Filamentous Fungus Trichoderma Reesei Research Articles

Evolution and Screening of Trichoderma reesei Mutants for Secreted Protein Production at Elevated Temperature.

The filamentous fungus Trichoderma reesei is a mesophilic ascomycete commercially used to produce industrial enzymes for a variety of applications. Strain improvement efforts over many years have resulted in more productive hosts, but also in undesirable traits such as the need for lower temperatures to achieve maximum protein secretion rates. Lower fermentation temperatures increase the need for cooling resulting in higher manufacturing costs. We used a droplet-based evolution strategy to increase the protein secretion temperature of a highly productive T. reesei whole cellulase strain from 25°C to 28°C by first isolating an improved mutant and subsequently tracing the causative high temperature mutation to one gene designated gef1. An industrial host with a gef1 deletion was found to be capable of improved productivity at higher temperature under industrially relevant fermentation conditions.

Transgressive phenotypes from outbreeding between the Trichoderma reesei hyper producer RutC30 and a natural isolate.

The filamentous fungus Trichoderma reesei produces cellulolytic enzymes that are essential for the hydrolysis of lignocellulosic biomass into monomerics sugars. The filamentous fungus T. reesei produces cellulolytic enzymes that are essential for the hydrolysis of lignocellulosic biomass into monomerics sugars, which can in turn be fermented to produce second-generation biofuels and bioproducts. Production performance improvement, which is essential to reduce production cost, relies on classical mutagenesis and genetic engineering techniques. Although sexual reproduction is a powerful tool for improving domesticated species, it is often difficult to apply to industrial fungi since most of them are considered asexual. In this study, we demonstrated that outbreeding is an efficient strategy to optimize T. reesei. Crossing between a natural isolate and a mutagenized strain generated a biodiverse progeny with some offspring displaying transgressive phenotype for cellulase activities.

Small GTPase Rab7 is involved in stress adaptation to carbon starvation to ensure the induced cellulase biosynthesis in Trichoderma reesei.

The saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers. The bulk production of lignocellulolytic enzymes by T. reesei not only relies on the efficient transcription of cellulase genes but also their efficient secretion after being translated. However, little attention has been paid to the functional roles of the involved secretory pathway in the high-level production of cellulases in T. reesei. Rab GTPases are key regulators in coordinating various vesicle trafficking associated with the eukaryotic secretory pathway. Specifically, Rab7 is a representative GTPase regulating the transition of the early endosome to the late endosome followed by its fusion to the vacuole as well as homotypic vacuole fusion. Although crosstalk between the endosomal/vacuolar pathway and the secretion pathway has been reported, the functional role of Rab7 in cellulase production in T. reesei remains unknown. A TrRab7 was identified and characterized in T. reesei. TrRab7 was shown to play important roles in T. reesei vegetative growth and vacuole morphology. Whereas knock-down of Trrab7 significantly compromised the induced production of T. reesei cellulases, overexpression of the key transcriptional activator, Xyr1, restored the production of cellulases in the Trrab7 knock-down strain (Ptcu-rab7KD) on glucose, indicating that the observed defective cellulase biosynthesis results from the compromised cellulase gene transcription. Down-regulation of Trrab7 was also found to make T. reesei more sensitive to various stresses including carbon starvation. Interestingly, overexpression of Snf1, a serine/threonine protein kinase known as an energetic sensor, partially restored the cellulase production of Ptcu-rab7KD on Avicel, implicating that TrRab7 is involved in an energetic adaptation to carbon starvation which contributes to the successful cellulase gene expression when T. reesei is transferred from glucose to cellulose. TrRab7 was shown to play important roles in T. reesei development and a stress response to carbon starvation resulting from nutrient shift. This adaptation may allow T. reesei to successfully initiate the inducing process leading to efficient cellulase production. The present study provides useful insights into the functional involvement of the endosomal/vacuolar pathway in T. reesei development and hydrolytic enzyme production.

Structure-guided engineering of transcriptional activator XYR1 for inducer-free production of lignocellulolytic enzymes in Trichoderma reesei

The filamentous fungus Trichoderma reesei is widely used for the production of lignocellulolytic enzymes in industry. XYR1 is the major transcriptional activator of cellulases and hemicellulases in T. reesei. However, rational engineering of XYR1 for improved lignocellulolytic enzymes production has been limited by the lack of structure information. Here, alanine 873 was identified as a new potential target for the engineering of XYR1 based on its structure predicted by AlphaFold2. The mutation of this residue to tyrosine enabled significantly enhanced production of xylanolytic enzymes in the medium with cellulose as the carbon source. Moreover, xylanase and cellulase production increased by 56.7- and 3.3-fold, respectively, when glucose was used as the sole carbon source. Under both conditions, the improvements of lignocellulolytic enzyme production were higher than those in the previously reported V821F mutant. With the enriched hemicellulases and cellulases, the crude enzymes secreted by the A873Y mutant strain produced 51 % more glucose and 52 % more xylose from pretreated corn stover than those of the parent strain. The results provide a novel strategy for engineering the lignocellulolytic enzyme-producing capacity of T. reesei, and would be helpful for understanding the molecular mechanisms of XYR1 regulation.

Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source

BackgroundThe filamentous fungus Trichoderma reesei has been used as a host organism for the production of lignocellulosic biomass-degrading enzymes. Although this microorganism has high potential for protein production, it has not yet been widely used for heterologous recombinant protein production. Transcriptional induction of the cellulase genes is essential for high-level protein production in T. reesei; however, glucose represses this transcriptional induction. Therefore, cellulose is commonly used as a carbon source for providing its degraded sugars such as cellobiose, which act as inducers to activate the strong promoters of the major cellulase (cellobiohydrolase 1 and 2 (cbh1 and cbh2) genes. However, replacement of cbh1 and/or cbh2 with a gene encoding the protein of interest (POI) for high productivity and occupancy of recombinant proteins remarkably impairs the ability to release soluble inducers from cellulose, consequently reducing the production of POI. To overcome this challenge, we first used an inducer-free biomass-degrading enzyme expression system, previously developed to produce cellulases and hemicellulases using glucose as the sole carbon source, for recombinant protein production using T. reesei.ResultsWe chose endogenous secretory enzymes and heterologous camelid small antibodies (nanobody) as model proteins. By using the inducer-free strain as a parent, replacement of cbh1 with genes encoding two intrinsic enzymes (aspartic protease and glucoamylase) and three different nanobodies (1ZVH, caplacizumab, and ozoralizumab) resulted in their high secretory productions using glucose medium without inducers such as cellulose. Based on signal sequences (carrier polypeptides) and protease inhibitors, additional replacement of cbh2 with the nanobody gene increased the percentage of POI to about 20% of total secreted proteins in T. reesei. This allowed the production of caplacizumab, a bivalent nanobody, to be increased to 9.49-fold (508 mg/L) compared to the initial inducer-free strain.ConclusionsIn general, whereas the replacement of major cellulase genes leads to extreme decrease in the degradation capacity of cellulose, our inducer-free system enabled it and achieved high secretory production of POI with increased occupancy in glucose medium. This system would be a novel platform for heterologous recombinant protein production in T. reesei.

Metabolic Engineering of Trichoderma reesei for l-Malic Acid Production.

l-Malic acid has various applications in the chemical and food industries. The filamentous fungus Trichoderma reesei is known to be an efficient enzyme producer. Here, through metabolic engineering, T. reesei was constructed for the first time as an excellent cell factory for l-malic acid production. The heterologous overexpression of genes encoding the C4-dicarboxylate transporter from Aspergillus oryzae and Schizosaccharomyces pombe initiated l-malic acid production. The overexpression of pyruvate carboxylase from A. oryzae in the reductive tricarboxylic acid pathway further increased both the titer and yield of l-malic acid, resulting in the highest titer reported in a shake-flask culture. Furthermore, the deletion of malate thiokinase blocked l-malic acid degradation. Finally, the engineered T. reesei strain produced 220.5 g/L of l-malic acid in a 5 L fed-batch culture (productivity of 1.15 g/L/h). A T. reesei cell factory was created for the efficient production of l-malic acid.

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.

Role of the Nitrogen Metabolism Regulator TAM1 in Regulation of Cellulase Gene Expression in Trichoderma reesei.

The filamentous fungus Trichoderma reesei is one of the most prolific cellulase producers and has been established as a model microorganism for investigating mechanisms modulating eukaryotic gene expression. Identification and functional characterization of transcriptional regulators involved in complex and stringent regulation of cellulase genes are, however, not yet complete. Here, a Zn(II)2Cys6-type transcriptional factor TAM1 that is homologous to Aspergillus nidulans TamA involved in nitrogen metabolism, was found not only to regulate ammonium utilization but also to control cellulase gene expression in T. reesei. Whereas Δtam1 cultivated with peptone as a nitrogen source did not exhibit a growth defect that was observed on ammonium, it was still significantly compromised in cellulase biosynthesis. The absence of TAM1 almost fully abrogated the rapid cellulase gene induction in a resting-cell-inducing system. Overexpression of gdh1 encoding the key ammonium assimilatory enzyme in Δtam1 rescued the growth defect on ammonium but not the defect in cellulase gene expression. Of note, mutation of the Zn(II)2Cys6 DNA-binding motif of TAM1 hardly affected cellulase gene expression, while a truncated ARE1 mutant lacking the C-terminal 12 amino acids that are required for the interaction with TAM1 interfered with cellulase biosynthesis. The defect in cellulase induction of Δtam1 was rescued by overexpression of the key transactivator for cellulase gene, XYR1. Our results thus identify a nitrogen metabolism regulator as a new modulator participating in the regulation of induced cellulase gene expression. IMPORTANCE Transcriptional regulators are able to integrate extracellular nutrient signals and exert a combinatorial control over various metabolic genes. A plethora of such factors therefore constitute a complex regulatory network ensuring rapid and accurate cellular response to acquire and utilize nutrients. Despite the in-depth mechanistic studies of functions of the Zn(II)2Cys6-type transcriptional regulator TamA and its orthologues in nitrogen utilization, their involvement in additional physiological processes remains unknown. In this study, we demonstrated that TAM1 exerts a dual regulatory role in mediating ammonium utilization and induced cellulase production in the well known cellulolytic fungus Trichoderma reesei, suggesting a potentially converged regulatory node between nitrogen utilization and cellulase biosynthesis. This study not only contributes to unveiling the intricate regulatory network underlying cellulase gene expression in cellulolytic fungus but also helps expand our knowledge of fungal strategies to achieve efficient and coordinated nutrient acquisition for rapid propagation.

Tailoring the expression of Xyr1 leads to efficient production of lignocellulolytic enzymes in Trichoderma reesei for improved saccharification of corncob residues

BackgroundThe filamentous fungus Trichoderma reesei is extensively used for the industrial-scale cellulase production. It has been well known that the transcription factor Xyr1 plays an important role in the regulatory network controlling cellulase gene expression. However, the role of Xyr1 in the regulation of cellulase expression has not been comprehensively elucidated, which hinders further improvement of lignocellulolytic enzyme production.ResultsHere, the expression dosage of xyr1 was tailored in T. reesei by differentially overexpressing the xyr1 gene under the control of three strong promoters (Pegl2, Pcbh1, and Pcdna1), and the transcript abundance of xyr1 was elevated 5.8-, 12.6-, and 47.2-fold, respectively. We found expression of cellulase genes was significantly increased in the Pegl2-driven xyr1 overexpression strain QE2X, whereas relatively low in the Pcbh1- and Pcdna1-driven overexpression strains. We also found that the Pegl2-driven overexpression of xyr1 caused a more significant opening of chromatin in the core promoter region of the prominent cellulase genes. Furthermore, the cellulase activity showed a 3.2-fold increase in the strain QE2X, while insignificant improvement in the Pcbh1- and Pcdna1-driven strains. Finally, the saccharification efficiency toward acid-pretreated corncob residues containing high-content lignin by the crude enzyme from QE2X was increased by 57.2% compared to that from the parental strain. Moreover, LC–MS/MS and RT-qPCR analysis revealed that expression of accessory proteins (Cip1, Cip2, Swo1, and LPMOs) was greatly improved in QE2X, which partly explained the promoting effect of the Pegl2-driven overexpression on enzymatic hydrolysis of lignocellulose biomass.ConclusionsOur results underpin that the precise tailoring expression of xyr1 is essential for highly efficient cellulase synthesis, which provide new insights into the role of Xyr1 in regulating cellulase expression in T. reesei. Moreover, these results also provides a prospective strategy for strain improvement to enhance the lignocellulolytic enzyme production for use in biorefinery applications.

Identification of a Bidirectional Promoter from Trichoderma reesei and Its Application in Dual Gene Expression.

The cellulolytic filamentous fungus Trichoderma reesei has a strong capability in protein synthesis and secretion and is increasingly used as a fungal chassis for the production of heterologous proteins or secondary metabolites. However, bidirectional promoters that would significantly facilitate multiple genes’ expression have not been characterized in T. reesei. Herein, we show that a 767-bp intergenic region between two polyketide synthase encoding genes that were involved in the biosynthesis of the typical yellow pigment served as a bidirectional promoter in T. reesei. This region was shown to be able to drive the simultaneous expression of two fluorescence reporter genes when fused to each end. Quantitative RT-PCR analysis demonstrated that the driving strength of this bidirectional promoter from each direction reached about half of that of the commonly used promoter PgpdA. Moreover, the co-expression of two cellulase genes driven by this bidirectional promoter enabled T. reesei to produce cellulases on glucose and improved the total cellulase activities with cellulose Avicel as the carbon source. Our work identified the first bidirectional promoter in T. reesei, which would facilitate gene co-expression and find applications in synthetic biology using fungal systems.

A histone H3K9 methyltransferase Dim5 mediates repression of sorbicillinoid biosynthesis in Trichoderma reesei.

Sorbicillinoids (also termed yellow pigment) are derived from either marine or terrestrial fungi, exhibit various biological activities and therefore show potential as commercial products for human or animal health. The cellulolytic filamentous fungus Trichoderma reesei is capable to biosynthesize sorbicillinoids, but the underlying regulatory mechanism is not yet completely clear. Herein, we identified a histone H3 lysine 9 (H3K9) methyltransferase, Dim5, in T. reesei. TrDIM5 deletion caused an impaired vegetative growth as well as conidiation, whereas the ∆Trdim5 strain displayed a remarkable increase in sorbicillinoid production. Post TrDIM5 deletion, the transcription of sorbicillinoid biosynthesis‐related (SOR) genes was significantly upregulated with a more open chromatin structure. Intriguingly, hardly any expression changes occurred amongst those genes located on both flanks of the SOR gene cluster. In addition, the assays provided evidence that H3K9 triple methylation (H3K9me3) modification acted as a repressive marker at the SOR gene cluster and thus directly mediated the repression of sorbicillinoid biosynthesis. Transcription factor Ypr1 activated the SOR gene cluster by antagonizing TrDim5‐mediated repression and therefore contributed to forming a relatively more open local chromatin environment, which further facilitated its binding and SOR gene expression. The results of this study will contribute to understanding the intricate regulatory network in sorbicillinoid biosynthesis and facilitate the endowment of T. reesei with preferred features for sorbicillinoid production by genetic engineering.

Trichoderma reesei.

The filamentous fungus Trichoderma reesei (teleomorph: Hypocrea jecorina) grows on rotting plant material in its natural habitat. It is among the most prolific producers of plant cell-wall-degrading enzymes and is frequently used in industry for production of those and other performance proteins. A complete telomere-to-telomere genome sequence is now available (34 Mb, 10 877 genes, 7 chromosomes). Sexual reproduction of the haploid T. reesei in the laboratory was achieved only about a decade ago and requires pheromones, but also other chemical signals. T. reesei is readily transformable, and a plethora of tools, including CRISPR/Cas, have been developed which facilitate functional genomics, genome-wide investigations, and live-cell imaging, and tools to investigate chemical communication. Studies focused on a detailed understanding of enzyme expression and its improvement revealed the interplay of numerous transcription factors, connections to signaling pathways, and a significant impact of light. Detailed understanding of the physiology of T. reesei will enable optimized enzyme expression and thereby support development of more sustainable, yet commercially viable, solutions for biofuel production, textile production and recycling, chemical conversions using enzymes, food processing, production of pharmaceuticals including antibodies, bioremediation, and agriculture.

Dual Regulatory Role of Chromatin Remodeler ISW1 in Coordinating Cellulase and Secondary Metabolite Biosynthesis in Trichoderma reesei.

ABSTRACTThe saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers isolated from nature. T. reesei also produces a typical yellow pigment identified as sorbicillinoids during cultivation. Here, we identified an evolutionarily conserved histone remodeling factor, ISW1, in T. reesei that simultaneously participates in regulating cellulase and the yellow pigment biosynthesis. Trisw1 deletion almost abolished vegetable growth, asexual spore formation, and cellulase gene expression. However, its absence significantly enhanced the production of the yellow pigment. The observed dual regulatory role of TrISW1 was dependent on its ATPase activity. We demonstrated that Trisw1 disruption elevated the transcription of ypr1 coding for the transcriptional activator of sor genes encoding the polyketide synthases catalyzing the biosynthesis of sorbicillinoids but compromised that of xyr1 encoding the key transcriptional activator of cellulase genes. Discrete T. reesei homologous ISW1 accessory factors were also found to exert differential effects on the expression of these two types of genes. Further analyses showed that TrISW1 was recruited to cellulase gene promoters, and its absence interfered with loss of histone H4 at the cbh1 and eg1 promoters upon cellulose induction. To the contrary, Trisw1 deletion facilitated loss of H4 at the sor locus. These data indicate that TrISW1 represents an important chromatin remodeler with a dual role in coordinating the cellulolytic response and biosynthesis of the major secondary metabolite in T. reesei.

Elimination of the Sugar Transporter GAT1 Increased Xylanase I Production in Trichoderma reesei.

The filamentous fungus Trichoderma reesei secretes large quantities of cellulases and hemicellulases that have found wide applications in industry. Compared with extensive studies on the mechanism controlling cellulase gene expression, less is known about the regulatory mechanism behind xylanase gene expression. Herein, several putative sugar transporter encoding genes that showed significant upregulation on xylan were identified in T. reesei. Deletion of one such gene, gat1, resulted in increased xylanase production but hardly affected cellulase induction. Further analyses demonstrated that deletion of gat1 markedly increased XYNI production at the transcriptional level and only exerted a minor effect on XYNII synthesis. In contrast, overexpressing gat1 caused a continuous decrease in xyn1 expression. Deletion of gat1 also affected the expression of xyn1 and pectinase genes when T. reesei was cultivated with galacturonic acid as the sole carbon source. Transcriptome analyses of Δgat1 and its parental strain identified 255 differentially expressed genes that are enriched in categories of glycoside hydrolases, lipid metabolism, transporters, and transcriptional factors. The results thus implicate a repressive role of the sugar transporter GAT1 in xyn1 expression and reveal that distinct regulatory mechanisms may exist in controlling the expression of different xylanase genes in T. reesei.

Development of a powerful synthetic hybrid promoter to improve the cellulase system of Trichoderma reesei for efficient saccharification of corncob residues

BackgroundThe filamentous fungus Trichoderma reesei is a widely used workhorse for cellulase production in industry due to its prominent secretion capacity of extracellular cellulolytic enzymes. However, some key components are not always sufficient in this cellulase cocktail, making the conversion of cellulose-based biomass costly on the industrial scale. Development of strong and efficient promoters would enable cellulase cocktail to be optimized for bioconversion of biomass.ResultsIn this study, a synthetic hybrid promoter was constructed and applied to optimize the cellulolytic system of T. reesei for efficient saccharification towards corncob residues. Firstly, a series of 5’ truncated promoters in different lengths were established based on the strong constitutive promoter Pcdna1. The strongest promoter amongst them was Pcdna1-3 (− 640 to − 1 bp upstream of the translation initiation codon ATG), exhibiting a 1.4-fold higher activity than that of the native cdna1 promoter. Meanwhile, the activation region (− 821 to − 622 bp upstream of the translation initiation codon ATG and devoid of the Cre1-binding sites) of the strong inducible promoter Pcbh1 was cloned and identified to be an amplifier in initiating gene expression. Finally, this activation region was fused to the strongest promoter Pcdna1-3, generating the novel synthetic hybrid promoter Pcc. This engineered promoter Pcc drove strong gene expression by displaying 1.6- and 1.8-fold stronger fluorescence intensity than Pcbh1 and Pcdna1 under the inducible condition using egfp as the reporter gene, respectively. Furthermore, Pcc was applied to overexpress the Aspergillus niger β-glucosidase BGLA coding gene bglA and the native endoglucanase EG2 coding gene eg2, achieving 43.5-fold BGL activity and 1.2-fold EG activity increase, respectively. Ultimately, to overcome the defects of the native cellulase system in T. reesei, the bglA and eg2 were co-overexpressed under the control of Pcc promoter. The bglA-eg2 double expression strain QPEB70 exhibited a 178% increase in total cellulase activity, whose cellulase system displayed 2.3- and 2.4-fold higher saccharification efficiency towards acid-pretreated and delignified corncob residues than the parental strain, respectively.ConclusionsThe synthetic hybrid promoter Pcc was generated and employed to improve the cellulase system of T. reesei by expressing specific components. Therefore, construction of synthetic hybrid promoters would allow particular cellulase genes to be expressed at desired levels, which is a viable strategy to optimize the cellulolytic enzyme system for efficient biomass bioconversion.

The novel repressor Rce2 competes with Ace3 to regulate cellulase gene expression in the filamentous fungus Trichoderma reesei.

The filamentous fungus Trichoderma reesei is widely used for industrial cellulase production. In T. reesei, cellulase gene expression is tightly controlled by a regulatory network involving multiple transcription factors. Here, we isolated a novel protein, Rce2, using a pull-down assay and mass spectrometry analysis, from a partial carbon catabolite de-repression mutant, T. reesei Rut-C30, cultured under glucose-repressing conditions. Deletion and overexpression of Rce2 in T. reesei wild-type QM6a and mutant Rut-C30 revealed that Rce2 acts as a repressor of cellulase gene expression. DNase I footprinting assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays revealed that Rce2 was located in the nucleus and bound to the consensus sequences 5'-(T/A)NNNNCCG-3' and 5'-CGGNNNN(T/A)-3' in the promoters of cellulase-related genes to repress their transcription. Additionally, Rce2 antagonized Ace3 binding to the cbh1 promoter to repress its transcription. However, Rce2 was not involved in Cre1-mediated carbon catabolite repression. These results demonstrate the mechanism through which Rce2 represses the expression of cellulase genes and provide novel insights into the regulatory system of cellulases and methods that can be used for the regulation of gene expression in T. reesei.

Engineering the endoplasmic reticulum secretory pathway in Trichoderma reesei for improved cellulase production

The filamentous fungus Trichoderma reesei is an extraordinarily efficient cell factory of industrial cellulase for production of biofuels and other bio-based products because of its excellent potential to secrete cellulolytic enzymes. Engineering the protein secretory pathway may be a powerful means for efficient protein production. However, it is uncertain whether this engineering approach could improve cellulase production in T. reesei. Herein, the endoplasmic reticulum (ER) secretory pathway was engineered for the production of cellulolytic enzymes by multiple strategies, including: (I) overexpression of the key components of protein folding (Pdi1, Ero1 and BiP); (II) overexpression of the glycosylation-related elements (Gpt1 and Gls2); (III) knockout of the ER mannosidase I (Mns1) encoding gene mns1. By utilizing these ER engineering strategies, the secretion of β-glucosidase was remarkably elevated in the engineered strains, ranging from 29.2 % to 112.5 %. Furthermore, it was found that engineering these components also regulated the ER stress resistance. More importantly, the total cellulase production was increased with varying degrees, which reached a maximum of 149.4 %, using the filter paper assay (FPA) as a characterization method. These results demonstrated that engineering the ER secretory pathway can enhance protein secretion, particularly for cellulase production, which shed light for the development of high-efficient cellulolytic enzymes for economically feasible bioethanol production from lignocellulosic biomass.

English

Enzymatic cellulose hydrolysis is an important step for the production of second-generation biofuels. The filamentous fungus Trichoderma reesei is among the most important organisms for obtaining cellulolytic enzymes. The Cel6A (CBH II) cellulase from T. ressei plays an important role in cellulose hydrolysis and acts on the non-reducing end of cellulose, in contrast to Cel7B (CBH I), which acts on the reducing end of cellulose thus releasing cellobiose. Therefore, Cel6A deficiency becomes a limiting factor in cellulose saccharification. This work attempted to use codon optimization to enhance Cel6A expression in Escherichia coli. A plasmid expression vector, pUCITD04, was designed; this vector contains: the cel6a gene, regulatory regions (the promoter and terminator T7 sequences), the OmpT signal peptide that allows the secretion of proteins into the culture medium, and a 6His tail to allow purification of the protein by affinity chromatography. The protein expression experiment using a strain of E. coli transformed with pUCITD04 resulted in a 31 kDa polypeptide being secreted into the culture medium that did not possess enzymatic activity, meanwhile, the control strain transformed with the empty plasmid did not secrete any protein fragments, indicating that a truncated Cel6A was being produced by the experimental strain. This phenomenon has been reported during the production of recombinant cellulases in E. coli. In this research, we discuss probable causes of this phenomenon, as well as the drawbacks in the production of cellulases by E. coli, directing efforts to elucidate the causes of the production of truncated cellulases by this bacterial factory.   Key words: Gene construct, recombinant cellobiohydrolase, Escherichia coli.

Molecular Characterization of Xyloglucanase cel74a from Trichoderma reesei.

Background: The filamentous fungus Trichoderma reesei is used on an industrial scale to produce enzymes of biotechnological interest. This fungus has a complex cellulolytic system involved in the degradation of lignocellulosic biomass. However, several aspects related to the regulation of the expression of holocellulolytic genes and the production of cellulases by this fungus are still understood. Methods: Here, we constructed a null mutant strain for the xyloglucanase cel74a gene and performed the characterization of the Δcel74a strain to evaluate the genetic regulation of the holocellulases during sugarcane bagasse (SCB) cultivation. Results: Our results demonstrate that the deletion of xyloglucanase cel74a may impact the regulation of holocellulase expression during SCB cultivation. The expression of cellulases cel7a, cel7b, and cel6a was reduced in Δcel74a strain, while the hemicellulases xyn1 and xyn2 were increased in the presence of SCB. The cel74a mutation also affected the xyloglucan hydrolysis patterns. In addition, CEL74A activity was modulated in the presence of calcium, suggesting that this ion may be required for efficient degradation of xyloglucan. Conclusions: CEL74A affects the regulation of holocellulolytic genes and the efficient degradation of SCB in T. reesei. This data makes a significant contribution to our understanding of the carbon utilization of fungal strains as a whole.

CAMP activates calcium signalling via phospholipase C to regulate cellulase production in the filamentous fungus Trichoderma reesei

BackgroundThe filamentous fungus Trichoderma reesei is one of the best producers of cellulase and has been widely studied for the production of cellulosic ethanol and bio-based products. We previously reported that Mn2+ and N,N-dimethylformamide (DMF) can stimulate cellulase overexpression via Ca2+ bursts and calcium signalling in T. reesei under cellulase-inducing conditions. To further understand the regulatory networks involved in cellulase overexpression in T. reesei, we characterised the Mn2+/DMF-induced calcium signalling pathway involved in the stimulation of cellulase overexpression.ResultsWe found that Mn2+/DMF stimulation significantly increased the intracellular levels of cAMP in an adenylate cyclase (ACY1)-dependent manner. Deletion of acy1 confirmed that cAMP is crucial for the Mn2+/DMF-stimulated cellulase overexpression in T. reesei. We further revealed that cAMP elevation induces a cytosolic Ca2+ burst, thereby initiating the Ca2+ signal transduction pathway in T. reesei, and that cAMP signalling causes the Ca2+ signalling pathway to regulate cellulase production in T. reesei. Furthermore, using a phospholipase C encoding gene plc-e deletion strain, we showed that the plc-e gene is vital for cellulase overexpression in response to stimulation by both Mn2+ and DMF, and that cAMP induces a Ca2+ burst through PLC-E.ConclusionsThe findings of this study reveal the presence of a signal transduction pathway in which Mn2+/DMF stimulation produces cAMP. Increase in the levels of cAMP activates the calcium signalling pathway via phospholipase C to regulate cellulase overexpression under cellulase-inducing conditions. These findings provide insights into the molecular mechanism of the cAMP–PLC–calcium signalling pathway underlying cellulase expression in T. reesei and highlight the potential applications of signal transduction in the regulation of gene expression in fungi.