scholarly journals Precision engineering of the transcription factor Cre1 in Hypocrea jecorina (Trichoderma reesei) for efficient cellulase production in the presence of glucose

2020 ◽  
Author(s):  
Lijuan Han ◽  
Yinshuang Tan ◽  
Wei Ma ◽  
Kangle Niu ◽  
Shaoli Hou ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Trichoderma Reesei ◽  
Parent Strain ◽  
Zinc Finger Protein ◽  
Cellulase Production ◽  
Fungal Species ◽  
Cellulolytic Enzymes ◽  
Phosphorylation Sites ◽  
Precision Engineering ◽  
C Terminus

SummaryIn Trichoderma reesei, carbon catabolite repression (CCR) significantly downregulates the transcription of cellulolytic enzymes, which is usually mediated by the zinc finger protein Cre1. It was found that there is a conserved region at the C-terminus of Cre1/CreA in several cellulase-producing fungi that contains up to three continuous S/T phosphorylation sites. Here, S387, S388, T389, and T390 at the C-terminus of Cre1 in T. reesei were mutated to valine for mimicking an unphosphorylated state, thereby generating the transformants Tr_Cre1S387V, Tr_Cre1S388V, Tr_Cre1T389V, and Tr_Cre1T390V, respectively. Transcription of cel7a in Tr_ Cre1S388V was markedly higher than that of the parent strain when grown in glucose-containing media. Under these conditions, both filter paperase (FPase) and p-nitrophenyl-β-D-cellobioside (pNPCase) activities, as well as soluble proteins from Tr_Cre1S388V were significantly increased by up to 2- to 3-fold compared with that of other transformants and the parent strain. To our knowledge, this is the first report demonstrating an improvement of cellulase production in fungal species under CCR by mimicking dephosphorylation at the C-terminus of Cre1. Taken together, we developed a precision engineering strategy based on the modification of phosphorylation sites of Cre1 transcription factor to enhance the production of cellulase in fungal species under CCR.

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 Cellulase Enzyme Production from Filamentous Fungi Trichoderma reesei and Aspergillus awamori in Submerged Fermentation with Rice Straw

2021 ◽  
Vol 7 (10) ◽  
pp. 868
Author(s):  
Laila Naher ◽  
Siti Noor Fatin ◽  
Md Abdul Halim Sheikh ◽  
Lateef Adebola Azeez ◽  
Shaiquzzaman Siddiquee ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Filamentous Fungi ◽  
Rice Straw ◽  
Submerged Fermentation ◽  
Enzyme Production ◽  
Dry Weight ◽  
Cellulase Production ◽  
Fungal Species ◽  
Aspergillus Awamori ◽  
Cellulase Enzyme

Fungi are a diverse group of microorganisms that play many roles in human livelihoods. However, the isolation of potential fungal species is the key factor to their utilization in different sectors, including the enzyme industry. Hence, in this study, we used two different fungal repositories—soil and weed leaves—to isolate filamentous fungi and evaluate their potential to produce the cellulase enzyme. The fungal strains were isolated using dichloran rose bengal agar (DRBA) and potato dextrose agar (PDA). For cellulase enzyme production, a rice straw submerged fermentation process was used. The enzyme production was carried out at the different incubation times of 3, 5, and 7 days of culture in submerged conditions with rice straw. Fungal identification studies by morphological and molecular methods showed that the soil colonies matched with Trichoderma reesei, and the weed leaf colonies matched with Aspergillus awamori. These species were coded as T. reesei UMK04 and A. awamori UMK02, respectively. This is the first report of A. awamori UMK02 isolation in Malaysian agriculture. The results of cellulase production using the two fungi incorporated with rice straw submerged fermentation showed that T. reesei produced a higher amount of cellulase at Day 5 (27.04 U/mg of dry weight) as compared with A. awamori (15.19 U/mg of dry weight), and the concentration was significantly different (p < 0.05). Our results imply that T. reesei can be utilized for cellulase production using rice straw.

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 ACE4, a novel transcriptional activator involved in the regulation of cellulase genes on cellulose in Trichoderma reesei

2021 ◽  
Author(s):  
Yumeng Chen ◽  
Aibo Lin ◽  
Pei Liu ◽  
Xingjia Fan ◽  
Chuan Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Trichoderma Reesei ◽  
Transcriptional Activator ◽  
Cellulase Production ◽  
Comparative Genomic ◽  
Transcriptional Level ◽  
Cellulase Gene ◽  
Cellulase Expression

The filamentous fungus Trichoderma reesei is a model strain for cellulase production. Cellulase gene expression in T. reesei is controlled by multiple transcription factors. Here, we identified by comparative genomic screening a novel transcriptional activator ACE4 ( A ctivator of c ellulase e xpression 4) that positively regulates cellulase gene expression on cellulose in T. reesei . Disruption of the ace4 gene significantly decreased expression of four main cellulase genes, and the essential cellulase transcription factor encoding gene ace3 . Overexpression of ace4 increased cellulase production by approximately 22% compared to that in the parental strain. Further investigations using electrophoretic mobility shift assays, DNase I footprinting assays, and chromatin immunoprecipitation assays indicated that ACE4 directly binds to the promoter of cellulase genes by recognizing the two adjacent 5′-GGCC-3′ sequences. Additionally, ACE4 directly binds to the promoter of ace3 and, in turn, regulates the expression of ACE3 to facilitate cellulase production. Collectively, these results demonstrate an important role for ACE4 in regulating cellulase gene expression, which will contribute to understanding the mechanism underlying cellulase expression in T. reesei . IMPORTANCE T. reesei is commonly utilized in industry to produce cellulases, enzymes that degrade lignocellulosic biomass for the production of bioethanol and bio-based products. T. reesei is capable of rapidly initiating the biosynthesis of cellulases in the presence of cellulose, which has made it useful as a model fungus for studying gene expression in eukaryotes. Cellulase gene expression is controlled through multiple transcription factors at the transcriptional level. However, the molecular mechanisms by which transcription is controlled remain unclear. In the present study, we identified a novel transcription factor, ACE4, which regulates cellulase expression on cellulose by binding to the promoters of cellulase genes and the cellulase activator ace3 . Our study not only expands the general functional understanding of the novel transcription factor ACE4 but also provides evidence for the regulatory mechanism mediating gene expression in T. reesei .

scholarly journals Studies on sugar transporter CRT1 reveal new characteristics that are critical for cellulase induction in Trichoderma reesei

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Sami Havukainen ◽  
Mari Valkonen ◽  
Kari Koivuranta ◽  
Christopher P. Landowski
Keyword(s):  
Trichoderma Reesei ◽  
Genome Annotation ◽  
Cellulase Production ◽  
Fungal Species ◽  
Extracellular Proteins ◽  
Sugar Transporter ◽  
Sugar Transporters ◽  
Cellulase Induction ◽  
The Many ◽  
Rut C30

Abstract Background Trichoderma reesei is an ascomycete fungus that has a tremendous capability of secreting extracellular proteins, mostly lignocellulose-degrading enzymes. Although many aspects of the biology of this organism have been unfolded, the roles of the many sugar transporters coded in its genome are still a mystery with a few exceptions. One of the most interesting sugar transporters that has thus far been discovered is the cellulose response transporter 1 (CRT1), which has been suggested to be either a sugar transporter or a sensor due to its seemingly important role in cellulase induction. Results Here we show that CRT1 is a high-affinity cellobiose transporter, whose function can be complemented by the expression of other known cellobiose transporters. Expression of two sequence variants of the crt1 gene in Saccharomyces cerevisiae revealed that only the variant listed in the RUT-C30 genome annotation has the capability to transport cellobiose and lactose. When expressed in the $$\Delta$$ Δ crt1 strain, the variant listed in the QM6a genome annotation offers partial complementation of the cellulase induction, while the expression of the RUT-C30 variant or cellobiose transporters from two other fungal species fully restore the cellulase induction. Conclusions These results add to our knowledge about the fungal metabolism of cellulose-derived oligosaccharides, which have the capability of inducing the cellulase production in many species. They also help us to deepen our understanding of the T. reesei lactose metabolism, which can have important consequences as this sugar is used as the inducer of protein secretion in many industrial processes which employ this species.

scholarly journals Disruption of the Trichoderma reesei gul1 gene stimulates hyphal branching and reduces broth viscosity in cellulase production

2021 ◽  
Author(s):  
Qinqin Zhao ◽  
Qin Liu ◽  
Qi Wang ◽  
Yuqi Qin ◽  
Yaohua Zhong ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Filamentous Fungi ◽  
Parent Strain ◽  
Fermentation Broth ◽  
Cellulase Production ◽  
Lower Viscosity ◽  
Close Relationship ◽  
Genes Encoding ◽  
Lateral Branches ◽  
Mrna Binding

Abstract Hyphal morphology is considered to have a close relationship with the production level of secreted proteins by filamentous fungi. In this study, the gul1 gene, which encodes a putative mRNA-binding protein, was disrupted in cellulase-producing fungus Trichoderma reesei. The hyphae of Δgul1 strain produced more lateral branches than the parent strain. Under the condition for cellulase production, disruption of gul1 resulted in smaller mycelial clumps and significantly lower viscosity of fermentation broth. In addition, cellulase production was improved by 22% relative to the parent strain. Transcriptome analysis revealed that a set of genes encoding cell wall remodeling enzymes as well as hydrophobins were differentially expressed in the Δgul1 strain. The results suggest that the regulatory role of gul1 in cell morphogenesis is likely conserved in filamentous fungi. To our knowledge, this is the first report on the engineering of gul1 in an industrially important fungus.

Sign in / Sign up

Export Citation Format

Share Document