Showing papers on "Trichoderma reesei published in 2021"
TL;DR: In this article, response surface methodology (RSM) is used for predictive model and optimization of the whole experimental methods of reducing sugar and energy in a Dred sunflower stalks were pretreated by sodium hydroxide (NaOH) and Trichoderma reesei as a function of two variables: concentration of NaOH (%) and time for pretreatment (Day).
Abstract: The present paper discusses response surface methodology (RSM) as an efficient tactic for predictive model and optimization of the whole experimental methods of reducing sugar and energy. In this work, the application of RSM presented for optimizing reducing sugar and energy as compared with production between chemical and biological pretreatments. All experiments applied statistical designs in order to develop a statistic multivariate analysis model that provides to consider the effect of different parameters on a process and describe the optimum values of these variables to optimize the response. Dred sunflower stalks were pretreated by sodium hydroxide (NaOH) and Trichoderma reesei as a function of two variables: concentration of NaOH (%) and T. reesei (%) and time for pretreatment (Day) to receive reducing sugar and energy. The chemical pretreatment model was characterized by 13 runs, varying the variables at two factors, NaOH (1, 1.5, 2%) and Day (1, 2, 3). The biological pretreatment model was characterized by 13 runs, varying the variables at two factors, T. reesei (1, 1.5, 2%) and Day (1, 2, 3), by central composite design experimental design. In the chemical pretreatment, experiments performed at 2% (w/v) of NaOH for 3 days were used. The chemical pretreatment model at 2% NaOH for a 3-day release reduced sugar by 5.812 g/L and energy by 92.992 kJ/L; on the other hand, biological pretreatment model at 2% T. reesei for a 3-day release reduced sugar by 3.891 g/L and energy by 62.256 kJ/L, reducing sugar starter for fermentation by 49.0670 ± 6.4660 g/L and fermentation efficiency by 71.60% at 48 h fermented time.
76 citations
TL;DR: This review shows the sequence and structural diversity of non-homologous cellulases and summarize cellulolytic enzyme systems used by cellulose degrading microorganisms and discusses recent progress in culture-independent metagenomic approaches as a powerful tool for the bioprospecting of novel cellulases.
28 citations
TL;DR: In this paper, a PET hydrolase from PET-assimilating bacterium Ideonella sakaiensis (IsPETase) that exhibits superior PET hydrolytic activity at mild conditions is attracting enormous attention in development of plastic biodegrading strategies.
27 citations
TL;DR: The results suggest that introducing product-activated cellulase genes is a simple and feasible method to alleviate the product inhibition of cellulase.
26 citations
TL;DR: Interestingly, their co-cultivation in sequential SoSF-SmF significantly improved overall enzyme production possibly due to synergism of the enzymes produced by both strains and combined advantages of SoSF and SmF.
21 citations
TL;DR: In this paper, the cellular function and distribution of 10 known β-glucosidases (CEL3B, CEL3E, Cel3F, Cel1B, Cel2G, Cel4G and Cel3G) in Trichoderma reesei were investigated.
Abstract: Trichoderma reesei has 11 putative β-glucosidases in its genome, playing key parts in the induction and production of cellulase. Nevertheless, the reason why the T. reesei genome encodes so many β-glucosidases and the distinct role each β-glucosidase plays in cellulase production remain unknown. In the present study, the cellular function and distribution of 10 known β-glucosidases (CEL3B, CEL3E, CEL3F, CEL3H, CEL3J, CEL1A, CEL3C, CEL1B, CEL3G, and CEL3D) were explored in T. reesei, leaving out BGL1 (CEL3A), which has been well investigated. We found that the overexpression of cel3b or cel3g significantly enhanced extracellular β-glucosidase production, whereas the overexpression of cel1b severely inhibited cellulase production by cellulose, resulting in nearly no growth of T. reesei Four types of cellular distribution patterns were observed for β-glucosidases in T. reesei: (i) CEL3B, CEL3E, CEL3F, and CEL3G forming clearly separated protein secretion vesicles in the cytoplasm; (ii) CEL3H and CEL3J diffusing the whole endomembrane as well as the cell membrane with protein aggregation, like a reticular network; (iii) CEL1A and CEL3D in vacuoles; (iv) and CEL3C in the nucleus. β-glucosidases CEL1A, CEL3B, CEL3E, CEL3F, CEL3G, CEL3H, and CEL3J were identified as extracellular, CEL3C and CEL3D as intracellular, and CEL1B as unknown. The extracellular β-glucosidases CEL3B, CEL3E, CEL3F, CEL3H, and CEL3G were secreted through a tip-directed conventional secretion pathway, and CEL1A, via a vacuole-mediated pathway that was achieved without any signal peptide, while CEL3J was secreted via an unconventional protein pathway bypassing the endoplasmic reticulum (ER) and Golgi.IMPORTANCE Although β-glucosidases play an important role in fungal cellulase induction and production, our current understanding does not provide a global perspective on β-glucosidase function. This work comprehensively studies all the β-glucosidases regarding their effect on cellulase production and their cellular distribution and secretion. Overexpression of cel3b or cel3g significantly enhanced β-glucosidase production, whereas overexpression of cel1b severely inhibited cellulase production on cellulose. In addition, overexpression of cel3b, cel3e, cel3f, cel3h, cel3j, cel3c, or cel3g delayed endoglucanase (EG) production. We first identified four cellular distribution patterns of β-glucosidases in Trichoderma reesei Specially, CEL3C was located in the nucleus. CEL3J was secreted through the nonclassical protein secretion pathway bypassing endoplasmic reticulum (ER) and Golgi. CEL1A was secreted via a vacuole-mediated conventional secretion route without a signal peptide. These findings advance our understanding of β-glucosidase properties and secretory pathways in filamentous fungi, holding key clues for future study.
19 citations
TL;DR: In this article, a novel transcriptional activator ACE4 was identified by comparative genomic screening that positively regulates cellulase gene expression on cellulose in T. reesei, and ACE4 directly binds to the promoter of cellulase genes by recognizing two adjacent 5'-GGCC-3' sequences.
Abstract: The filamentous fungus Trichoderma reesei is a model strain for cellulase production. Cellulase gene expression in T. reesei is controlled by multiple transcription factors. Here, we identified by comparative genomic screening a novel transcriptional activator ACE4 ( A ctivator of c ellulase e xpression 4) that positively regulates cellulase gene expression on cellulose in T. reesei. Disruption of the ace4 gene significantly decreased expression of four main cellulase genes, and the essential cellulase transcription factor encoding gene ace3. Overexpression of ace4 increased cellulase production by approximately 22% compared to that in the parental strain. Further investigations using electrophoretic mobility shift assays, DNase I footprinting assays, and chromatin immunoprecipitation assays indicated that ACE4 directly binds to the promoter of cellulase genes by recognizing the two adjacent 5'-GGCC-3' sequences. Additionally, ACE4 directly binds to the promoter of ace3 and, in turn, regulates the expression of ACE3 to facilitate cellulase production. Collectively, these results demonstrate an important role for ACE4 in regulating cellulase gene expression, which will contribute to understanding the mechanism underlying cellulase expression in T. reesei. IMPORTANCE T. reesei is commonly utilized in industry to produce cellulases, enzymes that degrade lignocellulosic biomass for the production of bioethanol and bio-based products. T. reesei is capable of rapidly initiating the biosynthesis of cellulases in the presence of cellulose, which has made it useful as a model fungus for studying gene expression in eukaryotes. Cellulase gene expression is controlled through multiple transcription factors at the transcriptional level. However, the molecular mechanisms by which transcription is controlled remain unclear. In the present study, we identified a novel transcription factor, ACE4, which regulates cellulase expression on cellulose by binding to the promoters of cellulase genes and the cellulase activator ace3. Our study not only expands the general functional understanding of the novel transcription factor ACE4 but also provides evidence for the regulatory mechanism mediating gene expression in T. reesei.
18 citations
TL;DR: In this paper, the authors provided kinetic characterization of cellulose peroxygenase and reductant peroxidase (oxidative cleavage of glycosidic bonds in cellulose) activities of the LPMO TrAA9A of the cellulose-degrading model fungus Trichoderma reesei.
18 citations
TL;DR: In this paper, a co-culture of Trichoderma reesei and Monascus purpureus was used for the enzymatic hydrolysis of wheat straw biomass.
Abstract: Wheat straw (Triticum aestivum) is one of the lignocellulosic materials largely available worldwide and could be potentially used for biofuel production. Aiming the cost-effective utilization of wheat straw in the sugar-based biorefineries, co-cultures of Trichoderma reesei and Monascus purpureus were used for the enzymatic hydrolysis of the wheat straw biomass. The enzymatic breakdown of the dual-fungi-treated wheat straw was chemically analyzed through different enzyme/compositional assays, and the structural modifications were studied through scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR). For hydrolytic enzyme assays, the co-culture treatments resulted in significantly higher values (carboxymethyl cellulase (212.3 U/ml; p = 0.0173*), total cellulase (202 U/ml; p < 0.0001****), and xylanase (96.7 U/ml; p < 0.0001****) when compared with the readings of pure cultures. This hydrolytic activity resulted in the enhanced breakdown of wheat straw exhibiting a significant loss of 45.2% in lignin, 19.18% in cellulase, and 21.84% in hemicellulose contents. Furthermore, SEM and FTIR analysis of the co-culture treatments verified the improved biodegradation of wheat straw. Accumulatively, these results suggest a better approach for the effective use of dual-fungi for the lignocellulosic biomass breakdown and may have applications in bioethanol biorefineries using wheat straw as a sugar feedstock.
18 citations
TL;DR: In this article, the performance of T. reesei during co-culturing has been assessed with several fungal partners, including Aspergillus niger, one of the most recurrent partners.
Abstract: Biorefineries are core facilities for implementing a sustainable circular bioeconomy. These facilities rely on microbial enzymes to hydrolyze lignocellulosic substrates into fermentable sugars. Fungal co-cultures mimic the process of natural biodegradation and have been shown to increase certain enzyme activities. Trichoderma reesei and its many mutant strains are major cellulase producers and are heavily utilized as a source of carbohydrate-active enzymes. Several reports have demonstrated that T. reesei co-cultures present higher enzyme activities compared with its monocultures, especially in the context of β-glucosidase activity. The performance of T. reesei during co-culturing has been assessed with several fungal partners, including Aspergillus niger, one of the most recurrent partners. Various aspects of co-cultivation still need further investigation, especially regarding the molecular interactions between fungi in controlled environments and the optimization of the resulting enzyme cocktails. Since plenty of genetic and physiological data on T. reesei is available, the species is an outstanding candidate for future co-culture investigations. Co-cultures are still a developing field for industrial enzyme production, and many aspects of the technique need further improvement before real applications. KEY POINTS: • T. reesei co-cultures are an alternative for producing lignocellulolytic enzymes. • Several reports suggest an increase in certain enzyme activities in co-cultures. • More in-depth investigations of co-cultures are necessary for advancing this field.
17 citations
TL;DR: In this article, the authors summarized recent progress and gave an overview of the cellular process of cellulase production in T. reesei, including the carbon source-dependent cellulase induction, complicated transcriptional regulation network, and efficient protein assembly and trafficking.
Abstract: The filamentous fungus Trichoderma reesei has been widely used for cellulase production that has extensive applications in green and sustainable development. Increasing costs and depletion of fossil fuels provoke the demand for hyper-cellulase production in this cellulolytic fungus. To better manipulate T. reesei for enhanced cellulase production and to lower the cost for large-scale fermentation, it is wise to have a comprehensive understanding of the crucial factors and complicated biological network of cellulase production that could provide new perspectives for further exploration and modification. In this review, we summarize recent progress and give an overview of the cellular process of cellulase production in T. reesei, including the carbon source-dependent cellulase induction, complicated transcriptional regulation network, and efficient protein assembly and trafficking. Among that, the key factors involved in cellulase production were emphasized, shedding light on potential perspectives for further engineering.
TL;DR: Efficient genome editing systems were developed in filamentous fungi P. oxalicum and T. reesei by using heterologous or native 5S rRNA promoters for guide RNA expression using a markerless donor DNA as editing template.
Abstract: To construct convenient CRISPR/Cas9-mediated genome editing systems in industrial enzyme-producing fungi Penicillium oxalicum and Trichoderma reesei. Employing the 5S rRNA promoter from Aspergillus niger for guide RNA expression, the β-glucosidase gene bgl2 in P. oxalicum was deleted using a donor DNA carrying 40-bp homology arms or a donor containing no selectable marker gene. Using a markerless donor DNA as editing template, precise replacement of a small region was achieved in the creA gene. In T. reesei, the A. niger 5S rRNA promoter was less efficient than that in P. oxalicum when used for gene editing. Using a native 5S rRNA promoter, stop codons were introduced into the lae1 coding region using a markerless donor DNA with an editing efficiency of 36.67%. Efficient genome editing systems were developed in filamentous fungi P. oxalicum and T. reesei by using heterologous or native 5S rRNA promoters for guide RNA expression.
TL;DR: In this paper, the authors summarized the biotechnological advances in engineering T. reesei cellulase for enhanced cellulase production and proposed innovative ideas of systematically optimizing cellulase composition at the transcriptional level and improving cellulase output at the regulation level.
Abstract: Cellulase has the biggest contribution to the high production costs of lignocellulose bioconversion and the substantial decrease of its production cost is the key to the commercialization of lignocellulosic biorefineries. Trichoderma reesei has the most robust cellulase among the candidates, which therefore is widely used for cellulase production in industry. This is not because of the size of its cellulase gene pool but its prodigious cargo of cellulase productivity. Still, T. reesei cellulase falls far short of perfection in real-world applications, especially for the composition. This review summarized the biotechnological advances in engineering T. reesei for enhanced cellulase production. Meanwhile, we proposed innovative ideas of systematically optimizing cellulase composition at the transcriptional level and improving cellulase production at the regulation level. Efficient genome editing is essential to achieving that target. Thus, the developments of the tools of multiple gene manipulations were discussed in detail here. This review provides ideas and/or inspirations to the future researches on T. reesei cellulase.
TL;DR: A solid screw feeder was designed and used to feed steam-exploded lignocellulosic biomass continuously for cellulase induction and production from Trichoderma reesei RUT C30 and a promising second-generation energy crop, Miscanthus lutarioriparius, was found to be a much better inducer than other types of biomass.
TL;DR: Force spectroscopy measurements of CBM1–cellulose interactions, along with molecular dynamics simulations, indicated that previous interpretations of classical binding assay results using multisite adsorption models may have complicated analysis, and instead suggest simpler single-site models should be used.
TL;DR: In this article, a xylanase hyper-producing system was established by tailoring two transcription factors, XYR1 and ACE1, and homologous overexpression of the major endo-xylanase XYNII.
Abstract: Filamentous fungus Trichoderma reesei has been widely used as a workhorse for cellulase and xylanase productions Xylanase has been reported as the crucial accessory enzyme in the degradation of lignocellulose for higher accessibility of cellulase In addition, the efficient hydrolysis of xylan needs the co-work of multiple xylanolytic enzymes, which rise an increasing demand for the high yield of xylanase for efficient biomass degradation In this study, a xylanase hyper-producing system in T reesei was established by tailoring two transcription factors, XYR1 and ACE1, and homologous overexpression of the major endo-xylanase XYNII The expressed xylanase cocktail contained 5256 U/mL xylanase activity and 925 U/mL β-xylosidase (pNPXase) activity Meanwhile, the transcription level of the xylanolytic genes in the strain with XYR1 overexpressed was upregulated, which was well correlated with the amount of XYR1-binding sites In addition, the higher expression of associated xylanolytic enzymes would result in more efficient xylan hydrolysis Besides, 2310–3085 U/mL of xylanase activities were achieved using soluble carbon source, which was more efficient and economical than the traditional strategy of xylan induction Unexpectedly, deletion of ace1 in C30OExyr1 did not give any improvement, which might be the result of the disturbed function of the complex formed between ACE1 and XYR1 The enzymatic hydrolysis of alkali pretreated corn stover using the crude xylanase cocktails as accessory enzymes resulted in a 3664% increase in saccharification efficiency with the ratio of xylanase activity vs FPase activity at 500, compared to that using cellulase alone An efficient and economical xylanase hyper-producing platform was developed in T reesei RUT-C30 The novel platform with outstanding ability for crude xylanase cocktail production would greatly fit in biomass degradation and give a new perspective of further engineering in T reesei for industrial purposes
TL;DR: In this article, a study of β-glucosidase production in solid state cultivation (SSC) of two filamentous fungi, thermophilic Thermoascus aurantiacus and mesophilic Trichoderma reesei, and application of the enzymatic extracts on non-pretreated sugarcane bagasse (SCB) saccharification was performed.
Abstract: For degradation of sugarcane bagasse (SCB), several enzymes are needed but β-glucosidase is rate limiting in cellulose hydrolysis. Since different microorganisms synthetize characteristic pool of enzymes, mixing extracts produced by different species may increase hydrolytic efficiency due to synergism between enzymes in cocktails. This paper reports the study of β-glucosidase production in solid state cultivation (SSC) of two filamentous fungi, thermophilic Thermoascus aurantiacus and mesophilic Trichoderma reesei, and application of the enzymatic extracts on non-pretreated SCB saccharification. Enzyme extract obtained from the thermophilic fungus presented higher β-glucosidase and FPU activities (1.8 U/mL and 10 FPU/mL) than the one from mesophilic (0.2 U/mL and 6 FPU/mL). Optimal SCB hydrolysis was achieved when applying enzymatic cocktail composed of equal volumes of both fungal extracts (3.6 FPU/gSCB, filter paper units per gram SCB, 2.25 FPU/gSCB provided by extract from T. aurantiacus and 1.35 FPU/gSCB from T. reesei) at 65 °C. The hydrolysis yield applying the enzyme cocktail, 124 mg total reducing sugars (TRS) per gSCB, was higher than any yield achieved when using the enzyme extracts separately (105 mgTRS/gSCB using 12.5 FPU per gSCB from T. aurantiacus at 65 °C; 79 mgTRS/gSCB using 7.5 FPU per gSCB from T. reesei at 45 °C). Therefore, the use of the cocktail (3.6 FPU/gSCB) at 65 °C released 18 and 57% more TRS respectively than when extracts from T. aurantiacus or from T. reesei were applied alone, respectively, even reducing enzyme load (FPU) by 70%, corroborating the synergistic effect when both extracts are used together.
TL;DR: In this article, the authors evaluated the effectiveness of pre-treatment and fermentation of wet corn distillers' grains and solubles (WDGS), soybean hull (SH), and their blend in swine and poultry diets.
TL;DR: The successful expression of laminarinase-encoding genes in S. cerevisiae is an important milestone in exploiting brown macroalgae for bioethanol production via consolidated bioprocessing.
Abstract: Increasing demands for fuel production have led to the identification of brown macroalgae as an alternative feedstock for biofuel production. However, the conversion of macroalgae to biofuel using yeast, such as Saccharomyces cerevisiae, requires the production of enzymes capable of hydrolysing macroalgal carbohydrates. In this study, three synthetic laminarinase-encoding genes from Rasamsonia emersonii (Relam1), Trichoderma viride (Tvlam1) and Trichoderma reesei (Trlam1), as well as a native laminarinase-like gene from S. cerevisiae (Sclam1) were cloned and expressed in the laboratory S. cerevisiae Y294 strain. Supernatant containing the Relam1 enzyme displayed the highest extracellular activity at 45 °C (0.2 U/mL), while supernatant from the strain co-expressing the Relam1 and Tvlam1 genes showed the highest extracellular activity at 37 °C (0.09 U/mL). The Relam1 and Tvlam1 laminarinases displayed optimum enzymatic activity at temperatures of 45 °C and 37 °C, respectively, and Relam1 retained more than 89% activity after 96 h at 45 °C. Large-scale fermentation of Ecklonia maxima macroalgae with S. cerevisiae Y294[Re/Tvlam1] in a benchtop bioreactor achieved 43% of the theoretic ethanol yield and 66% carbon conversion from laminarin. The successful expression of laminarinase-encoding genes in S. cerevisiae is an important milestone in exploiting brown macroalgae for bioethanol production via consolidated bioprocessing.
TL;DR: In this article, the authors identified the TrCYC8/TUP1 complex as a coactivator for XYR1 in filamentous fungus Trichoderma reesei, and showed that repressed Trtup1 or Trcyc8 expression caused a strong defect in Xyr1 occupancy and loss of histone H4 at cellulase gene promoters.
Abstract: Cellulase production in filamentous fungus Trichoderma reesei is highly responsive to various environmental cues involving multiple positive and negative regulators. XYR1 (Xylanase regulator 1) has been identified as the key transcriptional activator of cellulase gene expression in T. reesei. However, the precise mechanism by which XYR1 achieves transcriptional activation of cellulase genes is still not fully understood. Here, we identified the TrCYC8/TUP1 complex as a novel coactivator for XYR1 in T. reesei. CYC8/TUP1 is the first identified transcriptional corepressor complex mediating repression of diverse genes in Saccharomyces cerevisiae. Knockdown of Trcyc8 or Trtup1 resulted in markedly impaired cellulase gene expression in T. reesei. We found that TrCYC8/TUP1 was recruited to cellulase gene promoters upon cellulose induction and this recruitment is dependent on XYR1. We further observed that repressed Trtup1 or Trcyc8 expression caused a strong defect in XYR1 occupancy and loss of histone H4 at cellulase gene promoters. The defects in XYR1 binding and transcriptional activation of target genes in Trtup1 or Trcyc8 repressed cells could not be overcome by XYR1 overexpression. Our results reveal a novel coactivator function for TrCYC8/TUP1 at the level of activator binding, and suggest a mechanism in which interdependent recruitment of XYR1 and TrCYC8/TUP1 to cellulase gene promoters represents an important regulatory circuit in ensuring the induced cellulase gene expression. These findings thus contribute to unveiling the intricate regulatory mechanism underlying XYR1-mediated cellulase gene activation and also provide an important clue that will help further improve cellulase production by T. reesei.
TL;DR: In this article, the authors constructed the biosynthesis pathway of d-glucaric acid in S. cerevisiae INVSc1 opi1Δ and obtained two engineered strains, LGA-1 and LGA -C, producing record-breaking titers of dglucaranic acid: 9.53 ± 0.46
Abstract: The biomanufacturing of d-glucaric acid has attracted increasing interest because it is one of the top value-added chemicals produced from biomass. Saccharomyces cerevisiae is regarded as an excellent host for d-glucaric acid production. The opi1 gene was knocked out because of its negative regulation on myo-inositol synthesis, which is the limiting step of d-glucaric acid production by S. cerevisiae. We then constructed the biosynthesis pathway of d-glucaric acid in S. cerevisiae INVSc1 opi1Δ and obtained two engineered strains, LGA-1 and LGA-C, producing record-breaking titers of d-glucaric acid: 9.53 ± 0.46 g/L and 11.21 ± 0.63 g/L d-glucaric acid from 30 g/L glucose and 10.8 g/L myo-inositol in fed-batch fermentation mode, respectively. However, LGA-1 was preferable because of its genetic stability and its superior performance in practical applications. There have been no reports on d-glucaric acid production from lignocellulose. Therefore, the biorefinery processes, including separated hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF) and consolidated bioprocessing (CBP) were investigated and compared. CBP using an artificial microbial consortium composed of Trichoderma reesei (T. reesei) Rut-C30 and S. cerevisiae LGA-1 was found to have relatively high d-glucaric acid titers and yields after 7 d of fermentation, 0.54 ± 0.12 g/L d-glucaric acid from 15 g/L Avicel and 0.45 ± 0.06 g/L d-glucaric acid from 15 g/L steam-exploded corn stover (SECS), respectively. In an attempt to design the microbial consortium for more efficient CBP, the team consisting of T. reesei Rut-C30 and S. cerevisiae LGA-1 was found to be the best, with excellent work distribution and collaboration. Two engineered S. cerevisiae strains, LGA-1 and LGA-C, with high titers of d-glucaric acid were obtained. This indicated that S. cerevisiae INVSc1 is an excellent host for d-glucaric acid production. Lignocellulose is a preferable substrate over myo-inositol. SHF, SSF, and CBP were studied, and CBP using an artificial microbial consortium of T. reesei Rut-C30 and S. cerevisiae LGA-1 was found to be promising because of its relatively high titer and yield. T. reesei Rut-C30 and S. cerevisiae LGA-1were proven to be the best teammates for CBP. Further work should be done to improve the efficiency of this microbial consortium for d-glucaric acid production from lignocellulose.
TL;DR: In this article, the authors found that deletion of an α-tubulin (tubB) in T. reesei enhanced both the amount and rate of secretory protein production.
Abstract: Trichoderma reesei is a filamentous fungus that is important as an industrial producer of cellulases and hemicellulases due to its high secretion of these enzymes and outstanding performance in industrial fermenters. However, the reduction of enzyme production caused by carbon catabolite repression (CCR) has long been a problem. Disruption of a typical transcriptional regulator, Cre1, does not sufficiently suppress this reduction in the presence of glucose. We found that deletion of an α-tubulin (tubB) in T. reesei enhanced both the amount and rate of secretory protein production. Also, the tubulin-disrupted (ΔtubB) strain had high enzyme production and the same enzyme profile even if the strain was cultured in a glucose-containing medium. From transcriptome analysis, the ΔtubB strain exhibited upregulation of both cellulase and hemicellulase genes including some that were not originally induced by cellulose. Moreover, cellobiose transporter genes and the other sugar transporter genes were highly upregulated, and simultaneous uptake of glucose and cellobiose was also observed in the ΔtubB strain. These results suggested that the ΔtubB strain was released from CCR. Trichoderma reesei α-tubulin is involved in the transcription of cellulase and hemicellulase genes, as well as in CCR. This is the first report of overcoming CCR by disrupting α-tubulin gene in T. reesei. The disruption of α-tubulin is a promising approach for creating next-generation enzyme-producing strains of T. reesei.
TL;DR: In this article, the authors showed that Mn2+/DMF stimulation significantly increased the intracellular levels of cAMP in an adenylate cyclase (ACY1)-dependent manner, and that cAMP signalling causes the Ca2+ signalling pathway to regulate cellulase production in T. reesei.
Abstract: The 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. We 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. The 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.
TL;DR: In this paper, the authors analyzed the production profile of holocellulases from Aspergillus and Humicola species, Trichoderma reesei RP698, and Mycothermus thermophilus grown in sugarcane bagasse, culm of energy cane, and culm in sugar cane SP80-3280.
Abstract: Economic interest in sugarcane bagasse has significantly increased in recent years due to the worldwide demand for sustainable energy production. The use of sugarcane bagasse for holocellulase production has been a strategy for bioconversion of lignocellulosic residues into second-generation ethanol. The fungi secrete to the culture medium a cocktail of enzymes necessary to convert biomass into nutrients. Thus, this study aimed to analyze the production profile of holocellulases from Aspergillus and Humicola species, Trichoderma reesei RP698, and Mycothermus thermophilus grown in sugarcane bagasse, culm of energy cane, and culm of sugarcane SP80-3280. The capacity of the enzymatic pools in the hydrolysis of cell walls of these sugarcane varieties was also verified. M. thermophilus was the best producer of endoglucanase, cellobiohydrolase, β-glucosidase, xylanase, β-xylosidase, xyloglucanase, arabinanase, arabinofuranosidase, mannanase, and acetyl xylan esterase. T. reesei RP698 also produced and secreted a wide range of holocellulases to the medium. The saccharification of sugarcane bagasse, energy cane, and sugarcane SP80-3280 by the enzymatic cocktails obtained from M. thermophilus released 0.87 ± 0.05 mg.mL−1, 0.88 ± 0.07 mg.mL−1, and 1.10 ± 0.08 mg.mL−1 of reducing sugars, respectively. However, the application of T. reesei RP698 extracts showed a release of 0.85 ± 0.03 mg.mL−1, 0.40 ± 0.03 mg.mL−1, and 0.83 ± 0.03 mg.mL−1 of reducing sugars. Therefore, T. reesei RP698 and M. thermophilus showed to be good holocellulase producers, and their crude extracts presented a great capacity for the hydrolysis of the different kinds of sugarcane residues.
TL;DR: In this article, the gul1 gene, which encodes a putative mRNA-binding protein, was disrupted in cellulase-producing fungus Trichoderma reesei, resulting in smaller mycelial clumps and significantly lower viscosity of fermentation broth.
Abstract: Hyphal morphology is considered to have a close relationship with the production level of secreted proteins by filamentous fungi In this study, the gul1 gene, which encodes a putative mRNA-binding protein, was disrupted in cellulase-producing fungus Trichoderma reesei The hyphae of Δgul1 strain produced more lateral branches than the parent strain Under the condition for cellulase production, disruption of gul1 resulted in smaller mycelial clumps and significantly lower viscosity of fermentation broth In addition, cellulase production was improved by 22% relative to the parent strain Transcriptome analysis revealed that a set of genes encoding cell wall remodeling enzymes as well as hydrophobins were differentially expressed in the Δgul1 strain The results suggest that the regulatory role of gul1 in cell morphogenesis is likely conserved in filamentous fungi To our knowledge, this is the first report on the engineering of gul1 in an industrially important fungus
TL;DR: In this paper, four different fungi Aspergillus oryzae, Rhizopus oryzaa, Mucor indicus and Trichoderma reesei were employed to ferment the mixture of WDGS and soybean hull at 75/25 ratio at 28°C for 6-9 days.
TL;DR: Cultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates, suggesting that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specific substrate requirements.
Abstract: On-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharification by exploiting the fungal gene regulatory mechanism that enables it to continuously adapt enzyme secretion to the substrate used for cultivation. To harness this, the interrelation between substrate characteristics and fungal response must be understood. However, fungal morphology or gene expression studies often lack structural and chemical substrate characterization. Here, T. reesei QM6a was cultivated on three softwood substrates: northern bleached softwood Kraft pulp (NBSK) and lodgepole pine pretreated either by dilute-acid-catalyzed steam pretreatment (LP-STEX) or mild alkaline oxidation (LP-ALKOX). With different pretreatments of similar starting materials, we presented the fungus with systematically modified substrates. This allowed the elucidation of substrate-induced changes in the fungal response and the testing of the secreted enzymes’ hydrolytic strength towards the same substrates. Enzyme activity time courses correlated with hemicellulose content and cellulose accessibility. Specifically, increased amounts of side-chain-cleaving hemicellulolytic enzymes in the protein produced on the complex substrates (LP-STEX; LP-ALKOX) was observed by secretome analysis. Confocal laser scanning micrographs showed that fungal micromorphology responded to changes in cellulose accessibility and initial culture viscosity. The latter was caused by surface charge and fiber dimensions, and likely restricted mass transfer, resulting in morphologies of fungi in stress. Supplementing a basic cellulolytic enzyme mixture with concentrated T. reesei supernatant improved saccharification efficiencies of the three substrates, where cellulose, xylan, and mannan conversion was increased by up to 27, 45, and 2800%, respectively. The improvement was most pronounced for proteins produced on LP-STEX and LP-ALKOX on those same substrates, and in the best case, efficiencies reached those of a state-of-the-art commercial enzyme preparation. Cultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates. This suggests that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specific substrate requirements.
TL;DR: In this paper, the recombinant T. reesei PB-3 strain overexpressing the β-glucosidase gene was used to achieve a high cellobiase activity of 78.09 CBU/mL using glucose as the sole substrate using fedbatch fermentation.
TL;DR: Using cellulase enzyme in deinking process of MOW have a great potential as an alternative to chemical conventional deinking per se and properties of papers obtained from this deinks process were better than both enzymatic and chemical ones.
Abstract: The effects of cellulase enzymes (Trichoderma reesei) in flotation deinking of mixed office wastes (MOW) on the deinking efficiency, physical and optical properties of the papers were investigated....
TL;DR: In this paper, the authors evaluated the hydrolysis of a mixture of sugarcane bagasse varieties (SCB), energy cane fiber (EC), and sugarcan bagasse from the SP80-3280 variety, all in natura, using fungal enzymatic extracts obtained from Mycothermus thermophilus and Trichoderma reesei RP698 cultures supplemented with various agro-industrial residues.