meq-07-2015-0128 ◽

2016 ◽

Vol 27 (1) ◽

pp. 22-35 ◽

Cited By ~ 2

Author(s):

Elsa Cherian ◽

M. Dharmendira Kumar ◽

G. Baskar

Keyword(s):

Design Methodology ◽

Simultaneous Saccharification And Fermentation ◽

Agricultural Waste ◽

Cellulase Production ◽

Reducing Sugar ◽

Bioethanol Production ◽

Corn Cob ◽

Content Type ◽

Optimized Conditions ◽

Simultaneous Saccharification

Purpose – The purpose of this paper is to optimize production of cellulase enzyme from agricultural waste by using Aspergillus fumigatus JCF. The study also aims at the production of bioethanol using cellulase and yeast. Design/methodology/approach – Cellulase production was carried out using modified Mandel’s medium. The optimization of the cellulase production was carried out using Plackett-Burman and Response surface methodology. Bioethanol production was carried out using simultaneous saccharification and fermentation. Findings – Maximum cellulase production at optimized conditions was found to be 2.08 IU/ml. Cellulase was used for the saccharification of three different feed stocks, i.e. sugar cane leaves, corn cob and water hyacinth. Highest amount of reducing sugar was released was 29.1 gm/l from sugarcane leaves. Sugarcane leaves produced maximum bioethanol concentration of 9.43 g/l out of the three substrates studied for bioethanol production. Originality/value – The present study reveals that by using the agricultural wastes, cellulase production can be economically increased thereby bioethanol production.

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