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Showing papers on "Trichoderma reesei published in 2019"


Journal ArticleDOI
TL;DR: Proteomic analysis of fungal secretomes that significantly improved biomass degradation showed that the presence of proteins belonging to a putative LPMO family previously identified by genome analysis and awaiting experimental demonstration of activity was found, named AA16.
Abstract: Lignocellulosic biomass is considered as a promising alternative to fossil resources for the production of fuels, materials and chemicals. Efficient enzymatic systems are needed to degrade the plant cell wall and overcome its recalcitrance. A widely used producer of cellulolytic cocktails is the ascomycete Trichoderma reesei, but this organism secretes a limited set of enzymes. To improve the saccharification yields, one strategy is to upgrade the T. reesei enzyme cocktail with enzymes produced by other biomass-degrading filamentous fungi isolated from biodiversity. In this study, the enzymatic cocktails secreted by five strains from the genus Aspergillus (Aspergillus japonicus strains BRFM 405, 1487, 1489, 1490 and Aspergillus niger strain BRFM 430) were tested for their ability to boost a T. reesei reference cocktail for the saccharification of pretreated biomass. Proteomic analysis of fungal secretomes that significantly improved biomass degradation showed that the presence of proteins belonging to a putative LPMO family previously identified by genome analysis and awaiting experimental demonstration of activity. Members of this novel LPMO family, named AA16, are encountered in fungi and oomycetes with life styles oriented toward interactions with plant biomass. One AA16 protein from Aspergillus aculeatus (AaAA16) was produced to high level in Pichia pastoris. LPMO-type enzyme activity was demonstrated on cellulose with oxidative cleavage at the C1 position of the glucose unit. AaAA16 LPMO was found to significantly improve the activity of T. reesei CBHI on cellulosic substrates. Although Aspergillus spp. has been investigated for decades for their CAZymes diversity, we identified members of a new fungal LPMO family using secretomics and functional assays. Properties of the founding member of the AA16 family characterized herein could be of interest for use in biorefineries.

119 citations


Journal ArticleDOI
TL;DR: A comprehensive update on studies in developing high-performance cellulases for biorefineries is provided and the simplest minimal enzyme systems comprising selected sets of mono-component enzymes tailor-made for specific lignocellulosic substrates are provided.

74 citations


Journal ArticleDOI
TL;DR: It was shown that the hydrothermally (hemicellulose-deficient) pretreated and delignified biomass solids could be also successfully used for the production of crude cellulase from Trichoderma reesei cultures, providing a simple and low-cost method for the complementary production of cellulases by utilizing fractions of the integrated hydrolysis process.
Abstract: Acetone and ethanol extraction of lignin deposits from the surface of hydrothermally (liquid hot water) pretreated beech wood biomass alleviates the lignin inhibitory effects during enzymatic hydrolysis of cellulose to glucose and boosts the enzymatic digestibility to high values (≈70 %). Characterization of the extracted lignins (FTIR, pyrolysis/GC-MS, differential thermogravimetry, gel permeation chromatography) indicated high purity, low molecular weight, and features that suggest that it consists mainly of fragments of the native wood lignin partially depolymerized and recondensed on the biomass surface during the hydrothermal pretreatment. The pyrolysis products of the extracted surface lignins suggest their high potential as a feedstock for the production of high added value phenolic compounds. When the enzymatic hydrolysis of the pretreated and extracted biomass solids was assisted by mild wet milling, near complete cellulose digestibility (≥95 %) could be achieved. In the context of the biorefinery and whole-biomass valorization concept, it was also shown that the hydrothermally (hemicellulose-deficient) pretreated and delignified biomass solids could be also successfully used for the production of crude cellulase from Trichoderma reesei cultures, providing a simple and low-cost method for the complementary production of cellulases by utilizing fractions of the integrated hydrolysis process.

66 citations


Journal ArticleDOI
TL;DR: Insight is provided into the interrelation between the characteristics of the substrate and the enzyme production by T. reesei, which may help to advance integrated enzyme manufacturing of substrate-specific enzymes cocktails at scale.
Abstract: Biorefineries, designed for the production of lignocellulose-based chemicals and fuels, are receiving increasing attention from the public, governments, and industries. A major obstacle for biorefineries to advance to commercial scale is the high cost of the enzymes required to derive the fermentable sugars from the feedstock used. As summarized in this review, techno-economic studies suggest co-localization and integration of enzyme manufacturing with the cellulosic biorefinery as the most promising alternative to alleviate this problem. Thus, cultivation of Trichoderma reesei, the principal producer of lignocellulolytic enzymes, on the lignocellulosic biomass processed on-site can reduce the cost of enzyme manufacturing. Further, due to a complex gene regulation machinery, the fungus can adjust the gene expression of the lignocellulolytic enzymes towards the characteristics of the feedstock, increasing the hydrolytic efficiency of the produced enzyme cocktail. Despite extensive research over decades, the underlying regulatory mechanisms are not fully elucidated. One aspect that has received relatively little attention in literature is the influence the characteristics of a lignocellulosic substrate, i.e., its chemical and physical composition, has on the produced enzyme mixture. Considering that the fungus is dependent on efficient enzymatic degradation of the lignocellulose for continuous supply of carbon and energy, a relationship between feedstock characteristics and secretome composition can be expected. The aim of this review was to systematically collect, appraise, and aggregate data and integrate results from studies analyzing enzyme production by T. reesei on insoluble cellulosic model substrates and lignocellulosic biomass. The results show that there is a direct effect of the substrate’s complexity (rated by structure, composition of the lignin–carbohydrate complex, and recalcitrance in enzymatic saccharification) on enzyme titers and the composition of specific activities in the secretome. It further shows that process-related factors, such as substrate loading and cultivation set-up, are direct targets for increasing enzyme yields. The literature on transcriptome and secretome composition further supports the proposed influence of substrate-related factors on the expression of lignocellulolytic enzymes. This review provides insights into the interrelation between the characteristics of the substrate and the enzyme production by T. reesei, which may help to advance integrated enzyme manufacturing of substrate-specific enzymes cocktails at scale.

56 citations


Journal ArticleDOI
TL;DR: One-step fermentation proved to be a promising strategy for AXOS production from BSG, presenting a performance comparable with the use of commercial enzymes, enabling further developments of low-cost bioprocesses for the production of these valuable compounds.

56 citations


Journal ArticleDOI
TL;DR: RSM was used to identify the non-linear relationship and optimize various process parameters such as parameterssuch as pH, temperature, agitation rate, substrate concentration and inoculum size for bioethanol production.

52 citations


Journal ArticleDOI
TL;DR: Engineering of T. reesei for high-level production of highly enriched lipase B of Candida antarctica (calB) using glucose as a carbon source using multiplexed CRISPR/Cas9 in combination with the use of recently established synthetic expression system (SES) enabled accelerated construction of strains, which produced high amounts of highly pure calB.
Abstract: Trichoderma reesei is an established protein production host with high natural capacity to secrete enzymes. The lack of efficient genome engineering approaches and absence of robust constitutive gene expression systems limits exploitation of this organism in some protein production applications. Here we report engineering of T. reesei for high-level production of highly enriched lipase B of Candida antarctica (calB) using glucose as a carbon source. Multiplexed CRISPR/Cas9 in combination with the use of our recently established synthetic expression system (SES) enabled accelerated construction of strains, which produced high amounts of highly pure calB. Using SES, calB production levels in cellulase-inducing medium were comparable to the levels obtained by using the commonly employed inducible cbh1 promoter, where a wide spectrum of native enzymes were co-produced. Due to highly constitutive expression provided by the SES, it was possible to carry out the production in cellulase-repressing glucose medium leading to around 4 grams per liter of fully functional calB and simultaneous elimination of unwanted background enzymes.

51 citations


Journal ArticleDOI
TL;DR: A new AA9 LPMO from Talaromyces cellulolyticus, named TcAA9A, was identified and may offer an innovative solution for improving enzyme cocktail activity for lignocellulosic degradation.
Abstract: Auxiliary activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) show significant synergism with cellulase in cellulose degradation. In recent years, there have been many reports on AA9 LPMOs; however, the identification of efficient and thermostable AA9 LPMOs with broad potential for industrial applications remains necessary. In this study, a new AA9 LPMO from Talaromyces cellulolyticus, named TcAA9A, was identified. An analysis of the oxidation products of phosphoric acid-swollen cellulose categorized TcAA9A as a type 3 AA9 LPMO, and TcAA9A exhibited a better synergistic effect with cellulase from Trichoderma reesei than what is seen with TaAA9A, a well-studied AA9 LPMO from Thermoascus aurantiacus. Two AA9 LPMOs were successfully expressed in T. reesei, and the transformants were named TrTcAA9A and TrTaAA9A. The activities and thermostabilities of the AA9 LPMOs in TrTcAA9A were higher than those of the AA9 LPMOs in TrTaAA9A or the parent. The enzyme solution of TrTcAA9A was more efficient than that of the parent or TrTaAA9A for the degradation of Avicel and delignified corncob residue. Thus, TcAA9A showed a better performance than TaAA9A in T. reesei cellulase cocktails. This study may offer an innovative solution for improving enzyme cocktail activity for lignocellulosic degradation.

49 citations


Journal ArticleDOI
TL;DR: This work reports for the first time the isolation and characterization of T. reesei extracellular vesicles and points to important insights into a better understanding of the cellular mechanisms underlying the regulation of cellulolytic enzyme secretion in this fungus.
Abstract: Trichoderma reesei is the most important industrial producer of lignocellulolytic enzymes. These enzymes play an important role in biomass degradation leading to novel applications of this fungus in the biotechnology industry, specifically biofuel production. The secretory pathway of fungi is responsible for transporting proteins addressed to different cellular locations involving some cellular endomembrane systems. Although protein secretion is an extremely efficient process in T. reesei, the mechanisms underlying protein secretion have remained largely uncharacterized in this organism. Here, we report for the first time the isolation and characterization of T. reesei extracellular vesicles (EVs). Using proteomic analysis under cellulose culture condition, we have confidently identified 188 vesicular proteins belonging to different functional categories. Also, we characterized EVs production using transmission electron microscopy in combination with light scattering analysis. Biochemical assays revealed that T. reesei extracellular vesicles have an enrichment of filter paper (FPase) and β-glucosidase activities in purified vesicles from 24, 72 and 96, and 72 and 96 h, respectively. Furthermore, our results showed that there is a slight enrichment of small RNAs inside the vesicles after 96 h and 120 h, and presence of hsp proteins inside the vesicles purified from T. reesei grown in the presence of cellulose. This work points to important insights into a better understanding of the cellular mechanisms underlying the regulation of cellulolytic enzyme secretion in this fungus.

46 citations


Journal ArticleDOI
TL;DR: Transcriptional profiling with RNA-Seq revealed that ace3 deletion down-regulates not only the bulk of the major cellulase, hemicellulase, and related transcription factor genes, but also reduces the expression of lactose metabolism–related genes.

45 citations


Journal ArticleDOI
TL;DR: In this paper, an endophytic fungus (Trichoderma reesei) from Solanum surattense that promoted the wheat grasps was isolated and used to release salt stress.
Abstract: The use of plant endophytes to release salt stress is cheap and quick. In this respect, we have isolated an endophytic fungus (Trichoderma reesei) from Solanum surattense that promoted the wheat gr...

Journal ArticleDOI
TL;DR: Direct transformation of Cas9/gRNA complex into the cell is a fast means to disrupt a gene in T. reesei and may find wide applications in strain improvement and functional genomics study.
Abstract: CRISPR/Cas9 has wide application potentials in a variety of biological species including Trichoderma reesei, a filamentous fungus workhorse for cellulase production. However, expression of Cas9 heterologously in the host cell could be time-consuming and sometimes even troublesome. We tested two gene disruption methods in T. reesei using CRISPR/Cas9 in this study. The intracellularly expressed Cas9 led to unexpected off-target gene disruption in T. reesei QM9414, favoring inserting 9- or 12-bp at 70- and 100-bp downstream of the targeted ura5. An alternative method was, therefore, established by assembling Cas9 and gRNA in vitro, followed by transformation of the ribonucleoprotein complex with a plasmid containing the pyr4 marker gene into T. reesei TU-6. When the gRNA targeting cbh1 was used, eight among the twenty seven transformants were found to lose the ability to express CBH1, indicative of successful cbh1 disruption through genome editing. Large DNA fragments including the co-transformed plasmid, chromosomal genes, or a mixture of these nucleotides, were inserted in the disrupted cbh1 locus. Direct transformation of Cas9/gRNA complex into the cell is a fast means to disrupt a gene in T. reesei and may find wide applications in strain improvement and functional genomics study.

Journal ArticleDOI
TL;DR: In this paper, the authors sequenced the T. reesei NG14 strain using Illumina MiSeq to identify any genetic changes that occurred in SS-II and found 184 single nucleotide polymorphisms and 40 insertions and deletions were identified.
Abstract: Cellulolytic enzymes produced by the filamentous fungus Trichoderma reesei are commonly used in biomass conversion. The high cost of cellulase is still a significant challenge to commercial biofuel production. Improving cellulase production in T. reesei for application in the cellulosic biorefinery setting is an urgent priority. Trichoderma reesei hyper-cellulolytic mutant SS-II derived from the T. reesei NG14 strain exhibited faster growth rate and more efficient lignocellulosic biomass degradation than those of RUT-C30, another hyper-cellulolytic strain derived from NG14. To identify any genetic changes that occurred in SS-II, we sequenced its genome using Illumina MiSeq. In total, 184 single nucleotide polymorphisms and 40 insertions and deletions were identified. SS-II sequencing revealed 107 novel mutations and a full-length wild-type carbon catabolite repressor 1 gene (cre1). To combine the mutations of RUT-C30 and SS-II, the sequence of one confirmed beneficial mutation in RUT-C30, cre196, was introduced in SS-II to replace full-length cre1, forming the mutant SS-II-cre196. The total cellulase production of SS-II-cre196 was decreased owing to the limited growth of SS-II-cre196. In contrast, 57 genes mutated only in SS-II were selected and knocked out in RUT-C30. Of these, 31 were involved in T. reesei growth or cellulase production. Cellulase activity was significantly increased in five deletion strains compared with that in two starter strains, RUT-C30 and SS-II. Cellulase production of T. reesei Δ108642 and Δ56839 was significantly increased by 83.7% and 70.1%, respectively, compared with that of RUT-C30. The amount of glucose released from pretreated corn stover hydrolyzed by the crude enzyme from Δ108642 increased by 11.9%. The positive attribute confirmed in one cellulase hyper-producing strain does not always work efficiently in another cellulase hyper-producing strain, owing to the differences in genetic background. Genome re-sequencing revealed novel mutations that might affect cellulase production and other pathways indirectly related to cellulase formation. Our strategy of combining the mutations of two strains successfully identified a number of interesting phenotypes associated with cellulase production. These findings will contribute to the creation of a gene library that can be used to investigate the involvement of various genes in the regulation of cellulase production.

Journal ArticleDOI
TL;DR: This study constructed a Xyr1-deficient strain that can be fully reconstituted, which makes it an ideal platform strain for Xyr 1-related studies and gained new insights into the regulatory properties of transcription factors by constructing respective fusion proteins.
Abstract: The filamentous ascomycete T. reesei is industrially used to produce cellulases and xylanases. Cost-effective production of cellulases is a bottleneck for biofuel production. Previously, different strain and process optimizations were deployed to enhance enzyme production rates. One approach is the overexpression of the main activator Xyr1 and a second is the construction of synthetic transcription factors. Notably, these genetic manipulations were introduced into strains bearing the wild-type xyr1 gene and locus. Here, we constructed a Xyr1-deficient strain expressing a non-functional truncated version of Xyr1. This strain was successfully used as platform strain for overexpression of Xyr1, which enhanced the cellulase and xylanase production rates under inducing conditions, with the exception of lactose—there the cellulase production was severely reduced. Further, we introduced fusion transcription factors consisting of the DNA-binding domain of Xyr1 and the transactivation domain of either Ypr1 or Ypr2 (regulators of the sorbicillinoid biosynthesis gene cluster). The fusion of Xyr1 and Ypr2 yielded a moderately transactivating transcription factor, whereas the fusion of Xyr1 and Ypr1 yielded a highly transactivating transcription factor that induced xylanases and cellulases nearly carbon source independently. Especially, high production levels of xylanases were achieved on glycerol. During this study, we constructed a Xyr1-deficient strain that can be fully reconstituted, which makes it an ideal platform strain for Xyr1-related studies. The mere overexpression of Xyr1 turned out not to be a successful strategy for overall enhancement of the enzyme production rates. We gained new insights into the regulatory properties of transcription factors by constructing respective fusion proteins. The Xyr1–Ypr1-fusion transcription factor could induce xylanase production rates on glycerol to outstanding extents, and hence could be deployed in the future to utilize crude glycerol, the main co-product of the biodiesel production process.

Journal ArticleDOI
TL;DR: Analysis of a range of kinetic parameters consistently indicated that the B2 loop, covering the substrate-binding subsites −3 and −4 in TrC Cel7A, was a key determinant for the difference in CBH- or EG-like behavior between TrCel7A and TrCEL7B.

Journal ArticleDOI
TL;DR: In this paper, the possible effects of carbonaceous additives including Tween 80, betaine, carboxymethyl cellulose (CMC) and lactose on cellulases and xylanase production were investigated individually or in combination.

Journal ArticleDOI
TL;DR: This study has identified a novel transcriptional regulator controlling xyr1 and cellulase gene expression, which will contribute to elaborating the intricate network of cellulases gene regulation in T. reesei.
Abstract: XYR1 is the key transcription activator for cellulase gene expression in the model filamentous fungus Trichoderma reesei, which is widely applied in the industry due to its excellent capability of secreting a large quantity of cellulases. Despite the essential role of XYR1, the regulation of its expression in T. reesei cellulolytic response is poorly understood. In this study, we identified a transcription factor RXE1 exhibiting strong binding activity to the xyr1 promoter using yeast one-hybrid screen. RXE1 homologs exist in quite a few filamentous fungi but none of them have been assessed regarding their functional involvement in plant cell wall degradation. Knockdown of rxe1 in T. reesei using a copper-mediated RNAi system not only abrogated conidiation, but also remarkably compromised xyr1 and cellulase gene expression. The defective cellulase but not conidia production in the rxe1-knockdown strain was fully rescued by the constitutive expression of XYR1. Our study thus identified a novel transcriptional regulator controlling xyr1 and cellulase gene expression, which will contribute to elaborating the intricate network of cellulase gene regulation in T. reesei.

Journal ArticleDOI
TL;DR: A new mutant of Trichoderma reesei was used for cellulase production and the resulting variables including 74% for moisture content, 2 g/L for K2 HPO 4, 30°C of incubation temperature, and 4 days of incubations time were reported as the ultimate optimal condition for cellul enzyme production.
Abstract: Nowadays, the use of agricultural by-products, as the cheap substrate for the production of value-added products, is of high interest for the researchers and practitioners. Cellulase is a relatively expensive and a very important industrial enzyme where in this study was produced form rice by-products under solid-state fermentation. A new mutant of Trichoderma reesei was used for cellulase production. The effective variables were initially screened by "Plackett and Burman design." Afterward, the main variables including moisture content, P source, incubation temperature, and incubation time were optimized by "one factor at a time design." Finally, the resulting variables including 74% for moisture content, 2 g/L for K2 HPO 4, 30°C of incubation temperature, and 4 days of incubation time were reported as the ultimate optimal condition for cellulase production.

Journal ArticleDOI
TL;DR: A droplet-based microfluidic high-throughput screening platform for selection of high-cellulase producers from filamentous fungus Trichoderma reesei and two cellulase hyperproducers exhibiting improvements in cellulase activity were isolated from an atmospheric and room-temperature plasma (ARTP)-mutated library.
Abstract: The selection of improved producers among the huge number of variants in mutant libraries is a key issue in filamentous fungi of industrial biotechnology. Here, we developed a droplet-based microfluidic high-throughput screening platform for selection of high-cellulase producers from filamentous fungus Trichoderma reesei. The screening system used a fluorogenic assay to measure amount of cellulase and its activity. The key effectors such as cellulase-inducing medium, spore germination, droplet cultivation time, droplet fluorescence signal detection, and droplet cell sorting were studied. An artificial pre-mixed library of high- and low-cellulase-producing T. reesei strains was screened successfully to verify the feasibility of our method. Finally, two cellulase hyperproducers exhibiting improvements in cellulase activity of 27% and 46% were isolated from an atmospheric and room-temperature plasma (ARTP)-mutated library. This high-throughput screening system could be applied to the engineering of T. reesei strains and other industrially valuable protein-producing filamentous fungi.

Journal ArticleDOI
TL;DR: Eight new dimeric sorbicillinoids and 12 new monomeric sorbICillinoids, along with five known analogues, were isolated from the marine-derived fungus Trichoderma reesei 4670 and their structures were elucidated on the basis of extensive spectroscopic analyses and X-ray crystallography.
Abstract: Eight new dimeric sorbicillinoids (1–3, 5–9) and 12 new monomeric sorbicillinoids (10–20, 25), along with five known analogues (4 and 21–24), were isolated from the marine-derived fungus Trichoderma reesei 4670. Their structures were elucidated on the basis of extensive spectroscopic analyses (1D and 2D NMR, HR-ESIMS, and ECD) and X-ray crystallography. Compound 1, containing a pyrrolidin-2-one moiety, is reported for the first time in the sorbicillinoid family. Compounds 8 and 9 are the first examples of bisorbicillinoids possessing a benzofuro[2,3-h]chromene scaffold from a natural source. Compounds 3–11, 13–16, 18, 21, 22, 24, and 25 exhibited potent anti-inflammatory activity by inhibiting the production of NO in RAW264.7 cells activated by lipopolysaccharide with IC50 values in the range from 0.94 to 38 μM. Structure–activity relationships of the sorbicillinoids were discussed.

Journal ArticleDOI
TL;DR: YPR2 is an important regulator balancing secondary metabolism with carbon metabolism in darkness and depending on the carbon source, which reaches beyond the SOR cluster in which ypr2 is located and happens downstream of carbon catabolite repression mediated by CRE1.
Abstract: Filamentous fungi have evolved to succeed in nature by efficient growth and degradation of substrates, but also due to the production of secondary metabolites including mycotoxins. For Trichoderma reesei, as a biotechnological workhorse for homologous and heterologous protein production, secondary metabolite secretion is of particular importance for industrial application. Recent studies revealed an interconnected regulation of enzyme gene expression and carbon metabolism with secondary metabolism. Here, we investigated gene regulation by YPR2, one out of two transcription factors located within the SOR cluster of T. reesei, which is involved in biosynthesis of sorbicillinoids. Transcriptome analysis showed that YPR2 exerts its major function in constant darkness upon growth on cellulose. Targets (direct and indirect) of YPR2 overlap with induction specific genes as well as with targets of the carbon catabolite repressor CRE1 and a considerable proportion is regulated by photoreceptors as well. Functional category analysis revealed both effects on carbon metabolism and secondary metabolism. Further, we found indications for an involvement of YPR2 in regulation of siderophores. In agreement with transcriptome data, mass spectrometric analyses revealed a broad alteration in metabolite patterns in ∆ypr2. Additionally, YPR2 positively influenced alamethicin levels along with transcript levels of the alamethicin synthase tex1 and is essential for production of orsellinic acid in darkness. YPR2 is an important regulator balancing secondary metabolism with carbon metabolism in darkness and depending on the carbon source. The function of YPR2 reaches beyond the SOR cluster in which ypr2 is located and happens downstream of carbon catabolite repression mediated by CRE1.

Journal ArticleDOI
TL;DR: The results indicate that the production of cellulase from green pea hulls may provide a novel and economical solution for industrial waste disposal.
Abstract: Pea hulls have a significant contribution to agricultural waste and are left unattended. It has a rich nutrient media and may have application in waste utilization. In this investigation, the potential of pea hulls for the production of cellulase has been explored. The effect of H2O2 concentration, agitation speed and harvesting time on cellulase production by Trichoderma reesei QM9414 under submerged fermentation at constant pH of 5.0 and temperature 30 ± 0.1 °C were studied. The maximum filter paper (FP) cellulase activity of 0.372 ± 0.019 U/mL was obtained at 91 h incubation period and 120 rpm agitation speed. Based on the optimized results of fermentation parameters, 120 rpm agitation speed, 5% H2O2 concentration and 91 h harvesting time was recommended for efficient cellulase production. The effect of harvesting time on protein, reducing sugar and cellulase activity was pre-dominant. The purified cellulase enzyme specific activity was recorded as 13.8 U/mL. The results indicate that the production of cellulase from green pea hulls may provide a novel and economical solution for industrial waste disposal.

Journal ArticleDOI
10 Sep 2019
TL;DR: It is concluded that PKAc1 is involved in light dependent regulation of plant cell wall degradation, including carbon catabolite repression as well as secondary metabolism and development in T. reesei.
Abstract: Trichoderma reesei represents a model system for investigation of plant cell wall degradation and its connection to light response. The cyclic adenosine monophosphate pathway (cAMP pathway) plays an important role in both physiological outputs, being crucial for regulation of photoreceptor function as well as for cellulase regulation on different carbon sources. Phosphorylation of photoreceptors and of the carbon catabolite repressor CRE1 was shown in ascomycetes, indicating a relevance of protein kinase A in regulation of the target genes of these transcription factors as well as an impact on regulation of induction specific genes. Moreover, the cAMP pathway impacts growth and development. Here, we investigated gene regulation by the catalytic subunit of protein kinase A (PKAc1) upon growth on cellulose. We found distinct gene sets for regulation upon growth in light and darkness with an overlap of only 13 genes. PKAc1 regulates metabolic genes as well as transport and defense functions. The overlap of gene regulation by PKAc1 with the genes representing the cAMP dependent regulatory output of the photoreceptor ENV1 indicates an involvement of PKA in this pathway, which counteracts its effects by contrasting regulation. Moreover, we found considerable overlap with the gene sets regulated under cellulase inducing conditions and by the carbon catabolite repressor CRE1. Our analysis also showed that PKAc1 regulates the genes of the SOR cluster associated with the biosynthesis of sorbicillinoids. The homologue of gin4, encoding a CAMK type kinase, which is regulated by PKAc1, CRE1 and YPR2 showed a moderate impact on trichodimerol production. We isolated trichodimerol as representative sorbicillin compound and established a method for its quantification in large sample sets using high performance thin layer chromatography (HPTLC), which can be broadly applied for secondary metabolite screening of mutants or different growth conditions. Due to the high expression levels of the SOR cluster under conditions of sexual development we crosschecked the relevance of PKAc1 under these conditions. We could show that PKAc1 impacts biosynthesis of trichodimerol in axenic growth and upon mating. We conclude that PKAc1 is involved in light dependent regulation of plant cell wall degradation, including carbon catabolite repression as well as secondary metabolism and development in T. reesei.

Journal ArticleDOI
TL;DR: The feasibility of improvement of cellulase production is demonstrated by genetically disrupting the potential protease genes to construct the T. reesei strains with low extracellular protease secretion and this dataset provides an efficient approach for strain improvement by precise genetic engineering combined with "omics" strategy for high-production of industrial enzymes to reduce the cost of lignocellulose bioconversion.
Abstract: The filamentous fungus Trichoderma reesei is one of the most studied cellulolytic organisms and the major producer of cellulases for industrial applications. However, undesired degradation of cellulases often happens in culture filtrates and commercial enzyme preparations. Even studies have been reported about describing proteolytic degradation of heterologous proteins in T. reesei, there are few systematic explorations concerning the extracellular proteases responsible for degradation of cellulases. In this study, the cellulase activity was observed to rapidly decrease at late cultivation stages using corn steep liquor (CSL) as the nitrogen source in T. reesei. It was discovered that this decrease may be caused by proteases. To identify the proteases, comparative secretomics was performed to analyze the concomitant proteases during the cellulase production. 12 candidate proteases from the secretome of T. reesei were identified and their encoding genes were individually deleted via homologous recombination. Furthermore, three target proteases (tre81070, tre120998, and tre123234) were simultaneously deleted by one-step genetic transformation. The triple deletion strain ΔP70 showed a 78% decrease in protease activity and a six-fold increase in cellulase activity at late fermentation stages. These results demonstrated the feasibility of improvement of cellulase production by genetically disrupting the potential protease genes to construct the T. reesei strains with low extracellular protease secretion. This dataset also provides an efficient approach for strain improvement by precise genetic engineering combined with "omics" strategy for high-production of industrial enzymes to reduce the cost of lignocellulose bioconversion.

Book ChapterDOI
01 Jan 2019
TL;DR: The effects of environmental conditions, such as carbon and nitrogen sources, light and pH, on cellulase production from filamentous fungi are introduced and the recent progress on the novel regulatory network and the strategies for efficient cellulase induction and production in filamentous fungus are discussed.
Abstract: Lignocellulosic biomass serves as a renewable feedstock for production of biofuels and value-added biochemicals, and biorefinery of lignocellulosic feedstocks contributes greatly to the sustainable development of economy. However, release of fermentable sugars from lignocellulose is suffering from the high cost and low efficiency of lignocellulolytic enzymes. Filamentous fungi, especially Trichoderma reesei, are considered as the most efficient producers of lignocellulose-degrading enzymes, and improvement of enzyme production efficiency and optimization of enzyme composition are of great significance for biorefinery of lignocellulosic biomass. This review introduces the effects of environmental conditions, such as carbon and nitrogen sources, light and pH, on cellulase production from filamentous fungi. In addition, the recent progress on the novel regulatory network and the strategies for efficient cellulase induction and production in filamentous fungi are discussed. Optimization of cellulase ingredients and the effects of accessory enzymes on lignocellulose hydrolysis are also summarized. Efficient production of an optimized cocktail of lignocellulolytic enzymes with improved hydrolysis efficacy will greatly contribute to biofuels production from lignocellulosic biomass.

Journal ArticleDOI
Yanli Cao1, Fanglin Zheng1, Weixin Zhang1, Xiangfeng Meng1, Weifeng Liu1 
TL;DR: A model in which XYR1 recruits SWI/SNF through direct interactions with TrSNF12 to remodel chromatin at cellulase gene promoters, thereby activating cellulase genes expression to initiate the cellulolytic response in T. reesei is suggested.
Abstract: Cellulase gene expression in Trichoderma reesei is highly responsive to environmental cues and is under stringent regulation by multiple transcription factors XYR1 (Xylanase regulator 1) has been identified as the most important transcriptional activator of cellulase/hemicellulase gene expression although the precise transactivating mechanism remains largely elusive Here we show that the activation domain of XYR1 interacts with the T reesei homolog of the TrSNF12 subunit of SWI/SNF complex Deletion of Trsnf12 markedly impaired the induced cellulase gene expression Individual loss of other SWI/SNF subunits including the catalytic subunit also severely compromised cellulase gene expression and interfered with loss of histone H4 in the cbh1 and eg1 promoters upon cellulose induction In addition, we find that the SWI/SNF occupancy on cellulase gene promoters strictly required XYR1 and TrSNF12 but TrSNF12 was dispensable for the XYR1 binding to these promoters These data suggest a model in which XYR1 recruits SWI/SNF through direct interactions with TrSNF12 to remodel chromatin at cellulase gene promoters, thereby activating cellulase gene expression to initiate the cellulolytic response in T reesei

Journal ArticleDOI
TL;DR: The results confirmed the central role of catalytic and regulatory subunits of PP2A which regulates several aspects of cell growth and metabolism and showed that the additional homologue of PPH5 in Trichoderma spp.
Abstract: Trichoderma reesei represents one of the most prolific producers of plant cell wall degrading enzymes. Recent research showed broad regulation by phosphorylation in T. reesei, including important transcription factors involved in cellulase regulation. To evaluate factors crucial for changes in these phosphorylation events, we studied non-essential protein phosphatases (PPs) of T. reesei. Viable deletion strains were tested for growth on different carbon sources, osmotic and oxidative stress response, asexual and sexual development, cellulase and protease production as well as secondary metabolism. Six PPs were found to be positive or negative regulators for cellulase production. A correlation of the effects of PPs on protease activities and cellulase activities was not detected. Hierarchical clustering of regulation patterns and phenotypes of deletion indicated functional specialization within PP classes and common as well as variable effects. Our results confirmed the central role of catalytic and regulatory subunits of PP2A which regulates several aspects of cell growth and metabolism. Moreover we show that the additional homologue of PPH5 in Trichoderma spp., PPH5-2 assumes distinct functions in metabolism, development and stress response, different from PPH5. The influence of PPs on both cellulase gene expression and secondary metabolite production support an interrelationship in the underlying regulation mechanisms.

Journal ArticleDOI
TL;DR: The obtained results suggest that cellulase excretion majorly occurs via the conventional ER–Golgi secretory pathway, and might be assisted through unconventional protein secretion pathways.
Abstract: Filamentous fungi secret hydrolytic enzymes like cellulase and hemicellulase outside the cells, serving as important scavengers of plant biomass in nature and workhorses in the enzyme industry. Unlike the extensive study on the mechanism of cellulase production in fungi, research on spatiotemporal distribution and secretion of cellulase in fungi is lacking, retarding the deeper understanding of the molecular mechanism behind the fungal cellulase production. Recombinant Trichoderma reesei strains RBGL, RCBH, and RCMC were successfully constructed from T. reesei RUT-C30, expressing red fluorescent protein DsRed-tagged versions of β-glucosidase (BGL), cellobiohydrolase (CBH), and endoglucanase (CMC), respectively. With the assistance of these strains, we found that all three cellulase components BGL, CBH, and CMC diffused throughout the whole fungal mycelium with major accumulation at the hyphal apexes. These enzymes located in ER, Golgi, vacuoles and cell membrane/wall, but not septum, and secreted abundantly into the culture medium. Moreover, the major secretion of CBH and CMC started more early than that of BGL. Brefeldin A (BFA) completely blocked cellulase expression and secretion in T. reesei. Based on recombinant T. reesei RBGL, RCBH, and RCMC expressing DsRed-fused versions of BGL, CBH, and CMC, respectively, the distribution and secretion of cellulase production in T. reesei were first visualized directly in a dynamic way, preliminarily mapping the location and secretion of T. reesei cellulase and providing evidence for revealing the secretion pathways of cellulase in T. reesei. The obtained results suggest that cellulase excretion majorly occurs via the conventional ER–Golgi secretory pathway, and might be assisted through unconventional protein secretion pathways.

Journal ArticleDOI
TL;DR: This work has shown that filamentous fungi, including Trichoderma reesei, Aspergilli, and more recently Myceliophthora thermophile are the most widely used and promising ones which have powerful protein secretion capability.
Abstract: Filamentous fungi are important microorganisms used in industrial production of proteins and enzymes. Among these organisms, Trichoderma reesei, Aspergilli, and more recently Myceliophthora thermophile are the most widely used and promising ones which have powerful protein secretion capability. In recent years, there have been tremendous achievements in understanding the molecular mechanisms of the secretory pathways in filamentous fungi. The acquired pieces of knowledge can be harnessed to enhance protein production in filamentous fungi with assistance of state-of-the-art genetic engineering techniques.

Journal ArticleDOI
27 Aug 2019
TL;DR: This work identified a novel transcriptional regulator which plays a positive effect in the expression of cellulase-encoding genes in T. reesei and revealed novel forms of regulatory interaction in this model system for plant biomass deconstruction and also represented a new approach that could be easy applied to other organisms.
Abstract: Filamentous fungi are remarkable producers of enzymes dedicated to the degradation of sugar polymers found in the plant cell wall. Here, we integrated transcriptomic data to identify novel transcription factors (TFs) related to the control of gene expression of lignocellulosic hydrolases in Trichoderma reesei and Aspergillus nidulans. Using various sets of differentially expressed genes, we identified some putative cis-regulatory elements that were related to known binding sites for Saccharomyces cerevisiae TFs. Comparative genomics allowed the identification of six transcriptional factors in filamentous fungi that have corresponding S. cerevisiae homologs. Additionally, a knockout strain of T. reesei lacking one of these TFs (S. cerevisiaeAZF1 homolog) displayed strong reductions in the levels of expression of several cellulase-encoding genes in response to both Avicel and sugarcane bagasse, revealing a new player in the complex regulatory network operating in filamentous fungi during plant biomass degradation. Finally, RNA sequencing (RNA-seq) analysis showed the scope of the AZF1 homologue in regulating a number of processes in T. reesei, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) provided evidence for the direct interaction of this TF in the promoter regions of cel7a, cel45a, and swo. Therefore, we identified here a novel TF which plays a positive effect in the expression of cellulase-encoding genes in T. reesei. IMPORTANCE In this work, we used a systems biology approach to map new regulatory interactions in Trichoderma reesei controlling the expression of genes encoding cellulase and hemicellulase. By integrating transcriptomics related to complex biomass degradation, we were able to identify a novel transcriptional regulator which is able to activate the expression of these genes in response to two different cellulose sources. In vivo experimental validation confirmed the role of this new regulator in several other processes related to carbon source utilization and nutrient transport. Therefore, this work revealed novel forms of regulatory interaction in this model system for plant biomass deconstruction and also represented a new approach that could be easy applied to other organisms.