RESUMO
BACKGROUND: Prolyl endopeptidase from Aspergillus niger (AN-PEP) is a prominent serine proteinase with various potential applications in the food and pharmaceutical industries. However, the availability of efficient and low-cost AN-PEP remains a challenge owing to its low yield and high fermentation cost. RESULTS: Here, AN-PEP was recombinantly expressed in Trichoderma reesei (rAN-PEP) under the control of the cbh1 promoter and its secretion signal. After 4 days of shaking flask cultivation with the model cellulose Avicel PH101 as the sole carbon source, the extracellular prolyl endopeptidase activity reached up to 16.148 U/mL, which is the highest titer reported to date and the secretion of the enzyme is faster in T. reesei than in other eukaryotic expression systems including A. niger and Komagataella phaffii. Most importantly, when cultivated on the low-cost agricultural residue corn cob, the recombinant strain was found to secret a remarkable amount of rAN-PEP (37.125 U/mL) that is twice the activity under the pure cellulose condition. Furthermore, treatment with rAN-PEP during beer brewing lowered the content of gluten below the ELISA kit detection limit (< 10 mg/kg) and thereby, reduced turbidity, which would be beneficial for improving the non-biological stability of beer. CONCLUSION: Our research provides a promising approach for industrial production of AN-PEP and other enzymes (proteins) from renewable lignocellulosic biomass, which provides a new idea with relevant researchers for the utilization of agricultural residues.
Assuntos
Prolil Oligopeptidases , Trichoderma , Prolil Oligopeptidases/metabolismo , Aspergillus niger/metabolismo , Cerveja , Celulose/metabolismo , Fermentação , Trichoderma/metabolismoRESUMO
BACKGROUND: The filamentous fungus Trichoderma reesei has been used as a host organism for the production of lignocellulosic biomass-degrading enzymes. Although this microorganism has high potential for protein production, it has not yet been widely used for heterologous recombinant protein production. Transcriptional induction of the cellulase genes is essential for high-level protein production in T. reesei; however, glucose represses this transcriptional induction. Therefore, cellulose is commonly used as a carbon source for providing its degraded sugars such as cellobiose, which act as inducers to activate the strong promoters of the major cellulase (cellobiohydrolase 1 and 2 (cbh1 and cbh2) genes. However, replacement of cbh1 and/or cbh2 with a gene encoding the protein of interest (POI) for high productivity and occupancy of recombinant proteins remarkably impairs the ability to release soluble inducers from cellulose, consequently reducing the production of POI. To overcome this challenge, we first used an inducer-free biomass-degrading enzyme expression system, previously developed to produce cellulases and hemicellulases using glucose as the sole carbon source, for recombinant protein production using T. reesei. RESULTS: We chose endogenous secretory enzymes and heterologous camelid small antibodies (nanobody) as model proteins. By using the inducer-free strain as a parent, replacement of cbh1 with genes encoding two intrinsic enzymes (aspartic protease and glucoamylase) and three different nanobodies (1ZVH, caplacizumab, and ozoralizumab) resulted in their high secretory productions using glucose medium without inducers such as cellulose. Based on signal sequences (carrier polypeptides) and protease inhibitors, additional replacement of cbh2 with the nanobody gene increased the percentage of POI to about 20% of total secreted proteins in T. reesei. This allowed the production of caplacizumab, a bivalent nanobody, to be increased to 9.49-fold (508 mg/L) compared to the initial inducer-free strain. CONCLUSIONS: In general, whereas the replacement of major cellulase genes leads to extreme decrease in the degradation capacity of cellulose, our inducer-free system enabled it and achieved high secretory production of POI with increased occupancy in glucose medium. This system would be a novel platform for heterologous recombinant protein production in T. reesei.
Assuntos
Celulase , Anticorpos de Domínio Único , Trichoderma , Celulase/genética , Celulase/metabolismo , Glucose/metabolismo , Anticorpos de Domínio Único/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Celulose/metabolismo , Trichoderma/metabolismoRESUMO
Trichoderma is a genus of filamentous fungi widely studied and used as a biological control agent in agriculture. However, its ability to form fungal networks for inter-plant communication by means of the so-called inter-plant "wired communication" has not yet been addressed. In our study we used the model plant Arabidopsis thaliana, the fungus Trichoderma hamatum (isolated from Brassicaceae plants) and the pathogens Sclerotinia sclerotiorum and Xanthomonas campestris (necrotrophic fungus and hemibiotrophic bacteria, respectively). We performed different combinations of isolated/neighboring plants and root colonization/non-colonization by T. hamatum, as well as foliar infections with the pathogens. In this way, we were able to determine how, in the absence of T. hamatum, there is an inter-plant communication that induces systemic resistance in neighboring plants of plants infected by the pathogens. On the other hand, the plants colonized by T. hamatum roots show a greater systemic resistance against the pathogens. Regarding the role of T. hamatum as an inter-plant communicator, it is the result of an increase in foliar signaling by jasmonic acid (increased expression of LOX1 and VSP2 genes and decreased expression of ICS1 and PR-1 genes), antagonistically increasing root signaling by salicylic acid (increased expression of ICS1 and PR-1 genes and decreased expression of LOX1 and VSP2). This situation prevents root colonization by T. hamatum of the foliarly infected plant and leads to massive colonization of the neighboring plant, where jasmonic acid-mediated systemic defenses are induced.
Assuntos
Arabidopsis , Trichoderma , Trichoderma/genética , Trichoderma/metabolismo , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Arabidopsis/genética , Doenças das Plantas/microbiologia , Ácido Salicílico/metabolismoRESUMO
Candida albicans is the main causal pathogen of fungal infections in human beings. Although diverse anti-C. albicans drugs have been explored, the drug resistance and side effects of these drugs are intensifying. Thus, it is urgent to explore new anti-C. albicans compounds from natural products. In this study, we identified trichoderma acid (TA), a compound from Trichoderma spirale with a strong inhibitory effect on C. albicans. Transcriptomic and iTRAQ-based proteomic analyses of TA-treated C. albicans in combination with scanning electronic microscopy and reactive oxygen species (ROS) detection were performed to investigate the potential targets of TA. The most significant differentially expressed genes and proteins after TA treatment were verified through Western blot analysis. Our results revealed that mitochondrial membrane potential, endoplasmic reticulum, ribosomes in the mitochondria, and cell walls were disrupted in TA-treated C. albicans, leading to the accumulation of ROS. The impaired enzymatic activities of superoxide dismutase further contributed to the increase in ROS concentration. The high concentration of ROS led to DNA damage and cell skeleton destruction. The expression levels of Rho-related GTP-binding protein RhoE (RND3), asparagine synthetase (ASNS), glutathione S-transferase, and heat shock protein 70 were significantly up-regulated in response to apoptosis and toxin stimulation. These findings suggest that RND3, ASNS, and supereoxide dismutase 5 are the potential targets of TA, as further demonstrated through Western blot analysis. The combination of transcriptomic, proteomic, and cellular analyses would provide clues for the anti-C. albicans mechanism of TA and the defensive response mechanism of C. albicans. TA is thus recognized as a promising new anti-C. albicans leading compound that alleviates the hazard of C. albicans infection in human beings.
Assuntos
Candida albicans , Trichoderma , Humanos , Antifúngicos/farmacologia , Antifúngicos/metabolismo , Trichoderma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteômica , Testes de Sensibilidade MicrobianaRESUMO
In the large field of bioactive peptides, peptaibols represent a unique class of compounds. They are membrane-active peptides, produced by fungi of the genus Trichoderma and known to elicit plant defenses. Among the short-length peptaibols, trichogin GA IV is nonhemolytic, proteolysis-resistant, antibacterial, and cytotoxic. Several trichogin analogs are endowed with potent activity against phytopathogens, thus representing a sustainable alternative to copper for plant protection. In this work, we tested the activity of trichogin analogs against a breast cancer cell line and a normal cell line of the same derivation. Lys-containing trichogins showed an IC50 below 12 µM, a peptide concentration not significantly affecting the viability of normal cells. Two analogs were found to be membrane-active but noncytotoxic. They were anchored to gold nanoparticles (GNPs) and further investigated for their ability to act as targeting agents. GNP uptake by cancer cells increased with peptide decoration, while it decreased in the corresponding normal epithelial cells. This work highlights the promising biological properties of peptaibol analogs in the field of cancer therapy either as cytotoxic molecules or as active targeting agents in drug delivery.
Assuntos
Hypocreales , Nanopartículas Metálicas , Trichoderma , Ouro/farmacologia , Ouro/metabolismo , Peptaibols/farmacologia , Peptaibols/metabolismo , Hypocreales/metabolismo , Trichoderma/metabolismoRESUMO
In this study, bacterial BsEXLE1 gene was overexpressed into T. reesei (Rut-C30) to generate a desirable engineered TrEXLX10 strain. While incubated with alkali-pretreated Miscanthus straw as carbon source, the TrEXLX10 secreted the ß-glucosidases, cellobiohydrolases and xylanses with activities raised by 34%, 82% and 159% compared to the Rut-C30. Supplying EXLX10-secreted crude enzymes and commercial mixed-cellulases for two-step lignocellulose hydrolyses of corn and Miscanthus straws after mild alkali pretreatments, this work measured consistently higher hexoses yields released by the EXLX10-secreted enzymes for synergistic enhancements of biomass saccharification in all parallel experiments examined. Meanwhile, this study detected that the expansin, purified from EXLX10-secreted solution, was of exceptionally high binding activities with wall polymers, and further determined its independent enhancement for cellulose hydrolysis. Therefore, this study raised a mechanism model to highlight EXLX/expansin dual-activation roles for both secretion of stable biomass-degradation enzymes at high activity and biomass enzymatic saccharification in bioenergy crops.
Assuntos
Celulases , Trichoderma , Celulases/metabolismo , Zea mays/metabolismo , Biomassa , Poaceae/metabolismo , Trichoderma/metabolismo , HidróliseRESUMO
Increasing cellulase production in cellulolytic fungus Trichoderma reesei is of interest for biofuels and biorefineries. Previous studies indicated that secreted protein was occasionally accumulated in vacuoles; this phenomenon has also been reported in T. reesei. Therefore, alleviating vacuolar transport seems to be a promising strategy for improving cellulase production in T. reesei. Herein, we found that knockout of vps10, vps13, and vps21, among 11 vacuolar protein sorting factors, improved cellulase production in T. reesei. The filter paper activity in Δvps10, Δvps13, and Δvps21 increased by 1.28-, 2.45-, and 2.11-fold than that of the parent strain. Moreover, the ß-glucosidase activity in Δvps13 and Δvps21 increased by 3.22- and 3.56-fold after 6 days of fermentation. Furthermore, we also found that the vacuolar trafficking towards vacuoles was partially impaired in three knockout mutants, and disruption of vps13 alleviated the autophagy process. These results indicated that alleviated transport and degradation towards vacuole in Δvps10, Δvps13, and Δvps21 might improve cellulase production. Of note, the expression of cellulase genes in Δvps13 and Δvps21 was dramatically increased in the late induction phase compared to the parent. These results suggested that Vps13 and Vps21 might influence the cellulase production at transcription level. And further transcriptome analysis indicated that increased cellulase gene expression might be attributed to the differential expression of sugar transporters. Our study unravels the effect of alleviating vacuolar transport through knockout vps10, vps13, and vps21 for efficient cellulase secretion, providing new clues for higher cellulase production in T. reesei. KEY POINTS: ⢠Disruption of vps10, vps13 or vps21 improves cellulase production ⢠Vacuolar transport is impaired in three vps KO mutants ⢠Deletion of vps13 or vps21 increases the transcript of cellulase genes in late stage.
Assuntos
Celulase , Hypocreales , Trichoderma , Celulase/genética , Celulase/metabolismo , Trichoderma/genética , Trichoderma/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hypocreales/metabolismoRESUMO
Industrialization and human urbanization have led to an increase in heavy metal (HM) pollution which often cause negative/toxic effect on agricultural crops. The soil-HMs cannot be degraded biologically however, microbe-mediated detoxification of toxic HMs into lesser toxic forms are reported. Considering the potentiality of HMs-tolerant soil microbes in metal detoxification, Pseudomonas fluorescence PGPR-7 and Trichoderma sp. T-4 were recovered from HM-affected areas. Under both normal and cadmium stress, the ability of both microorganisms to produce different plant hormones and biologically active enzymes was examined. Strains PGPR-7 and T-4 tolerated cadmium (Cd) an up-to 1800 and 2000 µg mL-1, respectively, and produced various plant growth regulating substances (IAA, siderophore, ACC deaminase ammonia and HCN) in Cd-stressed condition. The growth promoting and metal detoxifying ability of both strains were evaluated (either singly/combined) by applying them in chickpea (Cicer arietinum L.) plants endogenously contaminated with different Cd levels (0-400 µg kg-1 soils). The higher Cd concentration (400 µg kg-1 soils) negatively influenced the plant parameters which, however, improved following single/combined inoculation of P. fluorescence PGPR-7 and Trichoderma sp. T-4. Both microbial strains increased the growth of Cd-treated chickpeas however, their combined inoculation (PGPR-7 + T-4) caused the most positive effect. For instance, 25 µg Cd Kg-1 + PGPR-7 + T4 treatment caused maximum increase in germination percentage (10%), root dry biomass (71.4%) and vigour index (33%), chl-a (38%), chl-b (41%) and carotenoid content (52%). Furthermore, combined inoculation of P. fluorescence PGPR-7 and Trichoderma sp. T-4 maximally decreased the proline, MDA content, POD and CAT activities by 50%, 43% and 62%, respectively following their application in 25 µg Cd kg-1 soils-treated chickpea. Additionally, microbial strains lowered the plant uptake of Cd. For example, Cd-uptake in root tissues was decreased by 42 and 34% when 25 µg Cd Kg-1- treated chickpea plants were inoculated with P. fluorescence PGPR-7, Trichoderma sp. T-4 and co-inoculation (PGPR-7 + T4) of both strains, respectively. Therefore, from the current observation, it is suggested that dual inoculation of metal tolerant P. fluorescence and Trichoderma sp. may potentially be used in detoxification and reclamation of metal-contaminated soils.
Assuntos
Cicer , Metais Pesados , Poluentes do Solo , Trichoderma , Humanos , Cádmio/metabolismo , Pseudomonas/metabolismo , Trichoderma/metabolismo , Sideróforos/metabolismo , Fluorescência , Metais Pesados/metabolismo , Solo/química , Poluentes do Solo/metabolismo , Raízes de Plantas/metabolismo , Biodegradação AmbientalRESUMO
In this study, the agricultural digestate from anaerobic biogas production mixed with food wastes was used as a substrate to grow Trichoderma reesei RUT-C30 and Trichoderma atroviride Ta13 in solid-state fermentation (SSF) and produce high-value bioproducts, such as bioactive molecules to be used as ingredients for biostimulants. The Trichoderma spp. reached their maximum growth after 6 and 3 SSF days, respectively. Both Trichoderma species were able to produce cellulase, esterase, and citric and malic acids, while T. atroviride also produced gibberellins and oxylipins as shown by ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) profiling. Experimental evaluation of germination parameters highlighted a significant promotion of tomato seed germination and root elongation induced by T. atroviride crude extracts from SSF. This study suggests an innovative sustainable use of the whole digestate mixed with agro-food waste as a valuable substrate in fungal biorefineries. Here, it has been applied to produce plant growth-promoting fungi and bioactive molecules for sustainable agriculture.
Assuntos
Celulase , Eliminação de Resíduos , Trichoderma , Fermentação , Trichoderma/metabolismo , Alimentos , Celulase/químicaRESUMO
Trichoderma reesei (T. reesei) is well-known for its excellent ability to secret a large quantity of cellulase. However, unlike the endogenous proteins, little is known about the molecular mechanisms governing heterologous protein production. Herein, we focused on the integration loci and the secretory pathway, and investigated their combinatorial effects on heterologous gene expression in T. reesei using a glucose oxidase from Aspergillus niger as a model protein. Integration in the cel3c locus was more efficient than the cbh1 locus in expressing the AnGOx by increasing the transcription of AnGOx in the early stage. In addition, we discovered that interruption of the cel3c locus has an additional effect by increasing the expression of the secretory pathway component genes. Accordingly, overexpressing three secretory pathway component genes, that were snc1, sso2, and rho3, increased AnGOx expression in the cbh1 transformant but not in the cel3c transformant.
Assuntos
Celulase , Trichoderma , Aspergillus niger/genética , Proteínas Fúngicas/metabolismo , Glucose Oxidase/genética , Glucose Oxidase/metabolismo , Via Secretória , Trichoderma/metabolismo , Celulase/metabolismoRESUMO
This study was aimed at exploring the effect of antagonism of Trichoderma reesei (T.r) and Phanerochaete chrysosporium (P.c) on humification during fermentation of rice (RS) and canola straw (CS). Results showed that exogeneous fungi accelerated straw degradation and enzyme activities of CMCase, xylanase and LiP. P.c inhibited the activity of LiP when co-existing with T.r beginning, it promoted the degradation of lignin and further increased the production of humus-like substances (HLS) and humic-like acid (HLA) in later fermentation when nutrients were insufficient. The HLS of RTP was 54.9 g/kg RS, higher than the other treatments, and displayed more complex structure and higher thermostability. Brucella and Bacillus were the main HLA bacterial producers. P.c was the HLA fungal producer, while T.r assisted FLA and polyphenol transformation. Therefore, RTP was recommended to advance technologies converting crop straw into humus resources.
Assuntos
Phanerochaete , Trichoderma , Phanerochaete/metabolismo , Solo , Antibiose , Lignina/metabolismo , Trichoderma/metabolismoRESUMO
In this study, biodelignification and enzymatic hydrolysis of elephant grass were performed by recombinant and native strain of Trichoderma reesei, respectively. Initially, rT. reesei displaying Lip8H and MnP1 gene was used for biodelignification with NiO nanoparticles. Saccharification was performed by combining hydrolytic enzyme produced with NiO nanoparticles. Elephant grass hydrolysate was used for bioethanol production using Kluyveromyces marxianus. Maximum lignolytic enzyme production was obtained with 15 µg/L of NiO nanoparticles and initial pH of 5 at 32 °C. Subsequently, about 54% of lignin degradation was achieved after 192 h. Hydrolytic enzymes showed elevated enzyme activity and resulted in 84.52 ± 3.5 g/L of total reducing sugar at 15 µg/mL NiO NPs. About 14.65 ± 1.75 g/L of ethanol was produced using K. marxianus after 24 h. Thus, dual strategy employed for conversion of elephant grass biomass into fermentable sugar and subsequent biofuel production could become potential platform for commercialization.
Assuntos
Açúcares , Trichoderma , Açúcares/metabolismo , Biomassa , Fermentação , Carboidratos , Hidrólise , Trichoderma/metabolismoRESUMO
The filamentous fungus Trichoderma reesei is a prolific producer of plant cell wall degrading enzymes, which are regulated in response to diverse environmental signals for optimal adaptation, but also produces a wide array of secondary metabolites. Available carbon source and light are the strongest cues currently known to impact secreted enzyme levels and an interplay with regulation of secondary metabolism became increasingly obvious in recent years. While cellulase regulation is already known to be modulated by different mitogen activated protein kinase (MAPK) pathways, the relevance of the light signal, which is transmitted by this pathway in other fungi as well, is still unknown in T. reesei as are interconnections to secondary metabolism and chemical communication under mating conditions. Here we show that MAPkinases differentially influence cellulase regulation in light and darkness and that the Hog1 homologue TMK3, but not TMK1 or TMK2 are required for the chemotropic response to glucose in T. reesei. Additionally, MAPkinases regulate production of specific secondary metabolites including trichodimerol and bisorbibutenolid, a bioactive compound with cytostatic effect on cancer cells and deterrent effect on larvae, under conditions facilitating mating, which reflects a defect in chemical communication. Strains lacking either of the MAPkinases become female sterile, indicating the conservation of the role of MAPkinases in sexual fertility also in T. reesei. In summary, our findings substantiate the previously detected interconnection of cellulase regulation with regulation of secondary metabolism as well as the involvement of MAPkinases in light dependent gene regulation of cellulase and secondary metabolite genes in fungi.
Assuntos
Celulase , Trichoderma , Celulase/metabolismo , Metabolismo Secundário , Trichoderma/metabolismo , Desenvolvimento Sexual , Regulação Fúngica da Expressão Gênica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
l-Malic acid has various applications in the chemical and food industries. The filamentous fungus Trichoderma reesei is known to be an efficient enzyme producer. Here, through metabolic engineering, T. reesei was constructed for the first time as an excellent cell factory for l-malic acid production. The heterologous overexpression of genes encoding the C4-dicarboxylate transporter from Aspergillus oryzae and Schizosaccharomyces pombe initiated l-malic acid production. The overexpression of pyruvate carboxylase from A. oryzae in the reductive tricarboxylic acid pathway further increased both the titer and yield of l-malic acid, resulting in the highest titer reported in a shake-flask culture. Furthermore, the deletion of malate thiokinase blocked l-malic acid degradation. Finally, the engineered T. reesei strain produced 220.5 g/L of l-malic acid in a 5 L fed-batch culture (productivity of 1.15 g/L/h). A T. reesei cell factory was created for the efficient production of l-malic acid.
Assuntos
Hypocreales , Trichoderma , Engenharia Metabólica/métodos , Malatos/metabolismo , Hypocreales/metabolismo , Trichoderma/genética , Trichoderma/metabolismoRESUMO
Although extensive research efforts have been dedicated to characterizing the laccase from white-rot fungus, little information is available on laccases from Trichoderma spp. A copper-tolerant strain with the ability to produce laccase was isolated and identified as Trichoderma asperellum Ts93. Under optimized conditions, the maximum laccase activity was 1.96 U/ml. The genome-wide survey of Ts93 revealed the presence of seven putative laccase genes that all contained the conserved domain and were divided into three different phylogenetic groups. Among these genes, three contained the four copper-binding conserved regions. The expression profiles acquired through real-time quantitative PCR analysis showed that five of the seven genes were significantly upregulated in response to laccase activity. Seven laccase genes in T. asperellum were identified for the first time by whole-genome sequencing followed by phylogenetic analysis. The findings of this work provide valuable information for the functional analysis of laccase genes in Trichoderma spp.
Assuntos
Hypocreales , Trichoderma , Lacase/química , Cobre/metabolismo , Filogenia , Hypocreales/metabolismo , Trichoderma/genética , Trichoderma/metabolismoRESUMO
The filamentous fungus Trichoderma reesei is one of the most prolific cellulase producers and has been established as a model microorganism for investigating mechanisms modulating eukaryotic gene expression. Identification and functional characterization of transcriptional regulators involved in complex and stringent regulation of cellulase genes are, however, not yet complete. Here, a Zn(II)2Cys6-type transcriptional factor TAM1 that is homologous to Aspergillus nidulans TamA involved in nitrogen metabolism, was found not only to regulate ammonium utilization but also to control cellulase gene expression in T. reesei. Whereas Δtam1 cultivated with peptone as a nitrogen source did not exhibit a growth defect that was observed on ammonium, it was still significantly compromised in cellulase biosynthesis. The absence of TAM1 almost fully abrogated the rapid cellulase gene induction in a resting-cell-inducing system. Overexpression of gdh1 encoding the key ammonium assimilatory enzyme in Δtam1 rescued the growth defect on ammonium but not the defect in cellulase gene expression. Of note, mutation of the Zn(II)2Cys6 DNA-binding motif of TAM1 hardly affected cellulase gene expression, while a truncated ARE1 mutant lacking the C-terminal 12 amino acids that are required for the interaction with TAM1 interfered with cellulase biosynthesis. The defect in cellulase induction of Δtam1 was rescued by overexpression of the key transactivator for cellulase gene, XYR1. Our results thus identify a nitrogen metabolism regulator as a new modulator participating in the regulation of induced cellulase gene expression. IMPORTANCE Transcriptional regulators are able to integrate extracellular nutrient signals and exert a combinatorial control over various metabolic genes. A plethora of such factors therefore constitute a complex regulatory network ensuring rapid and accurate cellular response to acquire and utilize nutrients. Despite the in-depth mechanistic studies of functions of the Zn(II)2Cys6-type transcriptional regulator TamA and its orthologues in nitrogen utilization, their involvement in additional physiological processes remains unknown. In this study, we demonstrated that TAM1 exerts a dual regulatory role in mediating ammonium utilization and induced cellulase production in the well known cellulolytic fungus Trichoderma reesei, suggesting a potentially converged regulatory node between nitrogen utilization and cellulase biosynthesis. This study not only contributes to unveiling the intricate regulatory network underlying cellulase gene expression in cellulolytic fungus but also helps expand our knowledge of fungal strategies to achieve efficient and coordinated nutrient acquisition for rapid propagation.
Assuntos
Celulase , Hypocreales , Trichoderma , Celulase/genética , Celulase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Hypocreales/genética , Expressão Gênica , Trichoderma/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
The laccases from white-rot fungi exhibit high redox potential in treating phenolic compounds. However, their application in commercial purposes has been limited because of the relatively low productivity of the native hosts. Here, the laccase A-encoding gene lacA of Trametes sp. AH28-2 was overexpressed under the control of the strong promoter of cbh1 (Pcbh1), the gene encoding the endogenous cellobiohydrolase 1 (CBH1), in the industrial workhorse fungus Trichoderma reesei. Firstly, the lacA expression cassette was randomly integrated into the T. reesei chromosome by genetic transformation. The lacA gene was successfully transcribed, but the laccase couldn't be detected in the liquid fermentation condition. Meanwhile, it was found that the endoplasmic reticulum-associated degradation (ERAD) was strongly activated, indicating that the expression of LacA probably triggered intense endoplasmic reticulum (ER) stress. Subsequently, the lacA expression cassette was added with the downstream region of cbh1 (Tcbh1) to construct the new expression cassette lacA::Δcbh1, which could replace the cbh1 locus in the genome via homologous recombination. After genetic transformation, the lacA gene was integrated into the cbh1 locus and transcribed. And the unfolded protein response (UPR) and ERAD were only slightly induced, for which the loss of endogenous cellulase CBH1 released the pressure of secretion. Finally, the maximum laccase activity of 168.3 U/l was obtained in the fermentation broth. These results demonstrated that the reduction of secretion pressure by deletion of endogenous protein-encoding genes would be an efficient strategy for the secretion of heterologous target proteins in industrial fungi. ONE-SENTENCE SUMMARY: The reduction of the secretion pressure by deletion of the endogenous cbh1 gene can contribute to heterologous expression of the laccase (LacA) from Trametes sp. AH28-2 in Trichoderma reesei.
Assuntos
Celulase , Trichoderma , Trametes/genética , Lacase/genética , Lacase/metabolismo , Celulose 1,4-beta-Celobiosidase/metabolismo , Celulase/genética , Celulase/metabolismo , Degradação Associada com o Retículo Endoplasmático , Trichoderma/genética , Trichoderma/metabolismoRESUMO
Advances in biocontrol potentials and fungicide resistance are highly desirable for Trichoderma. Thus, it is profitable to use mutagenic agents to develop superior strains with enhanced biocontrol properties and fungicide tolerance in Trichoderma. This study investigates the N-methyl-n-nitro-N-nitrosoguanidine (NTG) (100 mg/L) induced mutants of Trichoderma asperellum. Six NTG (3 each from 1st & 2nd round) induced mutants were developed and evaluated their biocontrol activities and carbendazim tolerance. Among the mutant N2-3, N2-1, N1 and N2-2 gave the best antagonistic and volatile metabolite activities on inhibition of chickpea F. oxysporum f. sp. ciceri, B. cinerea and R. bataticola mycelium under in vitro condition. Mutant N2-2 (5626.40 µg/ml) showed the highest EC50 value against carbendazim followed by N2-3 (206.36 µg/ml) and N2-1 (16.41 µg/ml); and succeeded to sporulate even at 2000 µg/ml of carbendazim. The biocontrol activity of N2-2 and N2 with half-dose of carbendazim was evaluated on chickpea dry root rot under controlled environment. Disease reduction and progress of the dry root rot was extremely low in T7 (N2-2 + with half-dose of carbendazim) treatment. Further, carbendazim resistant mutants demonstrated mutation in tub2 gene of ß-tubulin family which was suggested through the 37 and 183 residue changes in the superimposed protein structures encoded by tub2 gene in N2 and N2-2 with WT respectively. This study conclusively implies that the enhanced carbendazim tolerance in N2-2 mutant did not affect the mycoparasitism and plant growth activity of Trichoderma. These mutants were as good as the wild-type with respect to all inherent attributes.
Assuntos
Cicer , Fungicidas Industriais , Trichoderma , Fungicidas Industriais/farmacologia , Cicer/genética , Melhoramento Genético , Antibiose , Trichoderma/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/prevenção & controleRESUMO
Beneficial interactions between plant roots and Trichoderma species lead to both local and systemic enhancements of the plant immune system through a mechanism known as priming of defenses. Previously, we have reported a number of genes and proteins that are differentially regulated in distant tissues of maize plants following inoculation with Trichoderma atroviride. To further investigate the mechanisms involved in the systemic activation of plant responses, here we have further evaluated the regulatory aspects of a selected group of genes when priming is triggered in maize plants. Time-course experiments from the beginning of the interaction between T. atroviride and maize roots followed by leaf infection with Colletotrichum graminicola allowed us to identify a gene set regulated by priming in the leaf tissue. In the same experiment, phytohormone measurements revealed a decrease in jasmonic acid concentration while salicylic acid increased at 2 d and 6 d post-inoculation. In addition, chromatin structure and modification assays showed that chromatin was more open in the primed state compared with unprimed control conditions, and this allowed for quicker gene activation in response to pathogen attack. Overall, the results allowed us to gain insights on the interplay between the phytohormones and epigenetic regulatory events in the systemic and long-lasting regulation of maize plant defenses following Trichoderma inoculation.
Assuntos
Trichoderma , Zea mays , Zea mays/genética , Zea mays/metabolismo , Trichoderma/genética , Trichoderma/metabolismo , Ácido Salicílico/metabolismo , Folhas de Planta/metabolismo , Doenças das Plantas/genética , Raízes de Plantas/metabolismoRESUMO
To reduce the high cost of (hemi)cellulase production in lignocellulose biorefining, it is important to develop strategies to enhance enzyme productivity from economic and also readily manipulatable carbon sources. In this study, an artificial transcription factor XT was designed by fusing the DNA binding domain of Xyr1 to the transactivation domain of Tmac1. When overexpressed in Trichoderma reesei QM9414 Δxyr1, the XT recombinant strain (OEXT) greatly improved (hemi)cellulase production on repressing glucose compared with QM9414 on Avicel with 1.7- and 8.2-fold increases in pNPCase and xylanase activity, respectively. Both activities were even higher (0.9- and 33.8-fold higher, respectively) than the recombinant strain similarly overexpressing Xyr1. The dramatically enhanced xylanase activities in OEXT resulted from the elevated expression of various hemicellulases in the secretome. Moreover, the enzyme cocktail from OEXT improved the saccharification efficiency toward corn stover by 60% compared with enzymes from QM9414 with equal volume loading.
