Showing papers on "Trichoderma reesei published in 2020"

The synergy between LPMOs and cellulases in enzymatic saccharification of cellulose is both enzyme- and substrate-dependent

Abstract: The synergistic effects between cellulases and lytic polysaccharide monooxygenases (LPMOs) were investigated systematically in terms of their degree of synergy (DS) on amorphous and crystalline cellulose. Synergy curves were obtained for enzyme pairs containing a cellulase from Trichoderma reesei (Cel6A and Cel7A) and three LPMOs from Thermoascus aurantiacus (TaAA9A), Lentinus similis (LsAA9A) and Thielavia terrestris (TtAA9E). The synergistic experiments showed that the three LPMOs significantly improved the hydrolytic efficiency of Cel6A, on both cellulosic substrates; a more pronounced effect being seen for TtAA9E on amorphous cellulose at low cellulase:LPMO ratios. In contrast, the highly processive, reducing-end acting Cel7A synergised with the C1-C4 oxidising LPMOs, TaAA9A and LsAA9A, but was inhibited by the presence of C1-oxidizing TtAA9E. The degree of synergy exhibited by the cellulase-LPMO mixtures was enzyme- and substrate-specific. The observed Cel7A inhibition, rather than synergy, by the C1-oxidizing LPMO, TtAA9E, warrants further investigations.

Rational engineering of the Trichoderma reesei RUT-C30 strain into an industrially relevant platform for cellulase production.

Abstract: The path for the development of hypersecreting strains of Trichoderma reesei capable of producing industrially relevant enzyme titers remains elusive despite over 70 years of research and industrial utilization. Herein, we describe the rational engineering of the publicly available T. reesei RUT-C30 strain and a customized process for cellulase production based on agroindustrial by-products. A CRISPR/Cas9 system was used to introduce six genetic modifications in RUT-C30. Implemented changes included the constitutive expression of a mutated allele of the cellulase master regulator XYR1, the expression of two heterologous enzymes, the β-glucosidase CEL3A from Talaromyces emersonii and the invertase SUC1 from Aspergillus niger, and the deletion of genes encoding the cellulase repressor ACE1 and the extracellular proteases SLP1 and PEP1. These alterations resulted in a remarkable increase of protein secretion rates by RUT-C30 and amended its well described β-glucosidase deficiency while enabling the utilization of sucrose and eliminating the requirement of inducing sugars for enzyme production. With a developed sugarcane molasses-based bioprocess, the engineered strain reached an extracellular protein titer of 80.6 g L−1 (0.24 g L−1 h−1), which is the highest experimentally supported titer so far reported for T. reesei. The produced enzyme cocktail displayed increased levels of cellulase and hemicellulase activities, with particularly large increments being observed for the specific activities of β-glucosidase (72-fold) and xylanase (42-fold). Notably, it also exhibited a saccharification efficiency similar to that of a commercially available cellulase preparation in the deconstruction of industrially pretreated sugarcane straw. This work demonstrates the rational steps for the development of a cellulase hyperproducing strain from a well-characterized genetic background available in the public domain, the RUT-C30, associated with an industrially relevant bioprocess, paving new perspectives for Trichoderma research on cellulase production.

Quantitative comparison of the biomass-degrading enzyme repertoires of five filamentous fungi

Abstract: The efficiency of microorganisms to degrade lignified plants is of great importance in the Earth’s carbon cycle, but also in industrial biorefinery processes, such as for biofuel production. Here, we present a large-scale proteomics approach to investigate and compare the enzymatic response of five filamentous fungi when grown on five very different substrates: grass (sugarcane bagasse), hardwood (birch), softwood (spruce), cellulose and glucose. The five fungi included the ascomycetes Aspergillus terreus, Trichoderma reesei, Myceliophthora thermophila, Neurospora crassa and the white-rot basidiomycete Phanerochaete chrysosporium, all expressing a diverse repertoire of enzymes. In this study, we present comparable quantitative protein abundance values across five species and five diverse substrates. The results allow for direct comparison of fungal adaptation to the different substrates, give indications as to the substrate specificity of individual carbohydrate-active enzymes (CAZymes), and reveal proteins of unknown function that are co-expressed with CAZymes. Based on the results, we present a quantitative comparison of 34 lytic polysaccharide monooxygenases (LPMOs), which are crucial enzymes in biomass deconstruction.

Mycosynthesis of ZnO Nanoparticles Using Trichoderma spp. Isolated from Rhizosphere Soils and Its Synergistic Antibacterial Effect against Xanthomonas oryzae pv. oryzae.

Abstract: The Plant Growth Promoting Fungi (PGPF) is used as a source of biofertilizers due to their production of secondary metabolites and beneficial effects on plants. The present work is focused on the co-cultivation of Trichoderma spp. (T. harzianum (PGT4), T. reesei (PGT5) and T. reesei (PGT13)) and the production of secondary metabolites from mono and co-culture and mycosynthesis of zinc oxide nanoparticles (ZnO NPs), which were characterized by a UV visible spectrophotometer, Powder X-ray Diffraction (PXRD), Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM) with Energy Dispersive Spectroscopy (EDAX) and Transmission Electron Microscope (TEM) and Selected Area (Electron) Diffraction (SAED) patterns. The fungal secondary metabolite crude was extracted from the mono and co-culture of Trichoderma spp. And were analyzed by GC-MS, which was further subjected for antibacterial activity against Xanthomonas oryzae pv. Oryzae, the causative organism for Bacterial Leaf Blight (BLB) in rice. Our results showed that the maximum zone of inhibition was recorded from the co-culture of Trichoderma spp. rather than mono cultures, which indicates that co-cultivation of beneficial fungi can stimulate the synthesis of novel secondary metabolites better than in monocultures. ZnO NPs were synthesized from fungal secondary metabolites of mono cultures of Trichoderma harzianum (PGT4), Trichoderma reesei (PGT5), Trichoderma reesei (PGT13) and co-culture (PGT4 + PGT5 + PGT13). These ZnO NPs were checked for antibacterial activity against Xoo, which was found to be of a dose-dependent manner. In summary, the biosynthesized ZnO NPs and secondary metabolites from co-culture of Trichoderma spp. are ecofriendly and can be used as an alternative for chemical fertilizers in agriculture.

Engineering of Trichoderma reesei for enhanced degradation of lignocellulosic biomass by truncation of the cellulase activator ACE3

Abstract: The filamentous fungus Trichoderma reesei is a major workhorse employed to produce cellulase, which hydrolyzes lignocellulosic biomass for the production of cellulosic ethanol and bio-based products. However, the economic efficiency of biorefineries is still low. In this study, the truncation of cellulase activator ACE3 was identified and characterized in T. reesei classical mutant NG14 and its direct descendants for the first time. We demonstrated that the truncated ACE3 is the crucial cause of cellulase hyper-production in T. reesei NG14 branch. Replacing the native ACE3 with truncated ACE3 in other T. reesei strains remarkably improves cellulase production. By truncating ACE3, we engineered a T. reesei mutant, PC-3-7-A723, capable of producing more cellulase than other strains. In a 30-L fermenter, fed-batch fermentation with PC-3-7-A723 drastically increased the maximum cellulase titer (FPase) to 102.63 IU/mL at 240 h, which constitutes a 20–30% improvement to that of the parental strain PC-3-7. This work characterized the function of truncated ACE3 and demonstrated that analysis of classical mutants allows rational engineering of mutant strains with improved cellulase production necessary to process lignocellulosic biomass. Our rational engineering strategy might be useful for enhancing the production of other bio-based products.

Consolidated bioprocessing of cellulose to itaconic acid by a co-culture of Trichoderma reesei and Ustilago maydis.

Abstract: Itaconic acid is a bio-derived platform chemical with uses ranging from polymer synthesis to biofuel production. The efficient conversion of cellulosic waste streams into itaconic acid could thus enable the sustainable production of a variety of substitutes for fossil oil based products. However, the realization of such a process is currently hindered by an expensive conversion of cellulose into fermentable sugars. Here, we present the stepwise development of a fully consolidated bioprocess (CBP), which is capable of directly converting recalcitrant cellulose into itaconic acid without the need for separate cellulose hydrolysis including the application of commercial cellulases. The process is based on a synthetic microbial consortium of the cellulase producer Trichoderma reesei and the itaconic acid producing yeast Ustilago maydis. A method for process monitoring was developed to estimate cellulose consumption, itaconic acid formation as well as the actual itaconic acid production yield online during co-cultivation. The efficiency of the process was compared to a simultaneous saccharification and fermentation setup (SSF). Because of the additional substrate consumption of T. reesei in the CBP, the itaconic acid yield was significantly lower in the CBP than in the SSF. In order to increase yield and productivity of itaconic acid in the CBP, the population dynamics was manipulated by varying the inoculation delay between T. reesei and U. maydis. Surprisingly, neither inoculation delay nor inoculation density significantly affected the population development or the CBP performance. Instead, the substrate availability was the most important parameter. U. maydis was only able to grow and to produce itaconic acid when the cellulose concentration and thus, the sugar supply rate, was high. Finally, the metabolic processes during fed-batch CBP were analyzed in depth by online respiration measurements. Thereby, substrate availability was again identified as key factor also controlling itaconic acid yield. In summary, an itaconic acid titer of 34 g/L with a total productivity of up to 0.07 g/L/h and a yield of 0.16 g/g could be reached during fed-batch cultivation. This study demonstrates the feasibility of consortium-based CBP for itaconic acid production and also lays the fundamentals for the development and improvement of similar microbial consortia for cellulose-based organic acid production.

Identification of a novel repressor encoded by the putative gene ctf1 for cellulase biosynthesis in Trichoderma reesei through artificial zinc finger engineering

Abstract: Strains from Trichoderma reesei have been used for cellulase production with a long history. It has been well known that cellulase biosynthesis by the fungal species is controlled through regulators, and elucidation of their regulation network is of great importance for engineering T. reesei with robust cellulase production. However, progress in this regard is still very limited. In this study, T. reesei RUT-C30 was transformed with an artificial zinc finger protein (AZFP) library, and the mutant T. reesei M2 with improved cellulase production was screened. Compared to its parent strain, the filter paper activity and endo-β-glucanase activity in cellulases produced by T. reesei M2 increased 67.2% and 35.3%, respectively. Analysis by quantitative reverse transcription polymerase chain reaction indicated significant downregulation of the putative gene ctf1 in T. reesei M2, and its deletion mutants were thus developed for further studies. An increase of 36.9% in cellulase production was observed in the deletion mutants, but when ctf1 was constitutively overexpressed in T. reesei RUT-C30 under the control of the strong pdc1 promoter, cellulase production was substantially compromised. Comparative transcriptomic analysis revealed that the deletion of ctf1 upregulated transcription of gene encoding the regulator VIB1, but downregulated transcription of gene encoding another regulator RCE1, which consequently upregulated genes encoding the transcription factors XYR1 and ACE3 for the activation of genes encoding cellulolytic enzymes. As a result, ctf1 was characterized as a gene encoding a repressor for cellulase production in T. reesei RUT-C30, which is significant for further elucidating molecular mechanism underlying cellulase biosynthesis by the fungal species for rational design to develop robust strains for cellulase production. And in the meantime, AZFP transformation was validated to be an effective strategy for identifying functions of putative genes in the genome of T. reesei.

Optimised cellulase production from fungal co-culture of Trichoderma reesei NCIM 1186 and Penicillium citrinum NCIM 768 under solid state fermentation

Abstract: Microbial cellulases find applications in many industries and constitute a significant share of the world’s industrial enzyme market. In order to improve the cost function of the cellulose producing processes with enhanced yield and novel activities, superior bioprocesses are formulated these days. Intending to produce higher titre of cellulase enzyme, present study deals with fungal co-culture and to investigate its feasibility of producing higher cellulase titre via solid state fermentation (SSF). Various process parameters were optimized for induced cellulase production from the co-culture of Trichoderma reesei NCIM 1186 and Penicillium citrinum NCIM 768 in SSF. Among different low cost and easily available agricultural lignocellulosic waste, wheat bran was found to be the most suitable substrate. The highest cellulase yield of 6.71 FPU/gds is obtained on 6th day of fermentation using steam pre-treated wheat bran with 70 % moisture content when inoculated with 106 spores of mixed suspension at 30 °C and pH 5. A further possibility to use whole fermented matter for saccharification instead of extracted cellulase was also investigated with the aim of abolishing an extraction step. Akin sugar yield of 6.45 mg/mL and 6.58 mg/mL was obtained at 72 h of hydrolysis of steam pre-treated wheat bran, using cellulase extract and whole fermented matter, respectively. Fungal co-culture of T. reesei and P. citrinum in SSF results in the highest cellulase titre using low cost agricultural waste as a substrate, which can be further used for successful saccharification in the form of whole fermented matter or as extracted enzyme.

Biodegradable, lignin-based encapsulation enables delivery of Trichoderma reesei with programmed enzymatic release against grapevine trunk diseases.

Abstract: Antagonistic fungi such as Trichoderma reesei are promising alternatives to conventional fungicides in agriculture. This is especially true for worldwide occurring grapevine trunk diseases, causing losses of US$1.5 billion every year, at which conventional fungicides are mostly ineffective or prohibited by law. Yet, applications of Trichoderma against grapevine trunk diseases are limited to preventive measures, suffer from poor shelf life, or uncontrolled germination. Therefore, we developed a mild and spore-compatible layer-by-layer assembly to encapsulate spores of a new mycoparasitic strain of T. reesei IBWF 034-05 in a bio-based and biodegradable lignin shell. The encapsulation inhibits undesired premature germination and enables the application as an aqueous dispersion via trunk injection. First injected into a plant, the spores remain in a resting state. Second, when lignin-degrading fungi infect the plant, enzymatic degradation of the shell occurs and germination is selectively triggered by the pathogenic fungi itself, which was proven in vitro. Germinated Trichoderma antagonizes the fungal pathogens and finally supplants them from the plant. This concept enables Trichoderma spores for curative treatment of esca, one of the most infective grapevine trunk diseases worldwide.

Convergent evolution of processivity in bacterial and fungal cellulases.

Abstract: Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B and CfCel48A) slide on crystalline cellulose. The direction of processive movement of CfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei (TrCel7A), whose movement was first observed by this technique, while CfCel48A moves in the same direction as TrCel7A. When CfCel6B and TrCel7A were mixed on the same substrate, "traffic accidents" were observed, in which the two cellulases blocked each other's progress. The processivity of CfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.

Promoting and Impeding Effects of Lytic Polysaccharide Monooxygenases on Glycoside Hydrolase Activity

Abstract: Lytic polysaccharide monooxygenases (LPMOs) have attracted attention due to their ability to boost cellulolytic enzyme cocktails for application in biorefineries. However, the interplay between LPMOs and individual glycoside hydrolases remains poorly understood. We investigated how the activity of two cellobiohydrolases (Cel7A and Cel6A) and an endoglucanase (Cel7B) from Trichoderma reesei were affected by a C1-oxidizing LPMO from Thielavia terrestris (TtAA9). We quantified products from a mixture of LPMO and glycoside hydrolase and estimated separate contributions of products by each of the enzymes. Hereby, we assessed if an observed synergy reflected a promotion of the activity of hydrolase, LPMO, or both. We consistently found that TtAA9 affected the investigated hydrolases differently. It strongly impeded the turnover of the reducing end cellobiohydrolase, TrCel7A, moderately promoted the turnover of the nonreducing end cellobiohydrolase TrCel6A, and promoted the turnover of the endoglucanase, TrCel7B up to 5-fold. The promoting effect on the endoglucanase increased with hydrolysis extent, indicating that the promoting effect became more important as the recalcitrance of the substrate increased. Experiments with mixtures containing multiple glycoside hydrolases suggested that the LPMO primarily promoted the activity of the endoglucanase, whereas promotion of TrCel6A was secondary.

Studies on sugar transporter CRT1 reveal new characteristics that are critical for cellulase induction in Trichoderma reesei

Abstract: Trichoderma reesei is an ascomycete fungus that has a tremendous capability of secreting extracellular proteins, mostly lignocellulose-degrading enzymes. Although many aspects of the biology of this organism have been unfolded, the roles of the many sugar transporters coded in its genome are still a mystery with a few exceptions. One of the most interesting sugar transporters that has thus far been discovered is the cellulose response transporter 1 (CRT1), which has been suggested to be either a sugar transporter or a sensor due to its seemingly important role in cellulase induction. Here we show that CRT1 is a high-affinity cellobiose transporter, whose function can be complemented by the expression of other known cellobiose transporters. Expression of two sequence variants of the crt1 gene in Saccharomyces cerevisiae revealed that only the variant listed in the RUT-C30 genome annotation has the capability to transport cellobiose and lactose. When expressed in the $$\Delta$$ crt1 strain, the variant listed in the QM6a genome annotation offers partial complementation of the cellulase induction, while the expression of the RUT-C30 variant or cellobiose transporters from two other fungal species fully restore the cellulase induction. These results add to our knowledge about the fungal metabolism of cellulose-derived oligosaccharides, which have the capability of inducing the cellulase production in many species. They also help us to deepen our understanding of the T. reesei lactose metabolism, which can have important consequences as this sugar is used as the inducer of protein secretion in many industrial processes which employ this species.

Biological Management of Banana Fusarium Wilt Caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 Using Antagonistic Fungal Isolate CSR-T-3 (Trichoderma reesei).

Abstract: Fusarium wilt in bananas is one of the most devastating diseases that poses a serious threat to the banana industry globally. With no effective control measures available to date, biological control has been explored to restrict the spread and manage the outbreak. We studied the effective biological control potential of different Trichoderma spp. in the management of Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). Expression of the defense related genes and metabolites in banana plants inoculated with Foc TR4 and treated with effective Trichoderma sp interactions were also studied. The in vitro growth inhibition of Foc TR4 by Trichoderma reesei isolate CSR-T-3 was 85.19% indicating a higher antagonistic potential than other Trichoderma isolates used in the study. Further, in in vivo assays, the banana plants treated with the isolate CSR-T-3 T. reesei had a significant reduction in the disease severity index (0.75) and also had increased phenological indices with respect to Foc TR4 treated plants. Enhanced activity of defense enzymes, such as β-1, 3-glucanase, peroxidase, chitinase, polyphenol oxidase, and phenylalanine ammonia lyase with higher phenol contents were found in the Trichoderma isolate CSR-T-3 treated banana plants challenge-inoculated with Foc TR4. Fusarium toxins, such as fusaristatin A, fusarin C, chlamydosporal, and beauveric acid were identified by LC-MS in Foc TR4-infected banana plants while high intensity production of antifungal compounds, such as s-caryophyllene, catechin-o-gallate, soyasapogenol rhamnosyl glucoronide, peptaibols, fenigycin, iturin C19, anthocyanin, and gallocatechin-o-gallate were detected in T. reesei isolate CSR-T-3 treated plants previously inoculated with Foc TR4. Gene expression analysis indicated the upregulation of TrCBH1/TrCBH2, TrXYL1, TrEGL1, TrTMK1, TrTGA1, and TrVEL1 genes in CSR-T-3 treatment. LC-MS and gene expression analysis could ascertain the upregulation of genes involved in mycoparasitism and the signal transduction pathway leading to secondary metabolite production under CSR-T-3 treatment. The plants in the field study showed a reduced disease severity index (1.14) with high phenological growth and yield indices when treated with T. reesei isolate CSR-T-3 formulation. We report here an effective biocontrol-based management technological transformation from lab to the field for successful control of Fusarium wilt disease caused by Foc TR4 in bananas.

Modification of transcriptional factor ACE3 enhances protein production in Trichoderma reesei in the absence of cellulase gene inducer

Abstract: Trichoderma reesei is one of the best-known cellulolytic organisms, producing large quantities of a complete set of extracellular cellulases and hemicellulases for the degradation of lignocellulosic substances. Hence, T. reesei is a biotechnically important host and it is used commercially in enzyme production, of both native and foreign origin. Many strategies for producing enzymes in T. reesei rely on the cbh1 and other cellulase gene promoters for high-level expression and these promoters require induction by sophorose, lactose or other inducers for high productivity during manufacturing. We described an approach for producing high levels of secreted proteins by overexpression of a transcription factor ACE3 in T. reesei. We refined the ace3 gene structure and identified specific ACE3 variants that enable production of secreted cellulases and hemicellulases on glucose as a sole carbon source (i.e., in the absence of an inducer). These specific ACE3 variants contain a full-length Zn2Cys6 binuclear cluster domain at the N-terminus and a defined length of truncations at the C-terminus. When expressed at a moderate level in the fungal cells, the ACE3 variants can induce high-level expression of cellulases and hemicellulases on glucose (i.e., in the absence of an inducer), and further improve expression on lactose or glucose/sophorose (i.e., in the presence of an inducer). Finally, we demonstrated that this method is applicable to industrial strains and fermentation conditions, improving protein production both in the absence and in the presence of an inducer. This study demonstrates that overexpression of ACE3 variants enables a high level of protein production in the absence of an inducer, and boosts protein production in the presence of an inducer. It is an efficient approach to increase protein productivity and to reduce manufacturing costs.

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

Abstract: 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.

The effect of hemicellulose on the binding and activity of cellobiohydrolase I, Cel7A, from Trichoderma reesei to cellulose

Abstract: Hydrothermal pre-treatments decrease lignocellulose recalcitrance against enzymatic hydrolysis by removing the majority of the hemicellulose, thus increasing cellulase accessibility. However, a small amount of the hemicellulose may remain and become adsorbed to the cellulose, leading to cellulase inhibition. Here, we produced hemicellulose bound cellulose, using glucuronoxylan and galactomannan, to simulate hydrothermally pre-treated hardwoods and softwoods, respectively, and evaluated how this can affect cellulose hydrolysis by Trichoderma reesei derived cellobiohydrolase I (Cel7A). Based on X-ray powder diffraction (XRD), histochemistry, scanning electron microscopy and Simon’s staining, hemicellulose binding onto cellulose affected the physical properties of the biomass, which subsequently affected its hydrolysis rate. As a result of hemicellulose binding onto cellulose, the adsorption of Cel7A was significantly impacted (up to 45%), leading to lowered activities (a 40% reduction), especially for glucuronoxylan. The bound hemicellulose may be released from the cellulose during agitation and hydrolysis. We therefore evaluated the effect of free hemicellulose on Cel7A. Free xylan was more inhibitory to Cel7A than free mannan, demonstrating non-competitive inhibition, while mannan exhibited uncompetitive inhibition. The recalcitrant effect of both bound and free hemicellulose could be relieved by the addition of hemicellulolytic enzymes (i.e. XT6 and Man26A) during cellulose hydrolysis. During the degradation of cellulose in “realistic” woody biomasses by Cel7A, the addition of hemicellulases led to a significant improvement in cellulose hydrolysis. This study showed that hemicellulose remains a critical factor regarding biomass recalcitrance and that the addition of hemicellulolytic activities in commercial enzyme cocktails is required (especially the mannanolytic activities lacking from most commercial enzyme cocktails), in order to realise high sugar yields at low enzyme protein loadings for low-cost biofuel production.

Identification and Characterization of an Efficient d-Xylose Transporter in Saccharomyces cerevisiae

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

Interfacial molecular interactions of cellobiohydrolase Cel7A and its variants on cellulose

Abstract: Molecular-scale mechanisms of the enzymatic breakdown of cellulosic biomass into fermentable sugars are still poorly understood, with a need for independent measurements of enzyme kinetic parameters. We measured binding times of cellobiohydrolase Trichoderma reesei Cel7A (Cel7A) on celluloses using wild-type Cel7A (WTintact), the catalytically deficient mutant Cel7A E212Q (E212Qintact) and their proteolytically isolated catalytic domains (CD) (WTcore and E212Qcore, respectively). The binding time distributions were obtained from time-resolved, super-resolution images of fluorescently labeled enzymes on cellulose obtained with total internal reflection fluorescence microscopy. Binding of WTintact and E212Qintact on the recalcitrant algal cellulose (AC) showed two bound populations: ~ 85% bound with shorter residence times of < 15 s while ~ 15% were effectively immobilized. The similarity between binding times of the WT and E212Q suggests that the single point mutation in the enzyme active site does not affect the thermodynamics of binding of this enzyme. The isolated catalytic domains, WTcore and E212Qcore, exhibited three binding populations on AC: ~ 75% bound with short residence times of ~ 15 s (similar to the intact enzymes), ~ 20% bound for < 100 s and ~ 5% that were effectively immobilized. Cel7A binding to cellulose is driven by the interactions between the catalytic domain and cellulose. The cellulose-binding module (CBM) and linker increase the affinity of Cel7A to cellulose likely by facilitating recognition and complexation at the substrate interface. The increased affinity of Cel7A to cellulose by the CBM and linker comes at the cost of increasing the population of immobilized enzyme on cellulose. The residence time (or inversely the dissociation rates) of Cel7A on cellulose is not catalysis limited.

Precision Engineering of the Transcription Factor Cre1 in Hypocrea jecorina (Trichoderma reesei) for Efficient Cellulase Production in the Presence of Glucose.

Abstract: In Trichoderma reesei, carbon catabolite repression (CCR) significantly downregulates the transcription of cellulolytic enzymes, which is usually mediated by the zinc finger protein Cre1. It was found that there is a conserved region at the C-terminus of Cre1/CreA in several cellulase-producing fungi that contains up to three continuous S/T phosphorylation sites. Here, S387, S388, T389, and T390 at the C-terminus of Cre1 in T. reesei were mutated to valine for mimicking an unphosphorylated state, thereby generating the transformants Tr_Cre1S387V, Tr_Cre1S388V, Tr_Cre1T389V, and Tr_Cre1T390V, respectively. Transcription of cel7a in Tr_ Cre1S388V was markedly higher than that of the parent strain when grown in glucose-containing media. Under these conditions, both filter paperase (FPase) and p-nitrophenyl-β-D-cellobioside (pNPCase) activities, as well as soluble proteins from Tr_Cre1S388V were significantly increased by up to 2- to 3-fold compared with that of other transformants and the parent strain. The results suggested that S388 is critical site of phosphorylation for triggering CCR at the terminus of Cre1. To our knowledge, this is the first report demonstrating an improvement of cellulase production in T. reesei under CCR by mimicking dephosphorylation at the C-terminus of Cre1. Taken together, we developed a precision engineering strategy based on the modification of phosphorylation sites of Cre1 transcription factor to enhance the production of cellulase in T. reesei under CCR.

CRZ1 regulator and calcium cooperatively modulate holocellulases gene expression in Trichoderma reesei QM6a

Abstract: Trichoderma reesei is the main filamentous fungus used in industry to produce cellulases. Here we investigated the role of CRZ1 and Ca2+signaling in the fungus T. reesei QM6a concerning holocellulases production. For this, we first searched for potential CRZ1 binding sites in promoter regions of key genes coding holocellulases, as well as transcriptional regulators and sugar and calcium transporters. Using a nearly constructed T. reeseiAcrz1 strain, we demonstrated that most of the genes expected to be regulated by CRZ1 were affected in the mutant strain induced with sugarcane bagasse (SCB) and cellulose. In particular, our data demonstrate that Ca2+ acts synergistically with CRZ1 to modulate gene expression, but also exerts CRZ1-independent regulatory role in gene expression in T. reesei, highlighting the role of the major regulator Ca2+ on the signaling for holocellulases transcriptional control in the most part of cellulases genes here investigated. This work presents new evidence on the regulatory role of CRZ1 and Ca2+ sensing in the regulation of cellulolytic enzymes in T. reesei, evidencing significant and previously unknown function of this Ca2+sensing system in the control key transcriptional regulators (XYR1 and CRE1) and on the expression of genes related to sugar and Ca2+ transport.

Engineering the cbh1 Promoter of Trichoderma reesei for Enhanced Protein Production by Replacing the Binding Sites of a Transcription Repressor ACE1 to Those of the Activators.

Abstract: The strong and inducible cbh1 promoter is most widely used to express heterologous proteins, useful in food and feed industries, in Trichoderma reesei. Enhancing its ability to direct transcription provides a general strategy to improve protein production in T. reesei. The cbh1 promoter was engineered by replacing eight binding sites of the transcription repressor ACE1 to those of the activators ACE2, Hap2/3/5, and Xyr1. While changing ACE1 to Hap2/3/5-binding sites completely abolished the transcription ability, replacements with ACE2- and Xyr1-binding sites (designated cbh1pA and cbh1pX promoters, respectively) largely improved the promoter transcription efficiency, as reflected by expression of a reporter gene DsRed. The cbh1pA and cbh1pX promoters were applied to improve secretory expression of a codon-optimized mannanase from Aspergillus niger to 3.6- and 5.0-fold higher, respectively, which has high application potential in feed industry.

Penicillium janthinellum NCIM1366 shows improved biomass hydrolysis and a larger number of CAZymes with higher induction levels over Trichoderma reesei RUT-C30.

Abstract: Major cost of bioethanol is attributed to enzymes employed in biomass hydrolysis. Biomass hydrolyzing enzymes are predominantly produced from the hyper-cellulolytic mutant filamentous fungus Trichoderma reesei RUT-C30. Several decades of research have failed to provide an industrial grade organism other than T. reesei, capable of producing higher titers of an effective synergistic biomass hydrolyzing enzyme cocktail. Penicillium janthinellum NCIM1366 was reported as a cellulase hyper producer and a potential alternative to T. reesei, but a comparison of their hydrolytic performance was seldom attempted. Hydrolysis of acid or alkali-pretreated rice straw using cellulase enzyme preparations from P. janthinellum and T. reesei indicated 37 and 43% higher glucose release, respectively, with P. janthinellum enzymes. A comparison of these fungi with respect to their secreted enzymes indicated that the crude enzyme preparation from P. janthinellum showed 28% higher overall cellulase activity. It also had an exceptional tenfold higher beta-glucosidase activity compared to that of T. reesei, leading to a lower cellobiose accumulation and thus alleviating the feedback inhibition. P. janthinellum secreted more number of proteins to the extracellular medium whose total concentration was 1.8-fold higher than T. reesei. Secretome analyses of the two fungi revealed higher number of CAZymes and a higher relative abundance of cellulases upon cellulose induction in the fungus. The results revealed the ability of P. janthinellum for efficient biomass degradation through hyper cellulase production, and it outperformed the established industrial cellulase producer T. reesei in the hydrolysis experiments. A higher level of induction, larger number of secreted CAZymes and a high relative proportion of BGL to cellulases indicate the possible reasons for its performance advantage in biomass hydrolysis.

Improved Production of Majority Cellulases in Trichoderma reesei by Integration of cbh1 Gene From Chaetomium thermophilum.

Abstract: Lignocellulose is an abundant waste resource and has been considered as a promising material for production of biofuels or other valuable bio-products. Currently, one of the major bottlenecks in the economic utilization of lignocellulosic materials is the cost-efficiency of converting lignocellulose into soluble sugars for fermentation. One way to address this problem is to seek superior lignocellulose degradation enzymes or further improve current production yields of lignocellulases. In the present study, the lignocellulose degradation capacity of a thermophilic fungus Chaetomium thermophilum was firstly evaluated and compared to that of the biotechnological workhorse Trichoderma reesei. The data demonstrated that compared to T. reesei, C. thermophilum displayed substantially higher cellulose-utilizing efficiency with relatively lower production of cellulases, indicating that better cellulases might exist in C. thermophilum. Comparison of the protein secretome between C. thermophilum and T. reesei showed that the secreted protein categories were quite different in these two species. In addition, to prove that cellulases in C. thermophilum had better enzymatic properties, the major cellulase cellobiohydrolase I (CBH1) from C. thermophilum and T. reesei were firstly characterized, respectively. The data showed that the specific activity of C. thermophilum CBH1 was about 4.5-fold higher than T. reesei CBH1 in a wide range of temperatures and pH. To explore whether increasing CBH1 activity in T. reesei could contribute to improving the overall cellulose-utilizing efficiency of T. reesei, T. reesei cbh1 gene was replaced with C. thermophilum cbh1 gene by integration of C. thermophilum cbh1 gene into T. reesei cbh1 gene locus. The data surprisingly showed that this gene replacement not only increased the cellobiohydrolase activities by around 4.1-fold, but also resulted in stronger induction of other cellulases genes, which caused the filter paper activities, Azo-CMC activities and β-glucosidase activities increased by about 2.2, 1.9, and 2.3-fold, respectively. The study here not only provided new resources of superior cellulases genes and new strategy to improve the cellulase production in T. reesei, but also contribute to opening the path for fundamental research on C. thermophilum.