Showing papers on "Trichoderma reesei published in 2017"

Development of a low-cost cellulase production process using Trichoderma reesei for Brazilian biorefineries

Abstract: During the past few years, the first industrial-scale cellulosic ethanol plants have been inaugurated. Although the performance of the commercial cellulase enzymes used in this process has greatly improved over the past decade, cellulases still represent a very significant operational cost. Depending on the region, transport of cellulases from a central production facility to a biorefinery may significantly add to enzyme cost. The aim of the present study was to develop a simple, cost-efficient cellulase production process that could be employed locally at a Brazilian sugarcane biorefinery. Our work focused on two main topics: growth medium formulation and strain improvement. We evaluated several Brazilian low-cost industrial residues for their potential in cellulase production. Among the solid residues evaluated, soybean hulls were found to display clearly the most desirable characteristics. We engineered a Trichoderma reesei strain to secrete cellulase in the presence of repressing sugars, enabling the use of sugarcane molasses as an additional carbon source. In addition, we added a heterologous β-glucosidase to improve the performance of the produced enzymes in hydrolysis. Finally, the addition of an invertase gene from Aspegillus niger into our strain allowed it to consume sucrose from sugarcane molasses directly. Preliminary cost analysis showed that the overall process can provide for very low-cost enzyme with good hydrolysis performance on industrially pre-treated sugarcane straw. In this study, we showed that with relatively few genetic modifications and the right growth medium it is possible to produce considerable amounts of well-performing cellulase at very low cost in Brazil using T. reesei. With further enhancements and optimization, such a system could provide a viable alternative to delivered commercial cellulases.

Genetic engineering of Trichoderma reesei cellulases and their production

Abstract: Lignocellulosic biomass, which mainly consists of cellulose, hemicellulose and lignin, is the most abundant renewable source for production of biofuel and biorefinery products. The industrial use of plant biomass involves mechanical milling or chipping, followed by chemical or physicochemical pretreatment steps to make the material more susceptible to enzymatic hydrolysis. Thereby the cost of enzyme production still presents the major bottleneck, mostly because some of the produced enzymes have low catalytic activity under industrial conditions and/or because the rate of hydrolysis of some enzymes in the secreted enzyme mixture is limiting. Almost all of the lignocellulolytic enzyme cocktails needed for the hydrolysis step are produced by fermentation of the ascomycete Trichoderma reesei (Hypocreales). For this reason, the structure and mechanism of the enzymes involved, the regulation of their expression and the pathways of their formation and secretion have been investigated in T. reesei in considerable details. Several of the findings thereby obtained have been used to improve the formation of the T. reesei cellulases and their properties. In this article, we will review the achievements that have already been made and also show promising fields for further progress.

Cellulases immobilization on chitosan-coated magnetic nanoparticles: application for Agave Atrovirens lignocellulosic biomass hydrolysis

Abstract: In the present study, Trichoderma reesei cellulase was covalently immobilized on chitosan-coated magnetic nanoparticles using glutaraldehyde as a coupling agent. The average diameter of magnetic nanoparticles before and after enzyme immobilization was about 8 and 10 nm, respectively. The immobilized enzyme retained about 37 % of its initial activity, and also showed better thermal and storage stability than free enzyme. Immobilized cellulase retained about 80 % of its activity after 15 cycles of carboxymethylcellulose hydrolysis and was easily separated with the application of an external magnetic field. However, in this reaction, K m was increased eight times. The immobilized enzyme was able to hydrolyze lignocellulosic material from Agave atrovirens leaves with yield close to the amount detected with free enzyme and it was re-used in vegetal material conversion up to four cycles with 50 % of activity decrease. This provides an opportunity to reduce the enzyme consumption during lignocellulosic material saccharification for bioethanol production.

Multifunctional Cellulolytic Enzymes Outperform Processive Fungal Cellulases for Coproduction of Nanocellulose and Biofuels

Abstract: Producing fuels, chemicals, and materials from renewable resources to meet societal demands remains an important step in the transition to a sustainable, clean energy economy. The use of cellulolytic enzymes for the production of nanocellulose enables the coproduction of sugars for biofuels production in a format that is largely compatible with the process design employed by modern lignocellulosic (second generation) biorefineries. However, yields of enzymatically produced nanocellulose are typically much lower than those achieved by mineral acid production methods. In this study, we compare the capacity for coproduction of nanocellulose and fermentable sugars using two vastly different cellulase systems: the classical “free enzyme” system of the saprophytic fungus, Trichoderma reesei (T. reesei) and the complexed, multifunctional enzymes produced by the hot springs resident, Caldicellulosiruptor bescii (C. bescii). We demonstrate by comparative digestions that the C. bescii system outperforms the fungal ...

Single-molecule study of oxidative enzymatic deconstruction of cellulose.

Abstract: LPMO (lytic polysaccharide monooxygenase) represents a unique paradigm of cellulosic biomass degradation by an oxidative mechanism. Understanding the role of LPMO in deconstructing crystalline cellulose is fundamental to the enzyme’s biological function and will help to specify the use of LPMO in biorefinery applications. Here we show with real-time atomic force microscopy that C1 and C4 oxidizing types of LPMO from Neurospora crassa (NcLPMO9F, NcLPMO9C) bind to nanocrystalline cellulose with high preference for the very same substrate surfaces that are also used by a processive cellulase (Trichoderma reesei CBH I) to move along during hydrolytic cellulose degradation. The bound LPMOs, however, are immobile during their adsorbed residence time ( ~ 1.0 min for NcLPMO9F) on cellulose. Treatment with LPMO resulted in fibrillation of crystalline cellulose and strongly ( ≥ 2-fold) enhanced the cellulase adsorption. It also increased enzyme turnover on the cellulose surface, thus boosting the hydrolytic conversion. Understanding the role of enzymes in biomass depolymerization is essential for the development of more efficient biorefineries. Here, the authors show by atomic force microscopy the real-time mechanism of cellulose deconstruction by lytic polysaccharide monooxygenases.

Rce1, a novel transcriptional repressor, regulates cellulase gene expression by antagonizing the transactivator Xyr1 in Trichoderma reesei.

Abstract: Cellulase gene expression in the model cellulolytic fungus Trichoderma reesei is supposed to be controlled by an intricate regulatory network involving multiple transcription factors. Here, we identified a novel transcriptional repressor of cellulase gene expression, Rce1. Disruption of the rce1 gene not only facilitated the induced expression of cellulase genes but also led to a significant delay in terminating the induction process. However, Rce1 did not participate in Cre1-mediated catabolite repression. Electrophoretic mobility shift (EMSA) and DNase I footprinting assays in combination with chromatin immunoprecipitation (ChIP) demonstrated that Rce1 could bind directly to a cbh1 (cellobiohydrolase 1-encoding) gene promoter region containing a cluster of Xyr1 binding sites. Furthermore, competitive binding assays revealed that Rce1 antagonized Xyr1 from binding to the cbh1 promoter. These results indicate that intricate interactions exist between a variety of transcription factors to ensure tight and energy-efficient regulation of cellulase gene expression in T. reesei. This study also provides important clues regarding increased cellulase production in T. reesei.

Trichoderma reesei complete genome sequence, repeat-induced point mutation, and partitioning of CAZyme gene clusters.

Abstract: Trichoderma reesei (Ascomycota, Pezizomycotina) QM6a is a model fungus for a broad spectrum of physiological phenomena, including plant cell wall degradation, industrial production of enzymes, light responses, conidiation, sexual development, polyketide biosynthesis, and plant–fungal interactions. The genomes of QM6a and its high enzyme-producing mutants have been sequenced by second-generation-sequencing methods and are publicly available from the Joint Genome Institute. While these genome sequences have offered useful information for genomic and transcriptomic studies, their limitations and especially their short read lengths make them poorly suited for some particular biological problems, including assembly, genome-wide determination of chromosome architecture, and genetic modification or engineering. We integrated Pacific Biosciences and Illumina sequencing platforms for the highest-quality genome assembly yet achieved, revealing seven telomere-to-telomere chromosomes (34,922,528 bp; 10877 genes) with 1630 newly predicted genes and >1.5 Mb of new sequences. Most new sequences are located on AT-rich blocks, including 7 centromeres, 14 subtelomeres, and 2329 interspersed AT-rich blocks. The seven QM6a centromeres separately consist of 24 conserved repeats and 37 putative centromere-encoded genes. These findings open up a new perspective for future centromere and chromosome architecture studies. Next, we demonstrate that sexual crossing readily induced cytosine-to-thymine point mutations on both tandem and unlinked duplicated sequences. We also show by bioinformatic analysis that T. reesei has evolved a robust repeat-induced point mutation (RIP) system to accumulate AT-rich sequences, with longer AT-rich blocks having more RIP mutations. The widespread distribution of AT-rich blocks correlates genome-wide partitions with gene clusters, explaining why clustering of genes has been reported to not influence gene expression in T. reesei. Compartmentation of ancestral gene clusters by AT-rich blocks might promote flexibilities that are evolutionarily advantageous in this fungus’ soil habitats and other natural environments. Our analyses, together with the complete genome sequence, provide a better blueprint for biotechnological and industrial applications.

Transcription factor Xpp1 is a switch between primary and secondary fungal metabolism.

Abstract: Fungi can produce a wide range of chemical compounds via secondary metabolism. These compounds are of major interest because of their (potential) application in medicine and biotechnology and as a potential source for new therapeutic agents and drug leads. However, under laboratory conditions, most secondary metabolism genes remain silent. This circumstance is an obstacle for the production of known metabolites and the discovery of new secondary metabolites. In this study, we describe the dual role of the transcription factor Xylanase promoter binding protein 1 (Xpp1) in the regulation of both primary and secondary metabolism of Trichoderma reesei. Xpp1 was previously described as a repressor of xylanases. Here, we provide data from an RNA-sequencing analysis suggesting that Xpp1 is an activator of primary metabolism. This finding is supported by our results from a Biolog assay determining the carbon source assimilation behavior of an xpp1 deletion strain. Furthermore, the role of Xpp1 as a repressor of secondary metabolism is shown by gene expression analyses of polyketide synthases and the determination of the secondary metabolites of xpp1 deletion and overexpression strains using an untargeted metabolomics approach. The deletion of Xpp1 resulted in the enhanced secretion of secondary metabolites in terms of diversity and quantity. Homologs of Xpp1 are found among a broad range of fungi, including the biocontrol agent Trichoderma atroviride, the plant pathogens Fusarium graminearum and Colletotrichum graminicola, the model organism Neurospora crassa, the human pathogen Sporothrix schenckii, and the ergot fungus Claviceps purpurea.

Cellulase hyper-production by Trichoderma reesei mutant SEU-7 on lactose

Abstract: The induction of cellulase production by insoluble carbon source cellulose was a common and efficient strategy, but has some drawbacks, such as difficult fermentation operation, substantial cellulase loss, long fermentation time, and high energy-consumption, resulting in high cost of cellulase production in industry. These drawbacks can be overcome if soluble carbon sources are utilized as the inducers for cellulase production. However, until now the induction efficiency of most soluble carbon sources, especially lactose and glucose, is still inferior to cellulose despite extensive efforts have been made by either optimizing the fermentation process or constructing the recombinant strains. Therefore, strain improvement by metabolic engineering for high induction efficiency of soluble carbon sources is of great interest. Trichoderma reesei mutant SEU-7 was constructed from T. reesei RUT-C30 with the overexpression of endogenous gene β-glucosidase (BGL1) by insertional mutagenesis via Agrobacterium tumefaciens-mediated transformation (AMT). Compared to RUT-C30, SEU-7 displays substantially enhanced activities of both cellulase and hemicellulase when grown on either lactose or cellulose. The induction efficiency with lactose was found to be higher than cellulose in strain SEU-7. To the best of our knowledge, we achieved the highest FPase activity in SEU-7 in both batch culture (13.0 IU/mL) and fed-batch culture (47.0 IU/mL) on lactose. Moreover, SEU-7 displayed unrivaled pNPGase activity on lactose in both batch culture (81.0 IU/mL) and fed-batch culture (144.0 IU/mL) as compared to the other reported T. reesei strains in the literature grown in batch or fed-batch experiments on cellulose or lactose. This superiority of SEU-7 over RUT-C30 improves markedly the saccharification ability of SEU-7 on pretreated corn stover. The overexpression of gene BGL1 was found either at the mRNA or at the protein level in the mutant strains with increased cellulase production in comparison with RUT-C30, but only SEU-7 displayed much higher expression of gene BGL1 on lactose than on cellulose. Two copies of gene BGL1 were inserted into the chromosome of T. reesei SEU-7 between KI911141.1:347357 and KI911141.1:347979, replacing the original 623-bp fragment that is not within any genes’ coding region. The qRT-PCR analysis revealed that the mRNA levels of both cellulase and hemicellulase were upregulated significantly in SEU-7, together with the MFS transporter CRT1 and the XYR1 nuclear importer KAP8. Recombinant T. reesei SEU-7 displays hyper-production of both cellulase and hemicellulase on lactose with the highest FPase activity and pNPGase activity for T. reesei, enabling highly efficient saccharification of pretreated biomass. For the first time, the induction efficiency for cellulase production by lactose in T. reesei was reported to be higher than that by cellulose. This outperformance of T. reesei SEU-7, which is strain-specific, is attributed to both the overexpression of gene BGL and the collateral mutation. Moreover, the increased transcription levels of cellulase genes, the related transcription factors, and the MFS transporter CRT1 contribute to the outstanding cellulase production of SEU-7. Our research advances strain improvement to enhance the induction efficiency of soluble carbon sources to produce cost-effective cellulase and hemicellulase in industry.

In Vivo Study of the Sorbicillinoid Gene Cluster in Trichoderma reesei.

Abstract: Sorbicillinoids are a diverse group of yellow secondary metabolites that are produced by a range of not closely related ascomycetes, including Penicillium chrysogenum, Acremonium chrysogenum, and Trichoderma reesei. They share a similarity to the name-giving compound sorbicillin, a hexaketide. Previously, a conserved gene cluster containing two polyketide synthases has been identified as the source of sorbicillin, and a model for the biosynthesis of sorbicillin in P. chrysogenum has been proposed. In this study, we deleted the major genes of interest of the cluster in T. reesei, namely sor1, sor3, and sor4. Sor1 is the homolog of P. chrysogenum SorA, which is the first polyketide synthase of the proposed biosynthesis pathway. Sor3 is a flavin adenine dinucleotide (FAD)-dependent monooxygenase, and its homolog in P. chrysogenum, SorC, was shown to oxidize sorbicillin and 2',3'-dihydrosorbicillin to sorbicillinol and 2',3'-dihydrosorbicillinol, respectively, in vitro. Sor4 is an FAD/flavin mononucleotide-containing dehydrogenase with an unknown function. We measured the amounts of synthesized sorbicillinoids throughout growth and could verify the roles of Sor1 and Sor3 in vivo in T. reesei. In the absence of Sor4, two compounds annotated to dihydrosorbicillinol accumulate in the supernatant and only small amounts of sorbicillinol are synthesized. Therefore, we suggest extending the current biosynthesis model about Sor4 reducing 2',3'-dihydrosorbicillin and 2',3'-dihydrosorbicillinol to sorbicillinol and sorbicillinol, respectively. Sorbicillinol turned out to be the main chemical building block for most sorbicillinoids, including oxosorbicillinol, bisorbicillinol, and bisvertinolon. Further, we detected the sorbicillinol-dependent synthesis of 5-hydroxyvertinolide at early time points, which contradicts previous models for biosynthesis of 5-hydroxyvertinolide. Finally, we investigated whether sorbicillinoids from T. reesei have a growth limiting effect on the fungus itself or on plant pathogenic fungi or on pathogenic bacteria.

Comparative transcriptome analysis reveals different strategies for degradation of steam-exploded sugarcane bagasse by Aspergillus niger and Trichoderma reesei

Abstract: Second generation (2G) ethanol is produced by breaking down lignocellulosic biomass into fermentable sugars. In Brazil, sugarcane bagasse has been proposed as the lignocellulosic residue for this biofuel production. The enzymatic cocktails for the degradation of biomass-derived polysaccharides are mostly produced by fungi, such as Aspergillus niger and Trichoderma reesei. However, it is not yet fully understood how these microorganisms degrade plant biomass. In order to identify transcriptomic changes during steam-exploded bagasse (SEB) breakdown, we conducted a RNA-seq comparative transcriptome profiling of both fungi growing on SEB as carbon source. Particular attention was focused on CAZymes, sugar transporters, transcription factors (TFs) and other proteins related to lignocellulose degradation. Although genes coding for the main enzymes involved in biomass deconstruction were expressed by both fungal strains since the beginning of the growth in SEB, significant differences were found in their expression profiles. The expression of these enzymes is mainly regulated at the transcription level, and A. niger and T. reesei also showed differences in TFs content and in their expression. Several sugar transporters that were induced in both fungal strains could be new players on biomass degradation besides their role in sugar uptake. Interestingly, our findings revealed that in both strains several genes that code for proteins of unknown function and pro-oxidant, antioxidant, and detoxification enzymes were induced during growth in SEB as carbon source, but their specific roles on lignocellulose degradation remain to be elucidated. This is the first report of a time-course experiment monitoring the degradation of pretreated bagasse by two important fungi using the RNA-seq technology. It was possible to identify a set of genes that might be applied in several biotechnology fields. The data suggest that these two microorganisms employ different strategies for biomass breakdown. This knowledge can be exploited for the rational design of enzymatic cocktails and 2G ethanol production improvement.

Co-fermentation using recombinant Saccharomyces cerevisiae yeast strains hyper-secreting different cellulases for the production of cellulosic bioethanol

Abstract: To realize the economical production of ethanol and other bio-based chemicals from lignocellulosic biomass by consolidated bioprocessing (CBP), various cellulases from different sources were tested to improve the level of cellulase secretion in the yeast Saccharomyces cerevisiae by screening an optimal translational fusion partner (TFP) as both a secretion signal and fusion partner. Among them, four indispensable cellulases for cellulose hydrolysis, including Chaetomium thermophilum cellobiohydrolase (CtCBH1), Chrysosporium lucknowense cellobiohydrolase (ClCBH2), Trichoderma reesei endoglucanase (TrEGL2), and Saccharomycopsis fibuligera β-glucosidase (SfBGL1), were identified to be highly secreted in active form in yeast. Despite variability in the enzyme levels produced, each recombinant yeast could secrete approximately 0.6–2.0 g/L of cellulases into the fermentation broth. The synergistic effect of the mixed culture of the four strains expressing the essential cellulases with the insoluble substrate Avicel and several types of cellulosic biomass was demonstrated to be effective. Co-fermentation of these yeast strains produced approximately 14 g/L ethanol from the pre-treated rice straw containing 35 g/L glucan with 3-fold higher productivity than that of wild type yeast using a reduced amount of commercial cellulases. This process will contribute to the cost-effective production of bioenergy such as bioethanol and biochemicals from cellulosic biomass.

Analysis of Light- and Carbon-Specific Transcriptomes Implicates a Class of G-Protein-Coupled Receptors in Cellulose Sensing.

Abstract: In fungi, most metabolic processes are subject to regulation by light. Trichoderma reesei is adapted to degradation of plant cell walls and regulates production of the required enzymes in a manner dependent on the nutrient source and the light status. Here we investigated the interrelated relevance of two regulation levels of the transcriptome of T. reesei: light regulation and carbon source-dependent control. We show that the carbon source (cellulose, lactose, sophorose, glucose, or glycerol) is the major source of variation, with light having a modulating effect on transcript regulation. A total of 907 genes were regulated under cellulase-inducing conditions in light, and 947 genes were regulated in darkness, with 530 genes overlapping (1,324 in total). Only 218 of the 1,324 induction-specific genes were independent of light and not regulated by the BLR1, BLR2, and ENV1 photoreceptors. Analysis of the genomic distribution of genes regulated by light upon growth on cellulose revealed considerable overlap of light-regulated clusters with induction-specific clusters and carbohydrate-active enzyme (CAZyme) clusters. Further, we found evidence for the operation of a sensing mechanism for solid cellulosic substrates, with regulation of genes such as swo1, cip1, and cip2 or of genes encoding hydrophobins which is related to the cyclic AMP (cAMP)-dependent regulatory output of ENV1. We identified class XIII G-protein-coupled receptors (GPCRs) CSG1 and CSG2 in T. reesei as putative cellulose/glucose-sensing GPCRs. Our data indicate that the cellulase regulation pathway is bipartite, comprising a section corresponding to transcriptional regulation and one corresponding to posttranscriptional regulation, with the two connected by the function of CSG1. IMPORTANCE In fungi, most metabolic processes are subject to regulation by light. For Trichoderma reesei, light-dependent regulation of cellulase gene expression is specifically shown. Therefore, we intended to unravel the relationship between regulation of enzymes by the carbon source and regulation of enzymes by light. Our two-dimensional analysis included inducing and repressing carbon sources which we used to compare light-specific regulation to dark-specific regulation and to rule out effects specific for a single carbon source. We found close connections with respect to gene regulation as well as significant differences in dealing with carbon in the environment in light and darkness. Moreover, our analyses showed an intricate regulation mechanism for substrate degradation potentially involving surface sensing and provide a basis for knowledge-based screening for strain improvement.

Genome sequencing and transcriptome analysis of Trichoderma reesei QM9978 strain reveals a distal chromosome translocation to be responsible for loss of vib1 expression and loss of cellulase induction

Abstract: The hydrolysis of biomass to simple sugars used for the production of biofuels in biorefineries requires the action of cellulolytic enzyme mixtures. During the last 50 years, the ascomycete Trichoderma reesei, the main source of industrial cellulase and hemicellulase cocktails, has been subjected to several rounds of classical mutagenesis with the aim to obtain higher production levels. During these random genetic events, strains unable to produce cellulases were generated. Here, whole genome sequencing and transcriptomic analyses of the cellulase-negative strain QM9978 were used for the identification of mutations underlying this cellulase-negative phenotype. Sequence comparison of the cellulase-negative strain QM9978 to the reference strain QM6a identified a total of 43 mutations, of which 33 were located either close to or in coding regions. From those, we identified 23 single-nucleotide variants, nine InDels, and one translocation. The translocation occurred between chromosomes V and VII, is located upstream of the putative transcription factor vib1, and abolishes its expression in QM9978 as detected during the transcriptomic analyses. Ectopic expression of vib1 under the control of its native promoter as well as overexpression of vib1 under the control of a strong constitutive promoter restored cellulase expression in QM9978, thus confirming that the translocation event is the reason for the cellulase-negative phenotype. Gene deletion of vib1 in the moderate producer strain QM9414 and in the high producer strain Rut-C30 reduced cellulase expression in both cases. Overexpression of vib1 in QM9414 and Rut-C30 had no effect on cellulase production, most likely because vib1 is already expressed at an optimal level under normal conditions. We were able to establish a link between a chromosomal translocation in QM9978 and the cellulase-negative phenotype of the strain. We identified the transcription factor vib1 as a key regulator of cellulases in T. reesei whose expression is absent in QM9978. We propose that in T. reesei, as in Neurospora crassa, vib1 is involved in cellulase induction, although the exact mechanism remains to be elucidated. The data presented here show an example of a combined genome sequencing and transcriptomic approach to explain a specific trait, in this case the QM9978 cellulase-negative phenotype, and how it helps to better understand the mechanisms during cellulase gene regulation. When focusing on mutations on the single base-pair level, changes on the chromosome level can be easily overlooked and through this work we provide an example that stresses the importance of the big picture of the genomic landscape during analysis of sequencing data.

Production of the versatile cellulase for cellulose bioconversion and cellulase inducer synthesis by genetic improvement of Trichoderma reesei.

Abstract: The enzymes for efficient hydrolysis of lignocellulosic biomass are a major factor in the development of an economically feasible cellulose bioconversion process. Up to now, low hydrolysis efficiency and high production cost of cellulases remain the significant hurdles in this process. The aim of the present study was to develop a versatile cellulase system with the enhanced hydrolytic efficiency and the ability to synthesize powerful inducers by genetically engineering Trichoderma reesei. In our study, we employed a systematic genetic strategy to construct the carbon catabolite-derepressed strain T. reesei SCB18 to produce the cellulase complex that exhibited a strong cellulolytic capacity for biomass saccharification and an extraordinary high β-glucosidase (BGL) activity for cellulase-inducing disaccharides synthesis. We first identified the hypercellulolytic and uracil auxotrophic strain T. reesei SP4 as carbon catabolite repressed, and then deleted the carbon catabolite repressor gene cre1 in the genome. We found that the deletion of cre1 with the selectable marker pyrG led to a 72.6% increase in total cellulase activity, but a slight reduction in saccharification efficiency. To facilitate the following genetic modification, the marker pyrG was successfully removed by homologous recombination based on resistance to 5-FOA. Furthermore, the Aspergillus niger BGLA-encoding gene bglA was overexpressed, and the generated strain T. reesei SCB18 exhibited a 29.8% increase in total cellulase activity and a 51.3-fold enhancement in BGL activity (up to 103.9 IU/mL). We observed that the cellulase system of SCB18 showed significantly higher saccharification efficiency toward differently pretreated corncob residues than the control strains SDC11 and SP4. Moreover, the crude enzyme preparation from SCB18 with high BGL activity possessed strong transglycosylation ability to synthesize β-disaccharides from glucose. The transglycosylation product was finally utilized as the inducer for cellulase production, which provided a 63.0% increase in total cellulase activity compared to the frequently used soluble inducer, lactose. In summary, we constructed a versatile cellulase system in T. reesei for efficient biomass saccharification and powerful cellulase inducer synthesis by combinational genetic manipulation of three distinct types of genes to achieve the customized cellulase production, thus providing a viable strategy for further strain improvement to reduce the cost of biomass-based biofuel production.

Conservation and diversity of the regulators of cellulolytic enzyme genes in ascomycete fungi

Abstract: In the past decade, various transcriptional activators of cellulolytic enzyme genes have been identified in Ascomycete fungi. The regulatory system of cellulolytic enzymes is not only partially conserved, but also significantly diverse. For example, Trichoderma reesei has a system distinct from those of Aspergillus and Neurospora crassa-the former utilizes Xyr1 (the Aspergillus XlnR ortholog) as the major regulator of cellulolytic enzyme genes, while the latter uses CLR-2/ClrB/ManR orthologs. XlnR/Xyr1 and CLR-2/ClrB/ManR are evolutionarily distant from each other. Regulatory mechanisms that are controlled by CLR-2, ClrB, and ManR are also significantly different, although they are orthologous factors. Expression of clr-2 requires the activation of another transcription factor, CLR-1, by cellobiose, while CLR-2 is constitutively active for transactivation. By contrast, ClrB activation requires cellobiose. While ClrB mainly regulates cellulolytic genes, ManR is essential for the activation of not only cellulolytic but also mannanolytic enzyme genes. In this review, we summarize XlnR/Xyr1- and CLR-2/ClrB/ManR-dependent regulation in N. crassa, A. nidulans, A. oryzae, and T. reesei and emphasize the conservation and diversity of the regulatory systems for cellulolytic enzyme genes in these Ascomycete fungi. In addition, we discuss the role of McmA, another transcription factor that plays an important role in recruiting ClrB to the promoters in A. nidulans.

Optimization of enzymatic hydrolysis and fermentation conditions for improved bioethanol production from potato peel residues.

Abstract: The aim of this work was the optimization of the enzyme hydrolysis of potato peel residues (PPR) for bioethanol production. The process included a pretreatment step followed by an enzyme hydrolysis using crude enzyme system composed of cellulase, amylase and hemicellulase, produced by a mixed culture of Aspergillus niger and Trichoderma reesei. Hydrothermal, alkali and acid pretreatments were considered with regards to the enhancement of enzyme hydrolysis of potato peel residues. The obtained results showed that hydrothermal pretreatment lead to a higher enzyme hydrolysis yield compared to both acid and alkali pretreatments. Enzyme hydrolysis was also optimized for parameters such as temperature, pH, substrate loading and surfactant loading using a response surface methodology. Under optimized conditions, 77 g L(-1) of reducing sugars were obtained. Yeast fermentation of the released reducing sugars led to an ethanol titer of 30 g L(-1) after supplementation of the culture medium with ammonium sulfate. Moreover, a comparative study between acid and enzyme hydrolysis of potato peel residues was investigated. Results showed that enzyme hydrolysis offers higher yield of bioethanol production than acid hydrolysis. These results highlight the potential of second generation bioethanol production from potato peel residues treated with onsite produced hydrolytic enzymes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:397-406, 2017.

Role of Trichoderma reesei mitogen-activated protein kinases (MAPKs) in cellulase formation.

Abstract: Despite being the most important cellulase producer, the cellulase-regulating carbon source signal transduction processes in Trichoderma reesei are largely unknown. Elucidating these processes is the key for unveiling how external carbon sources regulate cellulase formation, and ultimately for the improvement of cellulase production and biofuel production from lignocellulose. In this work, the role of the mitogen-activated protein kinase (MAPK) signal transduction pathways on cellulase formation was investigated. The deletion of yeast FUS3-like tmk1 in T. reesei leads to improved growth and significantly improved cellulase formation. However, tmk1 deletion has no effect on the transcription of cellulase-coding genes. The involvement of the cell wall integrity maintenance governing yeast Slt2-like Tmk2 in cellulase formation was investigated by overexpressing tmk3 in T. reesei Δtmk2 to restore cell wall integrity. Transcriptional analysis found little changes in cellulase-coding genes between T. reesei parent, Δtmk2, and Δtmk2::OEtmk3 strains. Cell wall integrity decreased in T. reesei Δtmk2 over the parent strain and restored in Δtmk2::OEtmk3. Meanwhile, cellulase formation is increased in T. reesei Δtmk2 and then decreased in T. reesei Δtmk2::OEtmk3. These investigations elucidate the role of Tmk1 and Tmk2 on cellulase formation: they repress cellulase formation, respectively, by repressing growth and maintaining cell wall integrity, while neither MAPK regulates the transcription of cellulase-coding genes. This work, together with the previous investigations, suggests that all MAPKs are involved in cellulase formation, while Tmk3 is the only MAPK involved in signal transduction for the regulation of cellulase expression on the transcriptional level.

Cost-effective cellulase production using Parthenium hysterophorus biomass as an unconventional lignocellulosic substrate.

Abstract: The potential of untreated Parthenium hysterophorus weed biomass was evaluated as a substrate for cellulase production. The cellulose in the biomass was used as the main source of carbon. Solid-state fermentation was carried out using Trichoderma reesei, and optimization of cultural conditions was done for maximization of cellulase production. The results revealed that highest cellulase production was achieved on the 8th day of incubation, at 30 °C, keeping solid-to-liquid ratio 1:2 when two discs of inoculum were used per gram of the substrate. The optimized inoculum age was 96 h for CMCase and 120 h for FPase. On studying the enhancing effect of different carbon and nitrogen sources, lactose and ammonium molybdate were found suitable, respectively. The optimized concentration of lactose for the highest CMCase and FPase activities was 1.5 and 1%, respectively. Ammonium molybdate was best at 1% concentration for both CMCase and FPase. Maximum CMCase and FPase activities obtained were 20.49 and 2.42 U/gds, respectively.

Production of highly efficient cellulase mixtures by genetically exploiting the potentials of Trichoderma reesei endogenous cellulases for hydrolysis of corncob residues.

Abstract: Trichoderma reesei is one of the most important fungi utilized for cellulase production. However, its cellulase system has been proven to be present in suboptimal ratio for deconstruction of lignocellulosic substrates. Although previous enzymatic optimization studies have acquired different types of in vitro synthetic mixtures for efficient lignocellulose hydrolysis, production of in vivo optimized cellulase mixtures by industrial strains remains one of the obstacles to reduce enzyme cost in the biofuels production from lignocellulosic biomass. In this study, we used a systematic genetic strategy based on the pyrG marker to overexpress the major cellulase components in a hypercellulolytic T. reesei strain and produce the highly efficient cellulase mixture for saccharification of corncob residues. We found that overexpression of CBH2 exhibited a 32-fold increase in the transcription level and a comparable protein level to CBH1, the most abundant secreted protein in T. reesei, but did not contribute much to the cellulolytic ability. However, when EG2 was overexpressed with a 46-fold increase in the transcription level and a comparable protein level to CBH2, the engineered strain QPE36 showed a 1.5-fold enhancement in the total cellulase activity (up to 5.8 U/mL FPA) and a significant promotion of saccharification efficiency towards differently pretreated corncob residues. To assist the following genetic manipulations, the marker pyrG was successfully excised by homologous recombination based on resistance to 5-FOA. Furthermore, BGL1 was overexpressed in the EG2 overexpression strain QE51 (pyrG-excised) and a 11.6-fold increase in BGL activity was obtained. The EG2–BGL1 double overexpression strain QEB4 displayed a remarkable enhancement of cellulolytic ability on pretreated corncob residues. Especially, a nearly complete cellulose conversion (94.2%) was found for the delignified corncob residues after 48 h enzymatic saccharification. These results demonstrate that genetically exploiting the potentials of T. reesei endogenous cellulases to produce highly efficient cellulase mixtures is a powerful strategy to promote the saccharification efficiency, which will eventually facilitate cost reduction for lignocellulose-based biofuels.

Concurrent production of cellulase and xylanase from Trichoderma reesei NCIM 1186: enhancement of production by desirability-based multi-objective method

Abstract: Application of multiple response optimizations using desirability function in the production of microbial metabolites improves economy and efficiency. Concurrent production of cellulase and xylanase in Trichoderma reesei NCIM 1186 using an agricultural weed, Prosopis juliflora pods, was studied. The main aim of the study was to optimize significant medium nutrient parameters for maximization of cellulase and xylanase by multi-objective optimization strategy using biomass. Process parameters such as the nutrient concentrations (pods, sucrose, and yeast extract) and pH were investigated to improve cellulase and xylanase activities by one factor at a time approach, single response optimization and multi-objective optimization. At the corresponding optimized process parameters in single response optimization, the maximum cellulase activity observed was 3055.65 U/L where xylanase highest activity was 422.16 U/L. Similarly, the maximum xylanase activity, 444.94 U/L, was observed with the highest cellulase activity of 2804.40 U/L. The multi-objective optimization finds a tradeoff between the two objectives and optimal activity values in between the single-objective optima were achieved, 3033.74 and 439.13 U/L for cellulase and xylanase, respectively.

Expressing accessory proteins in cellulolytic Yarrowia lipolytica to improve the conversion yield of recalcitrant cellulose

Abstract: A recently constructed cellulolytic Yarrowia lipolytica is able to grow efficiently on an industrial organosolv cellulose pulp, but shows limited ability to degrade crystalline cellulose. In this work, we have further engineered this strain, adding accessory proteins xylanase II (XYNII), lytic polysaccharide monooxygenase (LPMO), and swollenin (SWO) from Trichoderma reesei in order to enhance the degradation of recalcitrant substrate. The production of EG I was enhanced using a promoter engineering strategy. This provided a new cellulolytic Y. lipolytica strain, which compared to the parent strain, exhibited higher hydrolytic activity on different cellulosic substrates. Furthermore, three accessory proteins, TrXYNII, TrLPMOA and TrSWO, were individually expressed in cellulolytic and non-cellulolytic Y. lipolytica. The amount of rhTrXYNII and rhTrLPMOA secreted by non-cellulolytic Y. lipolytica in YTD medium during batch cultivation in flasks was approximately 62 and 52 mg/L, respectively. The purified rhTrXYNII showed a specific activity of 532 U/mg-protein on beechwood xylan, while rhTrLPMOA exhibited a specific activity of 14.4 U/g-protein when using the Amplex Red/horseradish peroxidase assay. Characterization of rhTrLPMOA revealed that this protein displays broad specificity against β-(1,4)-linked glucans, but is inactive on xylan. Further studies showed that the presence of TrLPMOA synergistically enhanced enzymatic hydrolysis of cellulose by cellulases, while TrSWO1 boosted cellulose hydrolysis only when it was applied before the action of cellulases. The presence of rTrXYNII enhanced enzymatic hydrolysis of an industrial cellulose pulp and of wheat straw. Co-expressing TrXYNII and TrLPMOA in cellulolytic Y. lipolytica with enhanced EG I production procured a novel engineered Y. lipolytica strain that displayed enhanced ability to degrade both amorphous (CIMV-cellulose) and recalcitrant crystalline cellulose in complex biomass (wheat straw) by 16 and 90%, respectively. This study has provided a potent cellulose-degrading Y. lipolytica strain that co-expresses a core set of cellulolytic enzymes and some accessory proteins. Results reveal that the tuning of cellulase production and the production of accessory proteins leads to optimized performance. Accordingly, the beneficial effect of accessory proteins for cellulase-mediated degradation of cellulose is underlined, especially when crystalline cellulose and complex biomass are used as substrates. Findings specifically underline the benefits and specific properties of swollenin. Although in our study swollenin clearly promoted cellulase action, its use requires process redesign to accommodate its specific mode of action.