Cellulase Regulation Research Articles

Review of Recent Advances in the Physiology of the Regulation of Cellulase and Xylanase Production by Basidiomycetes

The potential of wood-rotting and litter-deconstructing basidiomycetes to convert lignocellulose into a wide variety of products has been extensively studied. In particular, wood-rotting basidiomycete secretomes are attracting much attention from researchers and biotechnology companies due to their ability to produce extracellular hydrolytic and oxidative enzymes that effectively degrade cellulose, hemicellulose, and lignin of plant biomass. An analysis of the available literature data shows that Basidiomycota fungi, which are most adapted to the depolymerization of plant polysaccharides, are promising but so far unexploited sources of new hydrolytic enzymes. The review summarizes the latest data on the great variety, common features, and unique properties of individual fungi and the production of cellulases and xylanases by various physiological and ecological groups of basidiomycetes. The most important microbial cellulase-producing strains for submerged and solid-phase fermentation, as well as the main substrates, including the use of agro-industrial waste, are considered. It highlights ways to increase both cellulase and xylanase expression levels and the cost-effectiveness of producing these enzymes for various biotechnological applications. It is anticipated that this review will be particularly useful to novice scientists working in the lignocellulose biorefinery, as it describes current knowledge and issues related to the production and regulation of polysaccharide hydrolyzing enzyme synthesis.

Novel Transcription Factor CXRD Regulates Cellulase and Xylanase Biosynthesis in Penicillium oxalicum under Solid-State Fermentation.

Penicillium oxalicum produces an integrated, extracellular cellulase and xylanase system, strictly regulated by several transcription factors. However, the understanding of the regulatory mechanism of cellulase and xylanase biosynthesis in P. oxalicum is limited, particularly under solid-state fermentation (SSF) conditions. In our study, deletion of a novel gene, cxrD (cellulolytic and xylanolytic regulator D), resulted in 49.3 to 2,230% enhanced production of cellulase and xylanase, except for 75.0% less xylanase at 2 days, compared with the P. oxalicum parental strain, when cultured on solid medium containing wheat bran plus rice straw for 2 to 4 days after transfer from glucose. In addition, the deletion of cxrD delayed conidiospore formation, leading to 45.1 to 81.8% reduced asexual spore production and altered mycelial accumulation to various extents. Comparative transcriptomics and real-time quantitative reverse transcription-PCR found that CXRD dynamically regulated the expression of major cellulase and xylanase genes and conidiation-regulatory gene brlA under SSF. In vitro electrophoretic mobility shift assays demonstrated that CXRD bound to the promoter regions of these genes. The core DNA sequence 5'-CYGTSW-3' was identified to be specifically bound by CXRD. These findings will contribute to understanding the molecular mechanism of negative regulation of fungal cellulase and xylanase biosynthesis under SSF. IMPORTANCE Application of plant cell wall-degrading enzymes (CWDEs) as catalysts in biorefining of lignocellulosic biomass into bioproducts and biofuels reduces both chemical waste production and carbon footprint. The filamentous fungus Penicillium oxalicum can secrete integrated CWDEs, with potential for industrial application. Solid-state fermentation (SSF), simulating the natural habitat of soil fungi, such as P. oxalicum, is used for CWDE production, but a limited understanding of CWDE biosynthesis hampers the improvement of CWDE yields through synthetic biology. Here, we identified a novel transcription factor CXRD, which negatively regulates the biosynthesis of cellulase and xylanase in P. oxalicum under SSF, providing a potential target for genetic engineering to improve CWDE production.

MAPkinases regulate secondary metabolism, sexual development and light dependent cellulase regulation in Trichoderma reesei

The filamentous fungus Trichoderma reesei is a prolific producer of plant cell wall degrading enzymes, which are regulated in response to diverse environmental signals for optimal adaptation, but also produces a wide array of secondary metabolites. Available carbon source and light are the strongest cues currently known to impact secreted enzyme levels and an interplay with regulation of secondary metabolism became increasingly obvious in recent years. While cellulase regulation is already known to be modulated by different mitogen activated protein kinase (MAPK) pathways, the relevance of the light signal, which is transmitted by this pathway in other fungi as well, is still unknown in T. reesei as are interconnections to secondary metabolism and chemical communication under mating conditions. Here we show that MAPkinases differentially influence cellulase regulation in light and darkness and that the Hog1 homologue TMK3, but not TMK1 or TMK2 are required for the chemotropic response to glucose in T. reesei. Additionally, MAPkinases regulate production of specific secondary metabolites including trichodimerol and bisorbibutenolid, a bioactive compound with cytostatic effect on cancer cells and deterrent effect on larvae, under conditions facilitating mating, which reflects a defect in chemical communication. Strains lacking either of the MAPkinases become female sterile, indicating the conservation of the role of MAPkinases in sexual fertility also in T. reesei. In summary, our findings substantiate the previously detected interconnection of cellulase regulation with regulation of secondary metabolism as well as the involvement of MAPkinases in light dependent gene regulation of cellulase and secondary metabolite genes in fungi.

Discovery of ER-localized sugar transporters for cellulase production with lac1 being essential

BackgroundIn the process of cellulose hydrolysis, carbohydrate hydrolysates are transported into cells through membrane transporters, and then affect the expression of cellulase-encoding genes. Sugar transporters play a crucial role in cellulase production in lignocellulolytic fungi, of which relatively few have been functionally validated to date and are all reported to be on cell membrane.ResultThrough transcriptome analysis and qRT-PCR, three putative MFS sugar transporters GST, MFS, and LAC1 were found to display significantly higher mRNA levels in T. reesei grown on cellulose than on glucose. The individual deletion of these three genes compromised cellulase production and delayed sugar absorption by 24 h in T. reesei. Nevertheless, they transported pretty low level of sugars, including galactose, lactose, and mannose, and did not transport glucose, when expressed in yeast system. Meanwhile, all three transporters were unexpectedly found to be intracellular, being located in endoplasmic reticulum (ER). Particularly, the knockout of lac1 almost abolished cellulase production, and significantly inhibited biomass generation regardless of sugar types, indicating that lac1 is essential for cellulase production and biomass formation. The absence of lac1 upregulated genes involved in ribosome biogenesis, while downregulated genes in cellulase production, protein processing in ER (particularly protein glycosylation), and lipid biosynthesis. The inhibition of lac1 deletion on the transcriptional levels of genes related to cellulase biosynthesis was restored after 72 h, but the cellulase production was still inhibited, indicating lac1 might pose a post-transcription regulation on cellulase production that are independent on the known cellulase regulation mediated by CRT1 and XYR1.ConclusionFor the first time, intracellular sugar transporters (mfs, gst, and lac1) facilitating cellulase production were identified, which was distributed in ER. Their sugar transporting ability was very weak, indicating that they might be related to sugar utilization inside cells rather than the cellular sugar uptake. More importantly, sugar transporter lac1 is first found to be essential for cellulase production and biomass formation by affecting protein processing in ER (particularly protein glycosylation) and lipid biosynthesis. The effect of LAC1 on cellulase production seems to be post-transcriptional at late stage of cellulase production, independent on the well-known cellulase regulation mediated by CRT1 and XYR1. These findings improve the understanding of intracellular sugar transporters in fungi and their important role in cellulase synthesis.

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.

ABSTRACTThe 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.

Development of an Efficient C-to-T Base-Editing System and Its Application to Cellulase Transcription Factor Precise Engineering in Thermophilic Fungus Myceliophthora thermophila.

ABSTRACTMyceliophthora thermophila is a thermophilic fungus with great potential in biorefineries and biotechnology. The base editor is an upgraded version of the clustered regularly interspaced short palindromic repeats (CRISPR)-dependent genome-editing tool that introduces precise point mutations without causing DNA double-strand breaks (DSBs) and has been used in various organisms but rarely in filamentous fungi, especially thermophilic filamentous fungi. Here, for the first time, we constructed three cytosine base editors (CBEs) in M. thermophila, namely, evolved apolipoprotein B mRNA-editing enzyme catalytic subunit 1 (APOBEC1) cytosine base editor 4 max (Mtevo-BE4max), bacteriophage Mu Gam protein cytosine base editor 4 max (MtGAM-BE4max), and evolved CDA1 deaminase cytosine base editor (Mtevo-CDA1), and efficiently inactivated genes by precisely converting three codons (CAA, CAG, and CGA) into stop codons without DSB formation. The Mtevo-CDA1 editor with up to 92.6% editing efficiency is a more suitable tool for cytosine base editing in thermophilic fungi. To investigate the function of each motif of the cellulase transcription factor M. thermophila CLR-2 (MtCLR-2), we used the Mtevo-CDA1 editor. The fungal-specific motif of MtCLR-2 was found to be strongly involved in cellulase secretion, conidium formation, hyphal branching, and colony formation. Mutation of the fungus-specific motif caused significant defects in these characteristics. Thus, we developed an efficient thermophilic fungus-compatible base-editing system that could also be used for genetic engineering in other relevant filamentous fungi.IMPORTANCE A CRISPR/Cas-based base-editing approach has been developed to introduce point mutations without inducing double-strand breaks (DSBs) and attracted substantial academic and industrial interest. Our study developed the deaminase-cytosine base-editing system to efficiently edit three target genes, amdS, cre-1, and the essential cellulase regulator gene Mtclr-2, in Myceliophthora thermophila. A variety of point mutations in the target loci of the DNA-binding domain and fungus-specific motif of M. thermophila CLR-2 (MtCLR-2) were successfully generated via our base editor Mtevo-CDA1 to elucidate its function. Here, we show that the DNA-binding domain of MtCLR-2 is important for the fungal response to cellulose conditions, while its fungus-specific motif is involved in fungal growth. These findings indicate that our base editor can be an effective tool for elucidating the functions of motifs of target genes in filamentous fungi and for metabolic engineering in the field of synthetic biology.

An actin-like protein PoARP9 involves in the regulation of development and cellulase and amylase expression in Penicillium oxalicum.

In eukaryotic cells, chromatin remodelling complexes are essential for the accessibility of transcription factors to the specific regulating regions of downstream genes. Here, we identified an actin-like protein PoARP9 in cellulase production strain Penicillium oxalicum 114-2, which was an essential member of SWI/SNF complex. To investigate the physiological function of PoARP9 in transcriptional regulation, the coding gene Poarp9 was deleted in P. oxalicum 114-2. The absence of PoARP9 affected the colony growth on medium with glucose, cellulose or starch as sole carbon source. Meanwhile, the expression levels of major cellulase genes were all upregulated in ΔPoarp9 under the cellulase-inducing condition. In addition, the expression levels of amylase transcription activator AmyR as well as two major amylase genes were also increased in ΔPoarp9. These results demonstrated that chromatin remodelling affects the development and expression of cellulase and amylase in P. oxalicum. And the SWI/SNF complex member PoARP9 plays essential roles in these processes. This study provided new insights into the regulation of cellulase and development in P. oxalicum. And the regulatory function of SWI/SNF complex member ARP9 towards cellulase and amylase expression in P. oxalicum was verified for the first time.

Screening for genes involved in cellulase regulation by expression under the control of a novel constitutive promoter in Trichoderma reesei

• We characterized the upstream region of cfe1 as promising promoter without upstream open reading frames (uORFs) or introns. • The cfe1 promoter enabled up to 400 fold overexpression. • We analyzed transcriptomic data with support from machine learning to select target genes. • We report three genes affecting cellulase regulation. The filamentous ascomycete Trichoderma reesei is a biotechnological workhorse used in the production of homologous and heterologous proteins for diverse applications including biofuel production, textile finishing and food additives. This fungus uses a complex adaptation machinery to regulate its cellulases in response to environmental conditions. Detailed understanding of this regulation allows for improvement of enzyme production using the strong enzyme gene promotors. Here, we selected six genes with characteristic transcript levels associated with cellulase production to be tested for their enzyme regulatory function. Machine learning for inference of a gene regulatory network (GRN) was applied to support the association of these genes with cellulase induction. Additionally, we screened available transcriptomic data for genes with strong constitutive transcript levels and selected the promoter of the gene cfe1 , whose transcript levels were above those of tef1 and cDNA1 and near those of cbh1 , for gene overexpression testing. Using this promoter, we explored the relevance to cellulose degradation efficiency of three transporters, two ferric reductases and one gene of unknown function, which were overexpressed in T. reesei grown on cellulose. This promoter enabled up to 400 fold overexpression and yielded transcript levels above those of tef1 or cDNA1. We provide evidence of effects of a ferric reductase, an ammonium permease and a gene of unknown function on the regulatory machinery of cellulase expression in T. reesei . In summary we identified the cfe1 promoter, a novel constitutive promoter with promising efficiency, as well as three genes relevant to cellulase regulation in T. reesei .

Engineering Trichoderma reesei for Hyperproduction of Cellulases on Glucose to Efficiently Saccharify Pretreated Corncobs.

The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is widely used as a cellulase producer in the industry. Herein, we describe the rational engineering of the publicly available T. reesei QM9414 strain to achieve a remarkable high-level production of cellulase on glucose. Overexpression of the key cellulase regulator XYR1 by the copper-repressible promoter Ptcu1 was first implemented to achieve a full cellulase production in the context of catabolite repression (CCR) while eliminating the requirement of inducing sugars for enzyme production. The T. reesei bgl1 gene was further overexpressed to compensate for its low β-glucosidase activity on glucose. This overexpression resulted in a 102% increase in FPase activity compared with the CCR-released RUT-C30 strain cultured on Avicel. Moreover, the saccharification efficiency toward pretreated corncob residues by crude enzymes from the engineered strain on glucose increased by 85% compared with that treated by enzymes from RUT-C30 cultivated on Avicel. The engineered T. reesei strain thus shows great potential as a viable alternative to deliver commercial cellulases after further optimization for efficient saccharification of agricultural waste.

Mini review: Advances in understanding regulation of cellulase enzyme in white-rot basidiomycetes

White-rot basidiomycetic fungi have gained a lot of scientific attention in recent years owing to their ability to produce cellulase enzymes that are of great importance in numerous industrial applications. This has seen a rise in number of studies seeking to comprehend both physical and molecular mechanisms that regulate the production of cellulase enzymes in these fungi. Cellulase has several applications in production of food and beverages, biofuel, biological detergents, pharmaceuticals, and deinking in paper and pulp industry. Enhanced understanding of genetic mechanisms that regulate cellulase production would have utility for optimal cellulase production in white-rot basidiomycetes using biotechnology approaches. Carbon catabolite repression and various transcriptional factors such as XlnR, Cre, Clr, Ace, and gna1 control expression of genes encoding cellobiohydrolase (CBH), endoglucanase (EGL) and β-glucosidase (BGL). In this review, we have consolidated and summarised some of recent findings on genetic regulation of cellulase with an aim of highlighting the general regulatory mechanisms that underlie cellulase expressions in white-rot fungi. This review further outlines some of important transcription factors that regulate cellulase genes, and key research gaps that may need to be addressed by future research.

Involvement of Xyr1 and Are1 for Trichodermapepsin Gene Expression in Response to Cellulose and Galactose in Trichoderma reesei.

The secretome of Trichoderma reesei contains a mixture of cellulases, hemicellulases, amylases, proteases, and lipases that synergistically degrade plant biomass. Trichodermapepsin (TrAsP), the most prominent protease of T. reesei, affects the stability of cellulases. Similar to cellulase production, TrAsP production also depends on carbon and nitrogen sources. Unlike the cellulase mechanism, the regulatory mechanism of TrAsP remains unknown. Therefore, this study aimed to determine the effect of the main cellulase regulator Xyr1 and nitrogen regulator Are1 on trasp regulation. Cellulase inducer Avicel and TrAsP inducer galactose were used as carbon sources. qRT-PCR analysis revealed that Xyr1 and Are1 acted as a repressor and an activator for trasp expression, respectively. Compared to Avicel, relative expression was higher in galactose. The binding motifs of Xyr1 and Are1 were located in upstream of the trasp promoter. From promoter deletant analysis using the β-glucuronidase reporter gene, the area from - 870bp to - 670bp was identified as the only region for positive regulation and there were both binding motifs of Xyr1 and Are1. Reporter assay of mutants confirmed functions of downregulation of Xyr1 and upregulation of Are1. Electrophoretic mobility shift assay demonstrated the binding ability of Xyr1 and Are1 to the particular binding motifs and their functionality was confirmed. Further, this study demonstrated that Cre1, Xpp1, and Pac1 downregulate trasp expression similar to that in cellulase regulation mechanism. These results demonstrate that transcriptional regulators of cellulase control trasp expression and suggest the possibility of the existence of specific protease regulators in T. reesei.

In Ganoderma lucidum, Glsnf1 regulates cellulose degradation by inhibiting GlCreA during the utilization of cellulose.

Cellulose is a by-product of agricultural production and an abundant waste. As a carbon source, cellulose can be degraded and utilized by fungi. Carbon sources, which act as nutrients, not only provide energy but also serve as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Nutrient-sensing pathways prioritize the use of preferred carbon sources and regulate the production of cellulose-degrading enzymes when necessary. Understanding the regulation of the fungal cellulolytic response will become increasingly important because we strive to increase the efficiency of the utilization of these renewable energy sources. Here, we show that Glsnf1, a sucrose-nonfermenting serine-threonine-protein kinase 1 (Snf1)/AMP-activated protein kinase homologue in medicinal macro basidiomycete Ganoderma lucidum, actively responds to carbon alterations and positively regulates cellulase activity and cellulase-related gene transcription. The carbon catabolite repressor CreA, a zinc binuclear cluster transcription factor that mediates the sensing of nutrients and suppression of the transcription of a number of genes necessary for the consumption of a less preferred carbon source, participates in the Glsnf1-mediated regulation of cellulases. Glsnf1 not only negatively regulates the transcription level of the CreA gene but also hinders its localization in the nucleus. Overall, our findings reveal a key nutrient-sensing mechanism that is critical for the modulation of carbon source adaptation in G. lucidum.

The role of PKAc1 in gene regulation and trichodimerol production in Trichoderma reesei

BackgroundTrichoderma reesei represents a model system for investigation of plant cell wall degradation and its connection to light response. The cyclic adenosine monophosphate pathway (cAMP pathway) plays an important role in both physiological outputs, being crucial for regulation of photoreceptor function as well as for cellulase regulation on different carbon sources. Phosphorylation of photoreceptors and of the carbon catabolite repressor CRE1 was shown in ascomycetes, indicating a relevance of protein kinase A in regulation of the target genes of these transcription factors as well as an impact on regulation of induction specific genes. Moreover, the cAMP pathway impacts growth and development.ResultsHere, we investigated gene regulation by the catalytic subunit of protein kinase A (PKAc1) upon growth on cellulose. We found distinct gene sets for regulation upon growth in light and darkness with an overlap of only 13 genes. PKAc1 regulates metabolic genes as well as transport and defense functions. The overlap of gene regulation by PKAc1 with the genes representing the cAMP dependent regulatory output of the photoreceptor ENV1 indicates an involvement of PKA in this pathway, which counteracts its effects by contrasting regulation. Moreover, we found considerable overlap with the gene sets regulated under cellulase inducing conditions and by the carbon catabolite repressor CRE1. Our analysis also showed that PKAc1 regulates the genes of the SOR cluster associated with the biosynthesis of sorbicillinoids. The homologue of gin4, encoding a CAMK type kinase, which is regulated by PKAc1, CRE1 and YPR2 showed a moderate impact on trichodimerol production. We isolated trichodimerol as representative sorbicillin compound and established a method for its quantification in large sample sets using high performance thin layer chromatography (HPTLC), which can be broadly applied for secondary metabolite screening of mutants or different growth conditions. Due to the high expression levels of the SOR cluster under conditions of sexual development we crosschecked the relevance of PKAc1 under these conditions. We could show that PKAc1 impacts biosynthesis of trichodimerol in axenic growth and upon mating.ConclusionsWe conclude that PKAc1 is involved in light dependent regulation of plant cell wall degradation, including carbon catabolite repression as well as secondary metabolism and development in T. reesei.

Enhanced cellulase production by decreasing intercellular pH through H+-ATPase gene deletion in Trichoderma reesei RUT-C30

BackgroundCellulolytic enzymes produced by Trichoderma reesei are widely used for the industrial production of biofuels and chemicals from lignocellulose. We speculated that intracellular pH during the fermentation process can affect cellulase induction.ResultsIn this study, two H+-ATPase genes, tre76238 and tre78757, were first identified in T. reesei. Deletion of tre76238 and tre78757 in T. reesei RUT-C30 confirmed that tre76238 has a major function in maintaining intracellular pH, whereas tre78757 has a minor function. The tre76238 deletion strain Δ76238 displayed a high level of cellulase production using cellulase-repressive glucose as a sole carbon source, along with intracellular acid accumulation and growth retardation. Our results indicated that intracellular acid accumulation in Δ76238 stimulated a significant increase in the cytosolic Ca2+ levels. Ca2+ channels were shown to be necessary for cellulase production using glucose as the carbon source in Δ76238. Delayed Δ76238 growth could be reversed by optimizing the medium’s nitrogen sources to produce ammonia for intracellular acid neutralization in the early phase. This may be useful for scale-up of cellulase production using glucose as the carbon source.ConclusionsThis study provides a new perspective for significant alterations in the cellulase expression pattern of T. reesei Δ76238, indicating a new mechanism for cellulase regulation under conditions of low intracellular pH.

CLP1, a Novel Plant Homeo Domain Protein, Participates in Regulating Cellulase Gene Expression in the Filamentous Fungus Trichoderma reesei.

The stringent regulatory network of cellulase gene expression in the filamentous fungus Trichoderma reesei involves multiple transcriptional regulators. However, identification and mechanistic investigation of these regulators are still insufficient. Here, we identified a novel transcriptional regulator, CLP1, a plant homeo domain (PHD) Protein that participates in regulating T. reesei cellulase gene expression. Phylogenetic analyses demonstrated that CLP1 homologs are widely distributed in filamentous fungi including Trichoderma, Penicillium, Fusarium, Neurospora, and Aspergillus species. We demonstrated that CLP1 is a nuclear protein and lack of CLP1 significantly impaired the induced expression of cellulase genes. ChIP experiments showed CLP1 binding to the cellulase gene promoters specifically under cellulose conditions and compromised XYR1 occupancy on the same promoters in the absence of CLP1 at the early induction stage. XYR1 overexpression fully rescued the defect in cellulase production but not the defect in conidia formation in the clp1 null mutant. Further analysis showed that the PHD is required for the CLP1 appropriate subcellular localization as well as the induced cellulase gene expression and conidiation. Taken together, these data demonstrated an important role of CLP1 in the regulation of cellulase and xylanase gene expression in T. reesei.

Protein phosphatases regulate growth, development, cellulases and secondary metabolism in Trichoderma reesei

Trichoderma reesei represents one of the most prolific producers of plant cell wall degrading enzymes. Recent research showed broad regulation by phosphorylation in T. reesei, including important transcription factors involved in cellulase regulation. To evaluate factors crucial for changes in these phosphorylation events, we studied non-essential protein phosphatases (PPs) of T. reesei. Viable deletion strains were tested for growth on different carbon sources, osmotic and oxidative stress response, asexual and sexual development, cellulase and protease production as well as secondary metabolism. Six PPs were found to be positive or negative regulators for cellulase production. A correlation of the effects of PPs on protease activities and cellulase activities was not detected. Hierarchical clustering of regulation patterns and phenotypes of deletion indicated functional specialization within PP classes and common as well as variable effects. Our results confirmed the central role of catalytic and regulatory subunits of PP2A which regulates several aspects of cell growth and metabolism. Moreover we show that the additional homologue of PPH5 in Trichoderma spp., PPH5-2 assumes distinct functions in metabolism, development and stress response, different from PPH5. The influence of PPs on both cellulase gene expression and secondary metabolite production support an interrelationship in the underlying regulation mechanisms.

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.

Regulation of the G\u03b1-cAMP/PKA signaling pathway in cellulose utilization of Chaetomium globosum

BackgroundThe canonical heterotrimeric G protein-cAMP/PKA pathway regulates numerous cellular processes in filamentous fungi. Chaetomium globosum, a saprophytic fungus, is known for producing many secondary metabolites, including cytotoxic chaetoglobosin A (ChA), as well as abundant cellulase and xylanase.ResultsHere we report on the functional characterization of this signaling pathway in C. globosum. We blocked the pathway by knocking down the putative Gα-encoding gene gna1 (in the pG14 mutant). This led to impaired cellulase production and significantly decreased transcription of the major cellulase and xylanase genes. Almost all the glycohydrolase family genes involved in cellulose degradation were downregulated, including the major cellulase genes, cel7a, cel6a, egl1, and egl2. Importantly, the expression of transcription factors was also found to be regulated by gna1, especially Ace1, Clr1/2 and Hap2/3/5 complex. Additionally, carbon metabolic processes including the starch and sucrose metabolism pathway were substantially diminished, as evidenced by RNA-Seq profiling and quantitative reverse transcription (qRT)-PCR. Interestingly, these defects could be restored by simultaneous knockdown of the pkaR gene encoding the regulatory subunit of cAMP-dependent PKA (in the pGP6 mutant) or supplement of the cAMP analog, 8-Br-cAMP. Moreover, the Gα-cAMP/PKA pathway regulating cellulase production is modulated by environmental signals including carbon sources and light, in which VelB/VeA/LaeA complex and ENVOY probably work as downstream effectors.ConclusionThese results revealed, for the first time, the positive role of the heterotrimeric Gα-cAMP/PKA pathway in the regulation of cellulase and xylanase utilization in C. globosum.

Analysis of the Transcriptome in Aspergillus tamarii During Enzymatic Degradation of Sugarcane Bagasse.

The production of bioethanol from non-food agricultural residues represents an alternative energy source to fossil fuels for incorporation into the world's economy. Within the context of bioconversion of plant biomass into renewable energy using improved enzymatic cocktails, Illumina RNA-seq transcriptome profiling was conducted on a strain of Aspergillus tamarii, efficient in biomass polysaccharide degradation, in order to identify genes encoding proteins involved in plant biomass saccharification. Enzyme production and gene expression was compared following growth in liquid and semi-solid culture with steam-exploded sugarcane bagasse (SB) (1% w/v) and glucose (1% w/v) employed as contrasting sole carbon sources. Enzyme production following growth in liquid minimum medium supplemented with SB resulted in 0.626 and 0.711 UI.mL−1 xylanases after 24 and 48 h incubation, respectively. Transcriptome profiling revealed expression of over 7120 genes, with groups of genes modulated according to solid or semi-solid culture, as well as according to carbon source. Gene ontology analysis of genes expressed following SB hydrolysis revealed enrichment in xyloglucan metabolic process and xylan, pectin and glucan catabolic process, indicating up-regulation of genes involved in xylanase secretion. According to carbohydrate-active enzyme (CAZy) classification, 209 CAZyme-encoding genes were identified with significant differential expression on liquid or semi-solid SB, in comparison to equivalent growth on glucose as carbon source. Up-regulated CAZyme-encoding genes related to cellulases (CelA, CelB, CelC, CelD) and hemicellulases (XynG1, XynG2, XynF1, XylA, AxeA, arabinofuranosidase) showed up to a 10-fold log2FoldChange in expression levels. Five genes from the AA9 (GH61) family, related to lytic polysaccharide monooxygenase (LPMO), were also identified with significant expression up-regulation. The transcription factor gene XlnR, involved in induction of hemicellulases, showed up-regulation on liquid and semi-solid SB culture. Similarly, the gene ClrA, responsible for regulation of cellulases, showed increased expression on liquid SB culture. Over 150 potential transporter genes were also identified with increased expression on liquid and semi-solid SB culture. This first comprehensive analysis of the transcriptome of A. tamarii contributes to our understanding of genes and regulatory systems involved in cellulose and hemicellulose degradation in this fungus, offering potential for application in improved enzymatic cocktail development for plant biomass degradation in biorefinery applications.

Abundance of Secreted Proteins of Trichoderma reesei Is Regulated by Light of Different Intensities.

In Trichoderma reesei light is an important factor in the regulation of glycoside hydrolase gene expression. We therefore investigated the influence of different light intensities on cellulase activity and protein secretion. Differentially secreted proteins in light and darkness as identified by mass spectrometry included members of different glycoside hydrolase families, such as CBH1, Cel3A, Cel61B, XYN2, and XYN4. Several of the associated genes showed light-dependent regulation on the transcript level. Deletion of the photoreceptor genes blr1 and blr2 resulted in a diminished difference of protein abundance between light and darkness. The amount of secreted proteins including that of the major exo-acting beta-1,4-glucanases CBH1 and CBH2 was generally lower in light-grown cultures than in darkness. In contrast, cbh1 transcript levels increased with increasing light intensity from 700 to 2,000 lux but dopped at high light intensity (5,000 lux). In the photoreceptor mutants Δblr1 and Δblr2 cellulase activity in light was reduced compared to activity in darkness, showing a discrepancy between transcript levels and secreted cellulase activity. Furthermore, evaluation of different light sensitivities revealed an increased light tolerance with respect to cellulase expression of QM9414 compared to its parental strain QM6a. Investigation of one of the differentially expressed proteins between light and darkness, CLF1, revealed its function as a factor involved in regulation of secreted protease activity. T. reesei secretes a different set of proteins in light compared to darkness, this difference being mainly due to the function of the major known photoreceptors. Moreover, cellulase regulation is adjusted to light intensity and improved light tolerance was correlated with increased cellulase production. Our findings further support the hypothesis of a light intensity dependent post-transcriptional regulation of cellulase gene expression in T. reesei.