scholarly journals A Novel CreA-Mediated Regulation Mechanism of Cellulase Expression in the Thermophilic Fungus Humicola insolens

2019 ◽  
Vol 20 (15) ◽  
pp. 3693 ◽  
Author(s):  
Xinxin Xu ◽  
Chao Fan ◽  
Liya Song ◽  
Jinyang Li ◽  
Yuan Chen ◽  
...  
Keyword(s):  
Xylanase Activity ◽  
Hyphal Growth ◽  
Expression Regulation ◽  
Thermophilic Fungus ◽  
Cellulolytic Enzymes ◽  
Regulation Mechanism ◽  
Thermophilic Fungi ◽  
Disruption Mutant ◽  
Humicola Insolens ◽  
Cellulase Expression

The thermophilic fungus Humicola insolens produces cellulolytic enzymes that are of great scientific and commercial interest; however, few reports have focused on its cellulase expression regulation mechanism. In this study, we constructed a creA gene (carbon catabolite repressor gene) disruption mutant strain of H. insolens that exhibited a reduced radial growth rate and stouter hyphae compared to the wild-type (WT) strain. The creA disruption mutant also expressed elevated pNPCase (cellobiohydrolase activities), pNPGase (β-glucosidase activities), and xylanase levels in non-inducing fermentation with glucose. Unlike other fungi, the H. insolens creA disruption mutant displayed lower FPase (filter paper activity), CMCase (carboxymethyl cellulose activity), pNPCase, and pNPGase activity than observed in the WT strain when fermentation was induced using Avicel, whereas its xylanase activity was higher than that of the parental strain. These results indicate that CreA acts as a crucial regulator of hyphal growth and is part of a unique cellulase expression regulation mechanism in H. insolens. These findings provide a new perspective to improve the understanding of carbon catabolite repression regulation mechanisms in cellulase expression, and enrich the knowledge of metabolism diversity and molecular regulation of carbon metabolism in thermophilic fungi.

SCREENING OF MYCELIAL FUNGI AS POTENTIAL PRODUCERS OF FODDER CELLULOLYTIC ENZYMES ON WASTE OIL PRODUCTION

2020 ◽  
pp. 330-331
Author(s):  
T. Kuzmicheva ◽  
I. Belsky
Keyword(s):  
Carbon Source ◽  
Crude Protein ◽  
Xylanase Activity ◽  
Oil Production ◽  
Cellulolytic Enzymes ◽  
Mycelial Fungi ◽  
Waste Oil ◽  
Primary Screening

The percentage of substances that make up the husks of sunflower is established. Primary screening of 17 strains of mycelial fungi was carried out on media having only crushed sunflower husk as a carbon source. The strains were selected that at the end of cultivation contained more than 44% crude protein, 15–20 U/g cellulolytic and more than 100 U/g xylanase activity.

scholarly journals MKL-1 regulates the stem cell marker. CD44 in breast cancer cells

2019 ◽  
Vol 78 ◽  
pp. 01002
Author(s):  
Zhou-Tong Dai ◽  
Ao Yao ◽  
Yuan Xiang ◽  
Jia Peng Li ◽  
Wei Guo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Malignant Tumors ◽  
Regulation Of Gene Expression ◽  
Gene Promoter ◽  
Expression Regulation ◽  
Stem Cell Marker ◽  
Luciferase Reporter ◽  
Regulation Mechanism ◽  
Luciferase Reporter Gene ◽  
Factors Affecting

CD44, cluster of differentiation 44 is a typical marker of stem cells. At present, it has been found that CD44 is prevalent in various human malignant tumors, but its expression regulation mechanism is still not clear. The initiation of gene expression, the modification of RNA levels, and the regulation of protein levels are the main factors affecting the expression level of genes, and the most critical one is the regulation of gene expression by signaling pathways. Up to now, there has been no report on the role of MKL-1 in the cloning of the cd44 promoter. Therefore, this study intends to clone the cd44 gene promoter, construct its luciferase reporter gene vector, transfect the MKL-1 overexpression vector, and analyze how it affects transcriptional activity, in order to further study the expression regulation of cd44. The mechanism provides a powerful tool in the future.

Crystal structure of a Cu,Zn superoxide dismutase from the thermophilic fungus Chaetomium thermophilum

2021 ◽  
Vol 28 ◽  
Author(s):  
Imran Mohsin ◽  
Li-Qing Zhang ◽  
Duo-Chuan Li ◽  
Anastassios C. Papageorgiou
Keyword(s):  
Crystal Structure ◽  
Thermophilic Fungus ◽  
Diffusion Method ◽  
Superoxide Dismutases ◽  
Therapeutic Effects ◽  
Thermophilic Fungi ◽  
Contributing Factors ◽  
Thermostable Enzymes ◽  
Chaetomium Thermophilum ◽  
Promising Source

Background: Thermophilic fungi have recently emerged as a promising source of thermostable enzymes. Superoxide dismutases are key antioxidant metalloenzymes with promising therapeutic effects in various diseases, both acute and chronic. However, structural heterogeneity and low thermostability limit their therapeutic efficacy. Objective: Although several studies from hypethermophilic superoxide dismutases (SODs) have been reported, information about Cu,Zn-SODs from thermophilic fungi is scarce. Chaetomium thermophilum is a thermophilic fungus that could provide proteins with thermophilic properties. Method: The enzyme was expressed in Pichia pastoris cells and crystallized using the vapor-diffusion method. X-ray data were collected, and the structure was determined and refined to 1.56 Å resolution. Structural analysis and comparisons were carried out. Results: The presence of 8 molecules (A through H) in the asymmetric unit resulted in four different interfaces. Molecules A and F form the typical homodimer which is also found in other Cu,Zn-SODs. Zinc was present in all subunits of the structure while copper was found in only four subunits with reduced occupancy (C, D, E and F). Conclusion: The ability of the enzyme to form oligomers and the elevated Thr:Ser ratio may be contributing factors to its thermal stability. Two hydrophobic residues that participate in interface formation and are not present in other CuZn-SODs may play a role in the formation of new interfaces and the oligomerization process. The CtSOD crystal structure reported here is the first Cu,Zn-SOD structure from a thermophilic fungus.

scholarly journals Crystal Structure of a GH3 β-Glucosidase from the Thermophilic Fungus Chaetomium thermophilum

2019 ◽  
Vol 20 (23) ◽  
pp. 5962 ◽  
Author(s):  
Imran Mohsin ◽  
Nirmal Poudel ◽  
Duo-Chuan Li ◽  
Anastassios C. Papageorgiou
Keyword(s):  
Crystal Structure ◽  
Elevated Temperatures ◽  
Cellulose Degradation ◽  
Thermophilic Fungus ◽  
Biofuel Production ◽  
Lignocellulose Degradation ◽  
Thermophilic Fungi ◽  
Biotechnological Applications ◽  
Chaetomium Thermophilum ◽  
Promising Source

Beta-glucosidases (β-glucosidases) have attracted considerable attention in recent years for use in various biotechnological applications. They are also essential enzymes for lignocellulose degradation in biofuel production. However, cost-effective biomass conversion requires the use of highly efficient enzymes. Thus, the search for new enzymes as better alternatives of the currently available enzyme preparations is highly important. Thermophilic fungi are nowadays considered as a promising source of enzymes with improved stability. Here, the crystal structure of a family GH3 β-glucosidase from the thermophilic fungus Chaetomium thermophilum (CtBGL) was determined at a resolution of 2.99 Å. The structure showed the three-domain architecture found in other β-glucosidases with variations in loops and linker regions. The active site catalytic residues in CtBGL were identified as Asp287 (nucleophile) and Glu517 (acid/base). Structural comparison of CtBGL with Protein Data Bank (PDB)-deposited structures revealed variations among glycosylated Asn residues. The enzyme displayed moderate glycosylation compared to other GH3 family β-glucosidases with similar structure. A new glycosylation site at position Asn504 was identified in CtBGL. Moreover, comparison with respect to several thermostability parameters suggested that glycosylation and charged residues involved in electrostatic interactions may contribute to the stability of the enzyme at elevated temperatures. The reported CtBGL structure provides additional insights into the family GH3 enzymes and could offer new ideas for further improvements in β-glucosidases for more efficient use in biotechnological applications regarding cellulose degradation.

scholarly journals Promotive Effects of Cellulolytic Enzymes Produced by Biomass-Degrading Bacteria on Saccharification of Different Pretreated Corn Stovers

2021 ◽  
Author(s):  
Yanwen Wu ◽  
Sarita Shrestha ◽  
Haipeng Guo ◽  
Jinchi Zhang ◽  
Haisong Wang ◽  
...  
Keyword(s):  
Corn Stover ◽  
Reducing Sugars ◽  
Synergistic Effects ◽  
Xylanase Activity ◽  
Enzymatic Saccharification ◽  
Crude Enzyme ◽  
Cellulolytic Enzymes ◽  
Degrading Bacteria ◽  
Commercial Enzymes ◽  
Cost Efficient

Abstract Enzymatic saccharification of corn stover can be enhanced by partially replacing commercial enzymes with bacterial crude enzyme extracts. Three bacteria, Bacillus sp. A0, Bacillus sp. CH20S1, and Exiguobacterium sp. AS2B, were cultured in a media with corn stover as the substrate. The cultural conditions were monitored and optimized to maximize CMCase and xylanase activity in the crude enzyme extracts. After 72 h of hydrolysis of corn stover with diluted crude enzymes (DCE) from the three strains, reducing sugars were released from non-pretreated and pretreated corn stovers. Values of the released sugars ranged from 48.23–71.69 mg g− 1, which were lower than those released by commercial cellulase (100–400 mg g− 1). The synergistic effects were observed when 12 FPU g− 1 and 4 FPU g− 1 of commercial cellulase were added to the DCE of the CH20S1 strain producing 315.90 mg g− 1 and 320.65 mg g− 1 reducing sugars, respectively. It was shown that an effective combination of bacterial DCE with commercial enzymes could achieve more cost-efficient saccharification of lignocellulosic biomass compared to either of the two enzymes used alone.

Overexpression and characterization of a novel endo-β-1,3(4)-glucanase from thermophilic fungus Humicola insolens Y1

2017 ◽  
Vol 138 ◽  
pp. 63-68 ◽  
Author(s):  
Jinyang Li ◽  
Xinxin Xu ◽  
Pengjun Shi ◽  
Bo Liu ◽  
Yuhong Zhang ◽  
...  
Keyword(s):  
Thermophilic Fungus ◽  
Humicola Insolens

Production of multiple xylanolytic and cellulolytic enzymes by thermophilic fungus Myceliophthora sp. IMI 387099

2007 ◽  
Vol 98 (3) ◽  
pp. 504-510 ◽  
Author(s):  
A.K. Badhan ◽  
B.S. Chadha ◽  
Jatinder Kaur ◽  
H.S. Saini ◽  
M.K. Bhat
Keyword(s):  
Thermophilic Fungus ◽  
Cellulolytic Enzymes

scholarly journals Upgrading of efficient and scalable CRISPR–Cas-mediated technology for genetic engineering in thermophilic fungus Myceliophthora thermophila

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Qian Liu ◽  
Yongli Zhang ◽  
Fangya Li ◽  
Jingen Li ◽  
Wenliang Sun ◽  
...  
Keyword(s):  
Genetic Engineering ◽  
Genome Editing ◽  
Plant Biomass ◽  
Thermophilic Fungus ◽  
Marker Genes ◽  
Thermophilic Fungi ◽  
Myceliophthora Thermophila ◽  
Dna Breaks ◽  
Direct Production ◽  
Marker Recycling

Abstract Background Thermophilic filamentous fungus Myceliophthora thermophila has great capacity for biomass degradation and is an attractive system for direct production of enzymes and chemicals from plant biomass. Its industrial importance inspired us to develop genome editing tools to speed up the genetic engineering of this fungus. First-generation CRISPR–Cas9 technology was developed in 2017 and, since then, some progress has been made in thermophilic fungi genetic engineering, but a number of limitations remain. They include the need for complex independent expression cassettes for targeting multiplex genomic loci and the limited number of available selectable marker genes. Results In this study, we developed an Acidaminococcus sp. Cas12a-based CRISPR system for efficient multiplex genome editing, using a single-array approach in M. thermophila. These CRISPR–Cas12a cassettes worked well for simultaneous multiple gene deletions/insertions. We also developed a new simple approach for marker recycling that relied on the novel cleavage activity of the CRISPR–Cas12a system to make DNA breaks in selected markers. We demonstrated its performance by targeting nine genes involved in the cellulase production pathway in M. thermophila via three transformation rounds, using two selectable markers neo and bar. We obtained the nonuple mutant M9 in which protein productivity and lignocellulase activity were 9.0- and 18.5-fold higher than in the wild type. We conducted a parallel investigation using our transient CRISPR–Cas9 system and found the two technologies were complementary. Together we called them CRISPR–Cas-assisted marker recycling technology (Camr technology). Conclusions Our study described new approaches (Camr technology) that allow easy and efficient marker recycling and iterative stacking of traits in the same thermophilic fungus strain either, using the newly established CRISPR–Cas12a system or the established CRISPR–Cas9 system. This Camr technology will be a versatile and efficient tool for engineering, theoretically, an unlimited number of genes in fungi. We expect this advance to accelerate biotechnology-oriented engineering processes in fungi.

Purification and characterization of a thermostable xylanase from a thermophilic fungus Thermoascus aurantiacus

1987 ◽  
Vol 33 (8) ◽  
pp. 689-692 ◽  
Author(s):  
Larry U. L. Tan ◽  
Paul Mayers ◽  
John N. Saddler
Keyword(s):  
Xylanase Activity ◽  
Barium Chloride ◽  
Thermophilic Fungus ◽  
Mercury Chloride ◽  
Thermoascus Aurantiacus ◽  
Purification And Characterization ◽  
Acting Mechanism ◽  
A Subunit ◽  
Temperature Optima

A thermostable endo-β-D-xylanase (1,4-β-D-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a thermophilic fungus Thermoascus aurantiacus C436, using a single chromatographic step on SP-Sephadex C50. The purified preparation was homogeneous based on denaturing polyacrylamide and isoelectric focusing gels. The xylanase had a subunit molecular mass of 32 000 daltons, isoelectric point at pH 7.1, apparent Km and Vmax of 0.17% (w/v) xylan and 61.3IU/mg protein, respectively, at 50 °C. The pH and temperature optima for xylan hydrolysis were pH 5.1 and 80 °C, respectively. The xylanase retained full activity following incubation at 60 °C for 97 h or 70 °C for 24 h. At 80 °C, the half-life of the enzyme was 54 min. The xylanase was not affected by copper sulfate, zinc sulfate, calcium chloride, cobalt chloride, barium chloride, magnesium sulfate, and EDTA at concentrations of 2 mM. Mercury chloride at 2 mM concentration abolished all xylanase activity, while lead acetate at the same concentration reduced xylanase activity by approximately 25%. From the initial hydrolysis products of xylan, the xylanase was deduced to hydrolyse xylan through an endo-acting mechanism.

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