Showing papers on "Trichoderma reesei published in 2016"

Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei.

Abstract: More than 70 years ago, the filamentous ascomycete Trichoderma reesei was isolated on the Solomon Islands due to its ability to degrade and thrive on cellulose containing fabrics. This trait that relies on its secreted cellulases is nowadays exploited by several industries. Most prominently in biorefineries which use T. reesei enzymes to saccharify lignocellulose from renewable plant biomass in order to produce biobased fuels and chemicals. In this review we summarize important milestones of the development of T. reesei as the leading production host for biorefinery enzymes, and discuss emerging trends in strain engineering. Trichoderma reesei has very recently also been proposed as a consolidated bioprocessing organism capable of direct conversion of biopolymeric substrates to desired products. We therefore cover this topic by reviewing novel approaches in metabolic engineering of T. reesei.

Enzymatic hydrolysis of cellulose: Visual characterization of the

Abstract: Cellulose from the Gram-negative bacterium Acetobacter xylinum has been used as a model substrate for visual- izing the action of cellulase enzymes from the fungus Trichoderma reesei. High-resolution electron microscopy reveals that A. xy- linum normally produces a ribbon of cellulose that is a composite of bundles of crystalline microfibrils. Visual patterns of the pro- cess of cellulose degradation have been established. Enzymes are initially observed bound to the cellulose ribbon. Within 10 min, the ribbon is split along its long axis into bundles of microfibrils which are subsequently thinned until they are completely dis- solved within 30 min. Incubations with purified components of the cellulase enzyme system produced less dramatic changes in ribbon structure. Purified 1,4-fi-D-glucan cellobiohydrolase I (D) (EC 3.2.1.91) produced no visible change in cellulose structure. Pu- rified endo-1,4-,B-D-glucanase IV (EC 3.2.1.4) produced some splaying of ribbons into microfibril bundles. In both cases, whole ribbons were present even after 60 min of incubation, visually confirming the synergistic mode of action of these enzymes.

Metagenomic and metaproteomic analyses of a corn stover-adapted microbial consortium EMSD5 reveal its taxonomic and enzymatic basis for degrading lignocellulose

Abstract: Microbial consortia represent promising candidates for aiding in the development of plant biomass conversion strategies for biofuel production. However, the interaction between different community members and the dynamics of enzyme complements during the lignocellulose deconstruction process remain poorly understood. We present here a comprehensive study on the community structure and enzyme systems of a lignocellulolytic microbial consortium EMSD5 during growth on corn stover, using metagenome sequencing in combination with quantitative metaproteomics. The taxonomic affiliation of the metagenomic data showed that EMSD5 was primarily composed of members from the phyla Proteobacteria, Firmicutes and Bacteroidetes. The carbohydrate-active enzyme (CAZyme) annotation revealed that representatives of Firmicutes encoded a broad array of enzymes responsible for hemicellulose and cellulose deconstruction. Extracellular metaproteome analysis further pinpointed the specific role and synergistic interaction of Firmicutes populations in plant polysaccharide breakdown. In particular, a wide range of xylan degradation-related enzymes, including xylanases, β-xylosidases, α-l-arabinofuranosidases, α-glucuronidases and acetyl xylan esterases, were secreted by diverse members from Firmicutes during growth on corn stover. Using label-free quantitative proteomics, we identified the differential secretion pattern of a core subset of enzymes, including xylanases and cellulases with multiple carbohydrate-binding modules (CBMs). In addition, analysis of the coordinate expression patterns indicated that transport proteins and hypothetical proteins may play a role in bacteria processing lignocellulose. Moreover, enzyme preparation from EMSD5 demonstrated synergistic activities in the hydrolysis of pretreated corn stover by commercial cellulases from Trichoderma reesei. These results demonstrate that the corn stover-adapted microbial consortium EMSD5 harbors a variety of lignocellulolytic anaerobic bacteria and degradative enzymes, especially those implicated in hemicellulose decomposition. The data in this study highlight the pivotal role and cooperative relationship of Firmicutes members in the biodegradation of plant lignocellulose by EMSD5. The differential expression patterns of enzymes reveal the strategy of sequential lignocellulose deconstruction by EMSD5. Our findings provide insights into the mechanism by which consortium members orchestrate their array of enzymes to degrade complex lignocellulosic biomass.

Production of a high-efficiency cellulase complex via β-glucosidase engineering in Penicillium oxalicum

Abstract: Trichoderma reesei is a widely used model cellulolytic fungus, supplying a highly effective cellulase production system. Recently, the biofuel industry discovered filamentous fungi from the Penicillium genus as a promising alternative to T. reesei. In our study, we present a systematic over-expression analysis of nine β-glucosidase encoding genes in the wild-type strain 114-2 of Penicillium oxalicum. We found that the over-expression of BGL1, BGL4, or BGL5 significantly enhanced both β-glucosidase activity and hydrolysis efficiency of the enzyme system on filter paper. We utilised two strategies to over-express β-glucosidase in the strain RE-10 that—although over-producing cellulase, does so at the cost of the cellulase mixture deficiency. The constitutive promoter of gene pde_02864 encoding 40S ribosomal protein S8 was used to over-express three β-glucosidases: BGL1, BGL4, and BGL5. We found that all mutants show significantly enhanced levels of β-glucosidase at transcriptional, protein, and activity levels. Furthermore, the inducible promoter from bgl2 was used to conditionally over-express the β-glucosidases BGL1 and BGL4. Surprisingly, this induced expression strategy enables significantly improved expression efficiency. The BGL1 over-expressing mutant I1-13 particularly improved the β-glucosidase activity at a factor of 65-folds, resulting in levels of up to 150 U/ml. All our BGL over-expression mutants displayed significant enhancement of cellulolytic ability on both microcrystalline cellulose and filter paper. In addition, they substantially reduced the enzyme loads in the saccharification of a natural lignocellulose material delignified corncob residue (DCCR). The mutant I4-32 with over-expression of BGL4 achieved the highest glucose yield in the saccharification of DCCR at only 25 % enzyme load compared to the parental strain RE-10. In summary, genetically engineering P. oxalicum to significantly improve β-glucosidase activity is a potent strategy to substantially boost the hydrolytic efficiency of the cellulase cocktail, which will ultimately lead to a considerable reduction of cost for biomass-based biofuel.

Identification of the Main Regulator Responsible for Synthesis of the Typical Yellow Pigment Produced by Trichoderma reesei.

Abstract: The industrially used ascomycete Trichoderma reesei secretes a typical yellow pigment during cultivation, while other Trichoderma species do not. A comparative genomic analysis suggested that a putative secondary metabolism cluster, containing two polyketide-synthase encoding genes, is responsible for the yellow pigment synthesis. This cluster is conserved in a set of rather distantly related fungi, including Acremonium chrysogenum and Penicillium chrysogenum. In an attempt to silence the cluster in T. reesei, two genes of the cluster encoding transcription factors were individually deleted. For a complete genetic proof-of-function, the genes were reinserted into the genomes of the respective deletion strains. The deletion of the first transcription factor (termed yellow pigment regulator 1 [Ypr1]) resulted in the full abolishment of the yellow pigment formation and the expression of most genes of this cluster. A comparative high-pressure liquid chromatography (HPLC) analysis of supernatants of the ypr1 deletion and its parent strain suggested the presence of several yellow compounds in T. reesei that are all derived from the same cluster. A subsequent gas chromatography/mass spectrometry analysis strongly indicated the presence of sorbicillin in the major HPLC peak. The presence of the second transcription factor, termed yellow pigment regulator 2 (Ypr2), reduces the yellow pigment formation and the expression of most cluster genes, including the gene encoding the activator Ypr1. IMPORTANCETrichoderma reesei is used for industry-scale production of carbohydrate-active enzymes. During growth, it secretes a typical yellow pigment. This is not favorable for industrial enzyme production because it makes the downstream process more complicated and thus increases operating costs. In this study, we demonstrate which regulators influence the synthesis of the yellow pigment. Based on these data, we also provide indication as to which genes are under the control of these regulators and are finally responsible for the biosynthesis of the yellow pigment. These genes are organized in a cluster that is also found in other industrially relevant fungi, such as the two antibiotic producers Penicillium chrysogenum and Acremonium chrysogenum. The targeted manipulation of a secondary metabolism cluster is an important option for any biotechnologically applied microorganism.

Understanding the Role of the Master Regulator XYR1 in Trichoderma reesei by Global Transcriptional Analysis.

Abstract: We defined the role of the transcriptional factor–XYR1–in the filamentous fungus Trichoderma reesei during cellulosic material degradation. In this regard, we performed a global transcriptome analysis using RNA-Seq of the ∆xyr1 mutant strain of T. reesei compared with the parental strain QM9414 grown in the presence of cellulose, sophorose, and glucose as sole carbon sources. We found that 5885 genes were expressed differentially under the three tested carbon sources. Of these, 322 genes were upregulated in the presence of cellulose, while 367 and 188 were upregulated in sophorose and glucose, respectively. With respect to genes under the direct regulation of XYR1, 30 and 33 are exclusive to cellulose and sophorose, respectively. The most modulated genes in the ∆xyr1 belong to Carbohydrate-Active Enzymes (CAZymes), transcription factors, and transporters families. Moreover, we highlight the downregulation of transporters belonging to the MFS and ABC transporter families. Of these, MFS members were mostly downregulated in the presence of cellulose. In sophorose and glucose, the expression of these transporters was mainly upregulated. Our results revealed that MFS and ABC transporters could be new players in cellulose degradation and their role was shown to be carbon source-dependent. Our findings contribute to a better understanding of the regulatory mechanisms of XYR1 to control cellulase gene expression in T. reesei in the presence of cellulosic material, thereby potentially enhancing its application in several biotechnology fields.

Proteomic analysis of the biomass hydrolytic potentials of Penicillium oxalicum lignocellulolytic enzyme system

Abstract: The mining of high-performance enzyme systems is necessary to develop industrial lignocellulose bioconversion. Large amounts of cellulases and hemicellulases can be produced by Penicillium oxalicum. Hence, the enzyme system of this hypercellulolytic fungus should be elucidated to help design optimum enzyme systems for effective biomass hydrolysis. The cellulolytic and xylanolytic activities of an SP enzyme system prepared from P. oxalicum JU-A10 were comparatively analyzed. Results indicated that the fungus possesses a complete cellulolytic-xylanolytic enzyme system. The cellobiohydrolase- and xylanase-specific activities of this system were higher than those of two other enzyme systems, i.e., ST from Trichoderma reesei SN1 and another commercial preparation Celluclast 1.5L. Delignified corncob residue (DCCR) could be hydrolyzed by SP to a greater extent than corncob residue (CCR). Beta-glucosidase (BG) supplemented in SP increased the ability of the system to hydrolyze DCCR and CCR, and resulted in a 64 % decrease in enzyme dosage with the same glucose yield. The behaviors of the enzyme components in the hydrolysis of CCR were further investigated by monitoring individual enzyme dynamics. The total protein concentrations and cellobiohydrolase (CBH), endoglucanase (EG), and filter paper activities in the supernatants significantly decreased during saccharification. These findings were more evident in SP than in the other enzyme systems. The comparative proteomic analysis of the enzyme systems revealed that both SP and ST were rich in carbohydrate-degrading enzymes and multiple non-hydrolytic proteins. A larger number of carbohydrate-binding modules 1 (CBM1) were also identified in SP than in ST. This difference might be linked to the greater adsorption to substrates and lower hydrolysis efficiency of SP enzymes than ST during lignocellulose saccharification, because CBM1 not only targets enzymes to insoluble cellulose but also leads to non-productive adsorption to lignin. Penicillium oxalicum can be applied to the biorefinery of lignocellulosic biomass. Its ability to degrade lignocellulosic substrates could be further improved by modifying its enzyme system on the basis of enzyme activity measurement and proteomic analysis. The proposed strategy may also be applied to other lignocellulolytic enzyme systems to enhance their hydrolytic performances rationally.

Characterization of the Ca(2+) -responsive signaling pathway in regulating the expression and secretion of cellulases in Trichoderma reesei Rut-C30.

Abstract: Calcium signaling plays pivotal roles in the hyphal growth, conidiation, and osmosis sensitivity of fungi through the Ca(2+) /calmodulin-calcineurin-dependent pathway. This study found that an appropriate extracellular Ca(2+) concentration markedly stimulated the hyphal growth, cellulase production, and total protein secretion of the cellulase hyper-producing strain, Trichoderma reesei Rut-C30. Transcription analysis revealed upregulation of not only encoding genes of cellulases and the transcriptional activator XYR1 but also several genes encoding endoplasmic reticulum-chaperones after Ca(2+) addition. The function of CRZ1, T. reesei calcineurin-responsive zinc finger transcription factor 1, was further characterized by gene disruption. Electrophoretic mobility shift assays (EMSAs) in combination with chromatin immunoprecipitation (ChIP) verified that CRZ1 could bind directly to the upstream regions of xyr1 and cbh1 (cellobiohydrolase I-encoding gene) in response to Ca(2+) . A DNase I footprinting assay identified its putative binding consensus site (5'-[T/G]GGCG-3' or 5'-GGGC[G/T]-3'). EMSAs confirmed that CRZ1 competed for occupancy of the xyr1 promoter with another transcription factor, ACE1. These results revealed putative signaling pathways downstream of calcineurin in response to extracellular Ca(2+) involved in upregulation of cellulose degradation-related genes, reflecting progress in the study of Ca(2+) signaling in filamentous fungi. This study also provides insight that will facilitate further improvement of (hemi-)cellulase production by T. reesei.

Comparison of three deep eutectic solvents and 1-ethyl-3-methylimidazolium acetate in the pretreatment of lignocellulose: effect on enzyme stability, lignocellulose digestibility and one-pot hydrolysis

Abstract: Certain ionic liquids (ILs) are well-known pretreatment chemicals for lignocellulosic substrates prior to enzymatic total hydrolysis. Deep eutectic solvents (DESs) are closely related to ILs in many properties, but are easier and on occasion cheaper to synthesize and have been claimed to be less inactivating to enzymes used in the hydrolysis, and less toxic for the environment and to micro-organisms used in fermentation. The use of DESs as lignocellulose pretreatment chemicals has not been studied to a similar extent as the use of ILs. In this study, the stability of three Trichoderma reesei cellulases (the endoglucanases Cel5A and Cel7B and the cellobiohydrolase Cel7A) and one T. reesei xylanase (Xyn11) was compared in concentrated solutions (85% w/w) of three DESs (choline chloride : boric acid in molar ratio 5 : 2, choline chloride : glycerol 1 : 1 and betaine : glycerol 1 : 1) and 1-ethyl-3-methylimidazolium acetate ([EMIM]AcO), a powerful lignocellulose-dissolving IL. The pretreatment efficiency of these chemicals was further compared in a mild pretreatment (90% w/w DES or [EMIM]AcO, 80 °C, 24 h, 5% (w/w) lignocellulose consistency) of four different substrates; microcrystalline cellulose, eucalyptus dissolving pulp, shredded wheat straw and spruce saw dust. After pretreatment, the enzymatic digestibility of the pretreated substrates was evaluated in the enzymatic total hydrolysis in three different setups, including hydrolysis of the washed pretreated substrates in buffer, and of the pretreated substrates in solutions containing 30% (w/w) and 80% (w/w) of DES or [EMIM]AcO. The stability analysis identified glycerol-containing DESs to be highly stabilizing for the cellulases, but their pretreatment efficiency was limited. [EMIM]AcO had a high pretreatment efficiency, but was highly inactivating for the used cellulases. The presence of DES or [EMIM]AcO led in all cases to decreased enzymatic hydrolysis yields. Thus, good enzymatic stability in a certain DES does not directly implicate good performance in the hydrolysis of solid lignocellulosic substrates in that DES.

Improvements in Glucose Sensitivity and Stability of Trichoderma reesei β-Glucosidase Using Site-Directed Mutagenesis

Abstract: Glucose sensitivity and pH and thermal stabilities of Trichoderma reesei Cel1A (Bgl II) were improved by site-directed mutagenesis of only two amino acid residues (L167W or P172L) at the entrance of the active site. The Cel1A mutant showed high glucose tolerance (50% of inhibitory concentration = 650 mM), glucose stimulation (2.0 fold at 50 mM glucose), and enhanced specific activity (2.4-fold) compared with those of the wild-type Cel1A. Furthermore, the mutant enzyme showed stability at a wide pH range of 4.5–9.0 and possessed high thermal stability up to 50°C with 80% of the residual activities compared with the stability seen at the pH range of 6.5–7.0 and temperatures of up to 40°C in the wild-type Cel1A. Kinetic studies for hydrolysis revealed that the Cel1A mutant was competitively inhibited by glucose at similar levels as the wild-type enzyme. Additionally, the mutant enzyme exhibited substrate inhibition, which gradually disappeared with an increasing glucose concentration. These data suggest that the glucose stimulation was caused by relieve the substrate inhibition in the presence of glucose. To conclude, all the properties improved by the mutagenesis would be great advantages in degradation of cellulosic biomass together with cellulases.

A β-glucosidase hyper-production Trichoderma reesei mutant reveals a potential role of cel3D in cellulase production

Abstract: The conversion of cellulose by cellulase to fermentable sugars for biomass-based products such as cellulosic biofuels, biobased fine chemicals and medicines is an environment-friendly and sustainable process, making wastes profitable and bringing economic benefits. Trichoderma reesei is the well-known major workhorse for cellulase production in industry, but the low β-glucosidase activity in T. reesei cellulase leads to inefficiency in biomass degradation and limits its industrial application. Thus, there are ongoing interests in research to develop methods to overcome this insufficiency. Moreover, although β-glucosidases have been demonstrated to influence cellulase production and participate in the regulation of cellulase production, the underlying mechanism remains unclear. The T. reesei recombinant strain TRB1 was constructed from T. reesei RUT-C30 by the T-DNA-based mutagenesis. Compared to RUT-C30, TRB1 displays a significant enhancement of extracellular β-glucosidase (BGL1) activity with 17-fold increase, a moderate increase of both the endoglucanase (EG) activity and the exoglucanase (CBH) activity, a minor improvement of the total filter paper activity, and a faster cellulase induction. This superiority of TRB1 over RUT-C30 is independent on carbon sources and improves the saccharification ability of TRB1 cellulase on pretreated corn stover. Furthermore, TRB1 shows better resistance to carbon catabolite repression than RUT-C30. Secretome characterization of TRB1 shows that the amount of CBH, EG and BGL in the supernatant of T. reesei TRB1 was indeed increased along with the enhanced activities of these three enzymes. Surprisingly, qRT-PCR and gene cloning showed that in TRB1 β-glucosidase cel3D was mutated through the random insertion by AMT and was not expressed. The T. reesei recombinant strain TRB1 constructed in this study is more desirable for industrial application than the parental strain RUT-C30, showing extracellular β-glucosidase hyper production, high cellulase production within a shorter time and a better resistance to carbon catabolite repression. Disruption of β-glucosidase cel3D in TRB1 was identified, which might contribute to the superiority of TRB1 over RUT-C30 and might play a role in the cellulase production. These results laid a foundation for future investigations to further improve cellulase enzymatic efficiency and reduce cost for T. reesei cellulase production.

Comparative genomic, transcriptomic and secretomic profiling of Penicillium oxalicum HP7-1 and its cellulase and xylanase hyper-producing mutant EU2106, and identification of two novel regulatory genes of cellulase and xylanase gene expression

Abstract: The filamentous fungus Penicillium oxalicum is a potential alternative to Trichoderma reesei for industrial production of a complete cellulolytic enzyme system for a bio-refinery. Comparative omics approaches can support rational genetic engineering and/or breeding of filamentous fungi with improved cellulase production capacity. In this study, comparative genomic, transcriptomic and secretomic profiling of P. oxalicum HP7-1 and its cellulase and xylanase hyper-producing mutant EU2106 were employed to screen for novel regulators of cellulase and xylanase gene expression. The 30.62 Mb P. oxalicum HP7-1 genome was sequenced, and 9834 protein-coding genes were annotated. Re-sequencing of the mutant EU2106 genome identified 274 single nucleotide variations and 12 insertion/deletions. Comparative genomic, transcriptomic and secretomic profiling of HP7-1 and EU2106 revealed four candidate regulators of cellulase and xylanase gene expression. Deletion of these candidate genes and measurement of the enzymatic activity of the resultant mutants confirmed the identity of three regulatory genes. POX02484 and POX08522, encoding a putative Zn(II)2Cys6 DNA-binding domain and forkhead protein, respectively, were found to be novel, while PoxClrB is an ortholog of ClrB, a key transcriptional regulator of cellulolytic enzyme gene expression in filamentous fungi. ΔPOX02484 and ΔPOX08522 mutants exhibited significantly reduced β-glucosidase activity, increased carboxymethylcellulose cellulase and xylanase activities, and altered transcription level of cellulase and xylanase genes compared with the parent strain ΔPoxKu70, with Avicel as the sole carbon source. Two novel genes, POX02484 and POX08522, were found and characterized to regulate the expression of cellulase and xylanase genes in P. oxalicum. These findings are important for engineering filamentous fungi to improve cellulase and xylanase production.

Production of Industrial Enzymes in Trichoderma reesei

Abstract: Trichoderma reesei (teleomorph Hypocrea jecorina) is one of the two major fungal platforms for industrial enzyme production, along with Aspergillus sp. Its use derives from its history as a model organism for studies on cellulose degradation and its cellulase enzyme complex since the 1940s, which suggested its use for industrial bioethanol manufacturing during the oil crisis in the mid 1970s. Extensive strain development campaigns by different laboratories proved that the wild type isolate QM6a can be developed into superior production strains, and later the genetic tools were established for the species, which allowed the strains to be tailored for maximum productivity with optimised backgrounds. T. reesei has now maintained its position as a highly productive, easy-to-handle, robust and safe cell factory for more than 40 years. The recently revived interest in lignocellulosic bioethanol paved the way for a comeback in the use of T. reesei native cellulase complex, whereas advances in the development of molecular biology tools—such as bioinformatics and mating—have provided further refinements in the modern strain development of T. reesei for enzyme and other protein production in virtually all segments of industrial biotechnology.

Identification and functional characterization of novel xylose transporters from the cell factories Aspergillus niger and Trichoderma reesei

Abstract: Global climate change and fossil fuels limitations have boosted the demand for robust and efficient microbial factories for the manufacturing of bio-based products from renewable feedstocks. In this regard, efforts have been done to enhance the enzyme-secreting ability of lignocellulose-degrading fungi, aiming to improve protein yields while taking advantage of their ability to use lignocellulosic feedstocks. Access to sugars in complex polysaccharides depends not only on their release by specific hydrolytic enzymes, but also on the presence of transporters capable of effectively transporting the constituent sugars into the cell. This study aims to identify and characterize xylose transporters from Aspergillus niger and Trichoderma reesei, two fungi that have been industrially exploited for decades for the production of lignocellulose-degrading hydrolytic enzymes. A hidden Markov model for the identification of xylose transporters was developed and used to analyze the A. niger and T. reesei in silico proteomes, yielding a list of candidate xylose transporters. From this list, three A. niger (XltA, XltB and XltC) and three T. reesei (Str1, Str2 and Str3) transporters were selected, functionally validated and biochemically characterized through their expression in a Saccharomyces cerevisiae hexose transport null mutant, engineered to be able to metabolize xylose but unable to transport this sugar. All six transporters were able to support growth of the engineered yeast on xylose but varied in affinities and efficiencies in the uptake of the pentose. Amino acid sequence analysis of the selected transporters showed the presence of specific residues and motifs recently associated to xylose transporters. Transcriptional analysis of A. niger and T. reesei showed that XltA and Str1 were specifically induced by xylose and dependent on the XlnR/Xyr1 regulators, signifying a biological role for these transporters in xylose utilization. This study revealed the existence of a variety of xylose transporters in the cell factories A. niger and T. reesei. The particular substrate specificity and biochemical properties displayed by A. niger XltA and XltB suggested a possible biological role for these transporters in xylose uptake. New insights were also gained into the molecular mechanisms regulating the pentose utilization, at inducer uptake level, in these fungi. Analysis of the A. niger and T. reesei predicted transportome with the newly developed hidden Markov model showed to be an efficient approach for the identification of new xylose transporting proteins.

Optimizing Cellulase Production from Municipal Solid Waste (MSW) using Solid State Fermentation (SSF)

Abstract: This paper explores the possibility of using an industrially processed municipal solid waste (MSW) for cellulase enzyme production via solid state fermentation (SSF) by Trichoderma reesei and Aspergillus niger. Both fungi grew well on the MSW substrate and production of cellulase enzymes was optimized for temperature, moisture content, inoculation and period of incubation. The effect of additional minerals, and alternative carbon and nitrogen sources were also examined. Following optimization a cellulase activity of 26.10 ± 3.09 FPU/g could be produced using T. reesei at 30°C with a moisture content of 60% with an inoculums of 0.5 million spores/g and incubation for 168 hours. Addition of extra nitrogen and/or carbon did not improve cellulase accumulation. Acid or alkali pretreatment of MSW led to reduced cellulase production. Crude enzymes produced from MSW by T. reesei were evaluated for their ability to release glucose from MSW. A cellulose hydrolysis yield of 24.7% was achieved, which was close to that obtained using a commercial enzyme. Results demonstrated that MSW can be used as an inexpensive lignocellulosic material for the production of cellulase enzymes.

Enhanced cell-surface display and secretory production of cellulolytic enzymes with Saccharomyces cerevisiae Sed1 signal peptide.

Abstract: Recombinant yeast strains displaying aheterologous cellulolytic enzymes on their cell surfaces using a glycosylphosphatidylinositol (GPI) anchoring system are a promising strategy for bioethanol production from lignocellulosic materials. A crucial step for cell wall localization of the enzymes is the intracellular transport of proteins in yeast cells. Therefore, the addition of a highly efficient secretion signal sequence is important to increase the amount of the enzymes on the yeast cell surface. In this study, we demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the Saccharomyces cerevisiae SED1 gene for cell-surface display and secretory production of cellulolytic enzymes. Gene cassettes with SP sequences derived from S. cerevisiae SED1 (SED1SP), Rhizopus oryzae glucoamylase (GLUASP), and S. cerevisiae α-mating pheromone (MFα1SP) were constructed for cell-surface display of Aspergillus aculeatus β-glucosidase (BGL1) and Trichoderma reesei endoglucanase II (EGII). These gene cassettes were integrated into the S. cerevisiae genome. The recombinant strains with the SED1SP showed higher cell-surface BGL and EG activities than those with the conventional SP sequences (GLUASP and MFα1SP). The novel SP sequence also improved the secretory production of BGL and EG in S. cerevisiae. The extracellular BGL activity of the recombinant strains with the SED1SP was 1.3- and 1.9-fold higher than the GLUASP and MFα1SP strains, respectively. Moreover, the utilization of SED1SP successfully enhanced the secretory production of BGL in Pichia pastoris. The utilization of the novel SP sequence is a promising option for highly efficient cell-surface display and secretory production of heterologous proteins in various yeast species. Biotechnol. Bioeng. 2016;113: 2358-2366. © 2016 Wiley Periodicals, Inc.

Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains

Abstract: Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (β-glucosidase), Talaromyces emersonii Cel7A (cellobiohydrolase), and Trichoderma reesei Cel5A (endoglucanase) were utilized as reporter proteins. Natural strain YI13 was identified to have a high secretory phenotype, demonstrating a 3.7- and 3.5-fold higher Cel7A and Cel5A activity, respectively, compared to the reference strain S288c. YI13 also demonstrated other industrially relevant characteristics such as growth vigor, high ethanol titer, multi-tolerance to high temperatures (37 and 40 °C), ethanol (10 % w/v), and towards various concentrations of a cocktail of inhibitory compounds commonly found in lignocellulose hydrolysates. This study accentuates the value of natural S. cerevisiae isolate strains to serve as potential robust and highly productive chassis organisms for CBP strain development.

Deciphering the molecular mechanisms behind cellulase production in Trichoderma reesei, the hyper-cellulolytic filamentous fungus

Abstract: The filamentous fungus Trichoderma reesei is a potent cellulase producer and the best-studied cellulolytic fungus. A lot of investigations not only on glycoside hydrolases produced by T. reesei, but also on the machinery controlling gene expression of these enzyme have made this fungus a model organism for cellulolytic fungi. We have investigated the T. reesei strain including mutants developed in Japan in detail to understand the molecular mechanisms that control the cellulase gene expression, the biochemical and morphological aspects that could favor this phenotype, and have attempted to generate novel strains that may be appropriate for industrial use. Subsequently, we developed recombinant strains by combination of these insights and the heterologous-efficient saccharifing enzymes. Resulting enzyme preparations were highly effective for saccharification of various biomass. In this review, we present some of the salient findings from the recent biochemical, morphological, and molecular analyses of this remarkable cellulase hyper-producing fungus.

Familiar Stranger: Ecological Genomics of the Model Saprotroph and Industrial Enzyme Producer Trichoderma reesei Breaks the Stereotypes.

Abstract: The filamentous fungus Trichoderma reesei (Hypocreales, Ascomycota) has properties of an efficient cell factory for protein production that is exploited by the enzyme industry, particularly with respect to cellulase and hemicellulase formation. Under conditions of industrial fermentations it yields more than 100 g secreted protein L −1 . Consequently, T. reesei has been intensively studied in the 20th century. Most of these investigations focused on the biochemical characteristics of its cellulases and hemicellulases, on the improvement of their properties by protein engineering, and on enhanced enzyme production by recombinant strategies. However, as the fungus is rare in nature, its ecology remained unknown. The breakthrough in the understanding of the fundamental biology of T. reesei only happened during 2000s–2010s. In this review, we compile the current knowledge on T. reesei ecology, physiology, and genomics to present a holistic view on the natural behavior of the organism. This is not only critical for science-driven further improvement of the biotechnological applications of this fungus, but also renders T. reesei as an attractive model of filamentous fungi with superior saprotrophic abilities.

Revisiting overexpression of a heterologous β-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities

Abstract: The filamentous fungus Trichoderma reesei has the capacity to secret large amounts of cellulase and is widely used in a variety of industries. However, the T. reesei cellulase is weak in β-glucosidase activity, which results in accumulation of cellobiose inhibiting the endo- and exo-cellulases. By expressing an exogenous β-glucosidase gene, the recombinant T. reesei cellulase is expected to degrade cellulose into glucose more efficiently. The thermophilic β-glucosidase NfBgl3A from Neosartorya fischeri is chosen for overexpression in T. reesei due to its robust activity. In vitro, the Pichia pastoris-expressed NfBgl3A aided the T. reesei cellulase in releasing much more glucose with significantly lower amounts of cellobiose from crystalline cellulose. The NfBgl3A gene was hence fused to the cbh1 structural gene and assembled between the strong cbh1 promoter and cbh1 terminator to obtain pRS-NfBgl3A by using the DNA assembler method. pRS-NfBgl3A was transformed into the T. reesei uridine auxotroph strain TU-6. Six positive transformants showed β-glucosidase activities of 2.3–69.7 U/mL (up to 175-fold higher than that of wild-type). The largely different β-glucosidase activities in the transformants may be ascribed to the gene copy numbers of NfBgl3A or its integration loci. The T. reesei-expressed NfBgl3A showed highly similar biochemical properties to that expressed in P. pastoris. As expected, overexpression of NfBgl3A enhanced the overall cellulase activity of T. reesei. The CBHI activity in all transformants increased, possibly due to the extra copies of cbh1 gene introduced, while the endoglucanase activity in three transformants also largely increased, which was not observed in any other studies overexpressing a β-glucosidase. NfBgl3A had significant transglycosylation activity, generating sophorose, a potent cellulase inducer, and other oligosaccharides from glucose and cellobiose. We report herein the successful overexpression of a thermophilic N. fischeri β-glucosidase in T. reesei. In the same time, the fusion of NfBgl3A to the cbh1 gene introduced extra copies of the cellobiohydrolase 1 gene. As a result, we observed improved β-glucosidase and cellobiohydrolase activity as well as the overall cellulase activity. In addition, the endoglucanase activity also increased in some of the transformants. Our results may shed light on design of more robust T. reesei cellulases.

Comparison of catalytic properties of multiple β-glucosidases of Trichoderma reesei.

Abstract: Ten putative Trichoderma reesei β-glucosidase (BGL) isozymes were heterologously expressed in Escherichia coli and Aspergillus oryzae and purified to homogeneity. Catalytic properties of nine enzymes which showed hydrolytic activity on cellobiose and p-nitrophenyl-β-D-glucopyranoside (pNPG) were investigated. Three BGLs, encoded by the genes cel3A, cel3B, and cel3E, contained a predicted signal peptide, showed higher hydrolytic activity on cello-oligosaccharides than on pNPG, and preferred longer oligosaccharides. Another three putative extracellular BGLs, Cel3B, Cel3F, and Cel3G, and two intracellular enzymes, Cel3C and Cel3D, exhibited preference for pNPG. Intracellular Cel1A showed the highest affinity for cellobiose as a typical cellobiase. Four BGLs, Cel3A, Cel3B, Cel3E, Cel1A, that showed high activity against cello-oligosaccharides were capable of catalyzing transglycosylation reactions from cellobiose, leading to formation of cellotriose and isomeric glucobioses. While Cel3A, Cel3B, and Cel3E synthesized mainly gentiobiose, glycosyl transfer reactions of Cel1A led mainly to sophorose and laminaribiose. Conversion of cellobiose to sophorose by Cel1A reached about 3.6 and 10 % at 1 and 10 % cellobiose concentration, respectively. The formation and persistence of individual cellobiose isomers in incubation mixtures of four BGLs (Cel3A, Cel3B, Cel3E, and Cel1A) with cellobiose correlated well with the k cat values for isomeric glucobioses. Cel1A also showed the lowest sensitivity to inhibition by glucose. Based on all studied catalytic properties, Cel1A appears to be unambiguously the best candidate for site-directed mutations or directed evolution toward improvement of activity, thermostability, and, eventually, efficiency of sophorose synthesis.

Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

Abstract: The filamentous fungus Trichoderma reesei has tremendous capability to secrete over 100 g/L of proteins and therefore it would make an excellent host system for production of high levels of therapeutic proteins at low cost. We have developed T. reesei strains suitable for production of therapeutic proteins by reducing the secreted protease activity. Protease activity has been the major hindrance to achieving high production levels. We have constructed a series of interferon alpha-2b (IFNα-2b) production strains with 9 protease deletions to gain knowledge for further strain development. We have identified two protease deletions that dramatically improved the production levels. Deletion of the subtilisin protease slp7 and the metalloprotease amp2 has enabled production levels of IFNα-2b up to 2.1 and 2.4 g/L, respectively. With addition of soybean trypsin protease inhibitor the level of production improved to 4.5 g/L, with an additional 1.8 g/L still bound to the secretion carrier protein. High levels of IFNα-2b were produced using T. reesei strains with reduced protease secretion. Further strain development can be done to improve the production system by reducing protease activity and improving carrier protein cleavage.

Functional characterization of the native swollenin from Trichoderma reesei: study of its possible role as C1 factor of enzymatic lignocellulose conversion.

Abstract: Through binding to cellulose, expansin-like proteins are thought to loosen the structural order of crystalline surface material, thus making it more accessible for degradation by hydrolytic enzymes. Swollenin SWO1 is the major expansin-like protein from the fungus Trichoderma reesei. Here, we have performed a detailed characterization of a recombinant native form of SWO1 with respect to its possible auxiliary role in the enzymatic saccharification of lignocellulosic substrates. The swo1 gene was overexpressed in T. reesei QM9414 Δxyr1 mutant, featuring downregulated cellulase production, and the protein was purified from culture supernatant. SWO1 was N-glycosylated and its circular dichroism spectrum suggested a folded protein. Adsorption isotherms (25 °C, pH 5.0, 1.0 mg substrate/mL) revealed SWO1 to be 120- and 20-fold more specific for binding to birchwood xylan and kraft lignin, respectively, than for binding to Avicel PH-101. The SWO1 binding capacity on lignin (25 µmol/g) exceeded 12-fold that on Avicel PH-101 (2.1 µmol/g). On xylan, not only the binding capacity (22 µmol/g) but also the affinity of SWO1 (K d = 0.08 µM) was enhanced compared to Avicel PH-101 (K d = 0.89 µM). SWO1 caused rapid release of a tiny amount of reducing sugars (<1 % of total) from different substrates (Avicel PH-101, nanocrystalline cellulose, steam-pretreated wheat straw, barley β-glucan, cellotetraose) but did not promote continued saccharification. Atomic force microscopy revealed that amorphous cellulose films were not affected by SWO1. Also with AFM, binding of SWO1 to cellulose nanocrystallites was demonstrated at the single-molecule level, but adsorption did not affect this cellulose. SWO1 exhibited no synergy with T. reesei cellulases in the hydrolysis of the different celluloses. However, SWO1 boosted slightly (1.5-fold) the reducing sugar release from a native grass substrate. SWO1 is a strongly glycosylated protein, which has implications for producing it in heterologous hosts. Although SWO1 binds to crystalline cellulose, its adsorption to xylan is much stronger. SWO1 is not an auxiliary factor of the enzymatic degradation of a variety of cellulosic substrates. Effect of SWO1 on sugar release from intact plant cell walls might be exploitable with certain (e.g., mildly pretreated) lignocellulosic feedstocks.