scholarly journals Inducer-Free Cellulase Production System Based on the Constitutive Expression of Mutated Xyr1 and Ace3 in the Industrial Fungus Trichoderma Reesei

2021 ◽  
Author(s):  
Toshiharu Arai ◽  
Sakurako Ichinose ◽  
Nozomu Shibata ◽  
Hiroshi Kakeshita ◽  
Hiroshi Kodama ◽  
...  
Keyword(s):  
Carbon Source ◽  
Trichoderma Reesei ◽  
Production System ◽  
Cellulose Degradation ◽  
Soluble Sugars ◽  
Constitutive Expression ◽  
Fold Increase ◽  
Production Method ◽  
Cellulase Production ◽  
Saccharification Efficiency

Abstract Background: Trichoderma reesei (Hypocrea jecorina) is a filamentous fungus that can produce extremely high levels of protein; consequently, it is utilized as a host for the production of cellulase and hemicellulase cocktails for lignocellulosic biomass degradation. Several hyper-producer strains of T. reesei have been bred for use in industrial production, but they generally require inducers to achieve high production capacities. The most commonly used inducers are soluble sugars produced by the degradation of cellulose; however, the dependence on cellulose degradation is problematic because cellulose is insoluble and has poor handling properties as a carbon source. Furthermore, once cellulose is decomposed, little cellulase is produced, making it difficult to produce the enzyme continuously and efficiently. The aim of this study was to establish a simple, inducer-free, cellulase production system using glucose as the sole carbon source.Results: Here, we focused on transcription factors that regulate both cellulase and hemicellulase genes. First, we verified that the previously reported Xylanase regulator 1 (Xyr1) mutation had a glucose-blind phenotype in T. reesei, and confirmed that constitutive expression of the V821F mutation in Xyr1 produced high levels of proteins, especially hemicellulase and cellulase, even in inducer-free conditions. However, the majority of proteins were hemicellulases. To reproduce cellulase/hemicellulase production similar to those observed under induced conditions, an activator of cellulase expression 3 (Ace3) was expressed in Xyr1V821F expressed strain additionally. As a result, the T. reesei strain constitutively expressing Xyr1V821F and Ace3 exhibited a 1.5-fold increase than Xyr1V821F expressed only in protein productivity under inducer-free conditions. Notably, the enzyme composition significantly improved for cellulases ratio and similar to that induced by cellulose. Furthermore, the enzymes exhibited a high saccharification efficiency when compared to that of produced by the strain expressing only the mutated Xyr1.Conclusions: This work shows that the constitutive expression of mutated Xyr1 and Ace3 can increase cellulase and hemicellulase production in T. reesei without inducers. This inducer-free enzyme production method could provide an effective system to reduce costs and simplify production processes, and is expected to be applied in the production of various proteins.

scholarly journals Use of fusion transcription factors to reprogram cellulase transcription and enable efficient cellulase production in Trichoderma reesei

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Fangzhong Wang ◽  
Ruiqin Zhang ◽  
Lijuan Han ◽  
Wei Guo ◽  
Zhiqiang Du ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Trichoderma Reesei ◽  
Parent Strain ◽  
Fungal Growth ◽  
Biomass Accumulation ◽  
Hyphal Growth ◽  
Fold Increase ◽  
Cellulase Production ◽  
Fungal Species ◽  
New Strategy

Abstract Background Trichoderma reesei is widely used for cellulase production and accepted as an example for cellulase research. Cre1-mediated carbon catabolite repression (CCR) can significantly inhibit the transcription of cellulase genes during cellulase fermentation in T. reesei. Early efforts have been undertaken to modify Cre1 for the release of CCR; however, this approach leads to arrested hyphal growth and decreased biomass accumulation, which negatively affects cellulase production. Results In this study, novel fusion transcription factors (fTFs) were designed to release or attenuate CCR inhibition in cellulase transcription, while Cre1 was left intact to maintain normal hyphal growth. Four designed fTFs were introduced into the T. reesei genome, which generated several transformants, named Kuace3, Kuclr2, Kuace2, and Kuxyr1. No obvious differences in growth were observed between the parent and transformant strains. However, the transcription levels of cel7a, a major cellulase gene, were significantly elevated in all the transformants, particularly in Kuace2 and Kuxyr1, when grown on lactose as a carbon source. This suggested that CCR inhibition was released or attenuated in the transformant strains. The growth of Kuace2 and Kuxyr1 was approximately equivalent to that of the parent strain in fed-batch fermentation process. However, we observed a 3.2- and 2.1-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of the parent strain. Moreover, we observed a 6.1- and 3.9-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of Δcre1 strain. Conclusions A new strategy based on fTFs was successfully established in T. reesei to improve cellulase titers without impairing fungal growth. This study will be valuable for lignocellulosic biorefining and for guiding the development of engineering strategies for producing other important biochemical compounds in fungal species.

scholarly journals Understanding the Role of Trichoderma reesei Vib1 in Gene Expression during Cellulose Degradation

2021 ◽  
Vol 7 (8) ◽  
pp. 613
Author(s):  
Xiuzhen Chen ◽  
Bingran Song ◽  
Minglu Liu ◽  
Lina Qin ◽  
Zhiyang Dong
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Trichoderma Reesei ◽  
Cellulose Degradation ◽  
Strain Improvement ◽  
Cellulase Production ◽  
Oxidoreductase Activity ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Almost All

Vib1, a member of the Ndt80/PhoG-like transcription factor family, has been shown to be essential for cellulase production of Trichoderma reesei. Here, we combined transcriptomic and genetic analyses to gain mechanistic insights into the roles of Vib1 during cellulose degradation. Our transcriptome analysis showed that the vib1 deletion caused 586 genes with decreased expression and 431 genes with increased expression on cellulose. The downregulated genes were enriched for Gene Ontology terms associated with carbohydrate metabolism, transmembrane transport, oxidoreductase activity, and transcription factor activity. Of the 258 genes induced by cellulose, 229 showed no or decreased expression in Δvib1 on cellulose, including almost all (hemi)cellulase genes, crucial sugar transporter genes (IDs:69957, 3405), and the genes encoding main transcriptional activators Xyr1 and Ace3. Additionally, Vib1 also regulated the expression of genes involved in secondary metabolism. Further comparison of the transcriptomes of Δvib1 and Δxyr1 in cellulose revealed that the genes regulated by Vib1 had much overlap with Xyr1 targets especially for the gene set induced by cellulose, presumably whose expression requires the cooperativity between Vib1 and Xyr1. Genetic evidence indicated that Vib1 regulates cellulase gene expression partially via Xyr1. Our results will provide new clues for strain improvement.

scholarly journals Deletion of the small GTPase rac1 in Trichoderma reesei provokes hyperbranching and impacts growth and cellulase production

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Elisabeth Fitz ◽  
Christian Gamauf ◽  
Bernhard Seiboth ◽  
Franziska Wanka
Keyword(s):  
Carbon Source ◽  
Trichoderma Reesei ◽  
Protein Secretion ◽  
Parental Strain ◽  
Rho Gtpase ◽  
Strain Engineering ◽  
Cellulase Production ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Biomass Formation

Abstract Background Trichoderma reesei is widely known for its enormous protein secretion capacity and as an industrially relevant producer of cellulases and hemicellulases. Over the last decades, rational strain engineering was applied to further enhance homologous and heterologous enzyme yields. The introduction of hyperbranching is believed to increase protein secretion, since most exocytosis is located at the hyphal apical tip. There are several genetic modifications which can cause hyperbranching, for example the deletion of the small Rho GTPase rac. Rac plays a crucial role in actin dynamics and is involved in polarisation of the cell during germination and apical extension of the hyphae. Results We deleted rac1 in a T. reesei strain with an ectopically overexpressed endoglucanase, CEL12A, under Pcdna1 control. This deletion provoked a hyperbranching phenotype and strong apolar growth during germination and in mature hyphae. The strains displayed dichotomous branching and shorter total mycelium length with a larger hyphal diameter. Δrac1 strains exhibited a decreased radial growth on solid media. Biomass formation in liquid cultures was carbon source dependent; similar to the reference strain during growth on lactose, increased on d-glucose and slightly enhanced on cellulose. While extracellular cellulase activities remained at parental strain levels on d-glucose and cellulose, the specific activity on lactose cultures was increased up to three times at 72 h accompanied by an upregulation of transcription of the main cellulases. Although the morphology of the Δrac1 strains was considerably altered, the viscosity of the culture broth in fed-batch cultivations were not significantly different in comparison to the parental strain. Conclusions Deletion of the small Rho GTPase rac1 changes the morphology of the hyphae and provokes hyperbranching without affecting viscosity, independent of the carbon source. In contrast, biomass formation and cellulase production are altered in a carbon source dependent manner in the Δrac1 strains.

scholarly journals Development of a Powerful Synthetic Hybrid Promoter to Improve the Cellulase System of Trichoderma Reesei for Efficient Saccharification of Corncob Residues

2021 ◽  
Author(s):  
Yifan Wang ◽  
Ruiyan Liu ◽  
Hong Liu ◽  
Xihai Li ◽  
Linjing Shen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Trichoderma Reesei ◽  
Cellulase Activity ◽  
Inducible Promoter ◽  
Cellulase Production ◽  
Cellulolytic Enzymes ◽  
Cellulolytic Enzyme ◽  
Activity Increase ◽  
Hybrid Promoter ◽  
Saccharification Efficiency

Abstract Background: The filamentous fungus Trichoderma reesei is a widely used workhorse for cellulase production in industry due to its prominent secretion capacity of extracellular cellulolytic enzymes. However, some key components are not always sufficient in this cellulase cocktail, making the conversion of cellulose-based biomass costly on the industrial scale. Development of strong and efficient promoters would enable cellulase cocktail to be optimized for bioconversion of biomass.Results: In this study, a synthetic hybrid promoter was constructed and applied to optimize the cellulolytic system of T. reesei for efficient saccharification towards corncob residues. Firstly, a promoter library was established by sequence truncation based on the strong constitutive promoter Pcdna1. The strongest promoter amongst them was Pcdna1-3, exhibiting a 1.4-fold higher activity than that of the native cdna1 promoter. Meanwhile, the activation region (-821 to -622 bp upstream of the translation initiation codon ATG and devoid of the Cre1-binding sites) of the strong inducible promoter Pcbh1 was cloned and identified to be an amplifier in initiating gene expression. Finally, this activation region was fused to the strongest promoter Pcdna1-3, generating the novel synthetic hybrid promoter Pcc. This engineered promoter Pcc drove strong gene expression by displaying 1.6- and 1.8-fold stronger fluorescence intensity than Pcbh1 and Pcdna1 under the inducible condition using egfp as the reporter gene, respectively. Furthermore, Pcc was applied to overexpress the Aspergillus niger β-glucosidase BGLA coding gene bglA and the native endoglucanase EG2 coding gene eg2, achieving a 43.5-fold BGL activity and 1.2-fold EG activity increase, respectively. Ultimately, to overcome the defects of the native cellulase system in T. reesei, the bglA and eg2 were co-overexpressed under the control of Pcc promoter. The bglA-eg2 double expression strain QPEB70 exhibited a 178% increase in total cellulase activity, whose cellulase system displayed 2.3- and 2.4-fold higher saccharification efficiency towards acid-pretreated and delignified corncob residues than the parental strain, respectively.Conclusions: The synthetic hybrid promoter Pcc was generated and employed to improve the cellulase system of T. reesei by expressing specific components. Therefore, construction of synthetic hybrid promoters would allow particular cellulase genes to be expressed at desired levels, which is a viable strategy to optimize the cellulolytic enzyme system for efficient biomass bioconversion.

Cellulase production and morphology of Trichoderma reesei in different experimental conditions

2018 ◽  
Vol 63 (2) ◽  
pp. 115-129
Author(s):  
Rahela Carpa ◽  
◽  
Alin Cândea ◽  
Alexei Remizovschi ◽  
Lucian Barbu-Tudoran ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Cellulase Production ◽  
Experimental Conditions

scholarly journals cAMP activates calcium signalling via phospholipase C to regulate cellulase production in the filamentous fungus Trichoderma reesei

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yumeng Chen ◽  
Xingjia Fan ◽  
Xinqing Zhao ◽  
Yaling Shen ◽  
Xiangyang Xu ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Phospholipase C ◽  
Trichoderma Reesei ◽  
Filamentous Fungus ◽  
Calcium Signalling ◽  
Signal Transduction Pathway ◽  
Cellulase Production ◽  
Signalling Pathway ◽  
Dependent Manner ◽  
Transduction Pathway

Abstract Background The filamentous fungus Trichoderma reesei is one of the best producers of cellulase and has been widely studied for the production of cellulosic ethanol and bio-based products. We previously reported that Mn2+ and N,N-dimethylformamide (DMF) can stimulate cellulase overexpression via Ca2+ bursts and calcium signalling in T. reesei under cellulase-inducing conditions. To further understand the regulatory networks involved in cellulase overexpression in T. reesei, we characterised the Mn2+/DMF-induced calcium signalling pathway involved in the stimulation of cellulase overexpression. Results We found that Mn2+/DMF stimulation significantly increased the intracellular levels of cAMP in an adenylate cyclase (ACY1)-dependent manner. Deletion of acy1 confirmed that cAMP is crucial for the Mn2+/DMF-stimulated cellulase overexpression in T. reesei. We further revealed that cAMP elevation induces a cytosolic Ca2+ burst, thereby initiating the Ca2+ signal transduction pathway in T. reesei, and that cAMP signalling causes the Ca2+ signalling pathway to regulate cellulase production in T. reesei. Furthermore, using a phospholipase C encoding gene plc-e deletion strain, we showed that the plc-e gene is vital for cellulase overexpression in response to stimulation by both Mn2+ and DMF, and that cAMP induces a Ca2+ burst through PLC-E. Conclusions The findings of this study reveal the presence of a signal transduction pathway in which Mn2+/DMF stimulation produces cAMP. Increase in the levels of cAMP activates the calcium signalling pathway via phospholipase C to regulate cellulase overexpression under cellulase-inducing conditions. These findings provide insights into the molecular mechanism of the cAMP–PLC–calcium signalling pathway underlying cellulase expression in T. reesei and highlight the potential applications of signal transduction in the regulation of gene expression in fungi.

scholarly journals Production of High Purity Biosurfactants Using Heavy Oil Residues as Carbon Source

Energies ◽  
2021 ◽  
Vol 14 (12) ◽  
pp. 3557
Author(s):  
Athina Mandalenaki ◽  
Nicolas Kalogerakis ◽  
Eleftheria Antoniou
Keyword(s):  
Carbon Source ◽  
Heavy Oil ◽  
Oil Pollution ◽  
Batch Reactor ◽  
Production Method ◽  
Promising Alternative ◽  
Heavy Oil Residues ◽  
Oil Residue ◽  
Increasing Demand ◽  
Heavy Oil Residue

Typically, oil pollution cleanup procedures following first response actions include dispersion. Crude oil is biodegradable, and its bioavailability can be increased when dispersed into very fine droplets by means of chemical surfactants. Although their use is widely spread in many applications, the latter may prove toxic, depending on the extent of use. The use of biological means, such as bioremediation and biosurfactants, has emerged over the past years as a very promising ‘green’ alternative technology. Biosurfactants (BSs) are amphiphilic molecules produced by microorganisms during biodegradation, thus increasing the bioavailability of the organic pollutants. It is their biodegradability and low toxicity that render BSs as a very promising alternative to the synthetic ones. Alcanivorax borkumensis SK2 strain ability to produce BSs, without any impurities from the substrate, was investigated. The biosurfactant production was scaled up by means of a sequencing batch reactor (SBR) and a heavy oil residue substrate as the carbon source. The product is free from substrate impurities, and its efficiency is tested on oil bioremediation in the marine environment. The product’s dispersion efficiency was determined by the baffled flask test. The production method proposed can have a significant impact to the market, given the ever-increasing demand for ecologically friendly, reliable, commercially viable and economically competitive environmental cleanup techniques.

The influence of culture conditions on mycelial structure and cellulase production by Trichoderma reesei Rut C-30

2000 ◽  
Vol 26 (5-6) ◽  
pp. 394-401 ◽  
Author(s):  
F.C. Domingues ◽  
J.A. Queiroz ◽  
J.M.S. Cabral ◽  
L.P. Fonseca
Keyword(s):  
Trichoderma Reesei ◽  
Cellulase Production ◽  
Culture Conditions ◽  
C 30
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