Strain Improvement of Trichoderma spp. Through Two Steps Protoplast Fusion for Cellulase Production Enhancement

2020 ◽  
Author(s):  
Zahra Papzan ◽  
Mojegan Kowsari ◽  
Mohammad Javan-Nikkhah ◽  
Amir Mirzadi Gohari ◽  
M. Carmen Limón
Keyword(s):  
Protoplast Fusion ◽  
Strain Improvement ◽  
Cellulase Activity ◽  
Cellulase Production ◽  
Mrna Levels ◽  
Hybrid Strain ◽  
Trichoderma Spp ◽  
Wide Range ◽  
Natural Isolates ◽  
Rut C30

Fungal protoplast fusion is an approach to introduce novel characteristics into industrially important strains. Cellulases, essential enzymes with a wide range of biotechnological applications, are produced by many species of the filamentous fungi Trichoderma. In this study, a collection of 60 natural isolates have been screened for Avicel and CMC degradation, and two cellulase producers of Trichoderma virens and Trichoderma harzianum were used for protoplast fusion. One of resulting hybrids with an improvement in cellulase activity, C1-3, was fused with the hyper producer Trichoderma reesei Rut-C30. A new selected hybrid, F7, increased its cellulase activity 1.8 and 5 times in comparison with Rut-C30 and C1-3, respectively. The increases in enzyme activity correlated with an upregulation of cellulolytic genes cbh1, cbh2, egl3, and bgl1 in the parents. Amount of mRNA of cbh1 and cbh2 in F7 resembled Rut-C30 while the bgl1 mRNA levels were similar to C1-3. AFLP fingerprinting and GC-MS analysis represented variations in parental strains and fusants. In conclusion, results demonstrate that a 3-interspecific hybrid strain has been isolated with improved characteristics for cellulase degradation showing genetic polymorphisms and differences in the volatile profile which suggests reorganizations at genetic level.

scholarly journals Quantitative Proteome Profiling Reveals Cellobiose-Dependent Protein Processing and Export Pathways for the Lignocellulolytic Response in Neurospora crassa

2020 ◽  
Vol 86 (15) ◽  
Author(s):  
Dan Liu ◽  
Yisong Liu ◽  
Duoduo Zhang ◽  
Xiaoting Chen ◽  
Qian Liu ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Filamentous Fungi ◽  
Ribosome Biogenesis ◽  
Strain Improvement ◽  
Cellulase Production ◽  
Protein Processing ◽  
Mrna Levels ◽  
Industrial Enzymes ◽  
Content Type ◽  
Proteome Profiling

ABSTRACT Filamentous fungi are intensively used for producing industrial enzymes, including lignocellulases. Employing insoluble cellulose to induce the production of lignocellulases causes some drawbacks, e.g., a complex fermentation operation, which can be overcome by using soluble inducers such as cellobiose. Here, a triple β-glucosidase mutant of Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to profile the proteome responses to cellobiose and cellulose (Avicel). Our results revealed a shared proteomic response to cellobiose and Avicel, whose elements included lignocellulases and cellulolytic product transporters. While the cellulolytic proteins showed a correlated increase in protein and mRNA levels, only a moderate correlation was observed on a proteomic scale between protein and mRNA levels (R2 = 0.31). Ribosome biogenesis and rRNA processing were significantly overrepresented in the protein set with increased protein but unchanged mRNA abundances in response to Avicel. Ribosome biogenesis, as well as protein processing and protein export, was also enriched in the protein set that showed increased abundance in response to cellobiose. NCU05895, a homolog of yeast CWH43, is potentially involved in transferring a glycosylphosphatidylinositol (GPI) anchor to nascent proteins. This protein showed increased abundance but no significant change in mRNA levels. Disruption of CWH43 resulted in a significant decrease in cellulase activities and secreted protein levels in cultures grown on Avicel, suggesting a positive regulatory role for CWH43 in cellulase production. The findings should have an impact on a systems engineering approach for strain improvement for the production of lignocellulases. IMPORTANCE Lignocellulases are important industrial enzymes for sustainable production of biofuels and bio-products. Insoluble cellulose has been commonly used to induce the production of lignocellulases in filamentous fungi, which causes a difficult fermentation operation and enzyme loss due to adsorption to cellulose. The disadvantages can be overcome by using soluble inducers, such as the disaccharide cellobiose. Quantitative proteome profiling of the model filamentous fungus Neurospora crassa revealed cellobiose-dependent pathways for cellulase production, including protein processing and export. A protein (CWH43) potentially involved in protein processing was found to be a positive regulator of lignocellulase production. The cellobiose-dependent mechanisms provide new opportunities to improve the production of lignocellulases in filamentous fungi.

scholarly journals High-dose rapamycin exerts a temporary impact on T. reesei RUT-C30 through gene trFKBP12

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Ai-Ping Pang ◽  
Haiyan Wang ◽  
Funing Zhang ◽  
Xin Hu ◽  
Fu-Gen Wu ◽  
...  
Keyword(s):  
Cell Growth ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Cellulase Production ◽  
High Dose ◽  
Mrna Levels ◽  
Tor Signaling ◽  
Inhibition Effect ◽  
Temporary Inhibition ◽  
Rut C30

Abstract Background Knowledge with respect to regulatory systems for cellulase production is prerequisite for exploitation of such regulatory networks to increase cellulase production, improve fermentation efficiency and reduce the relevant production cost. The target of rapamycin (TOR) signaling pathway is considered as a central signaling hub coordinating eukaryotic cell growth and metabolism with environmental inputs. However, how and to what extent the TOR signaling pathway and rapamycin are involved in cellulase production remain elusive. Result At the early fermentation stage, high-dose rapamycin (100 μM) caused a temporary inhibition effect on cellulase production, cell growth and sporulation of Trichoderma reesei RUT-C30 independently of the carbon sources, and specifically caused a tentative morphology defect in RUT-C30 grown on cellulose. On the contrary, the lipid content of T. reesei RUT-C30 was not affected by rapamycin. Accordingly, the transcriptional levels of genes involved in the cellulase production were downregulated notably with the addition of rapamycin. Although the mRNA levels of the putative rapamycin receptor trFKBP12 was upregulated significantly by rapamycin, gene trTOR (the downstream effector of the rapamycin–FKBP12 complex) and genes associated with the TOR signaling pathways were not changed markedly. With the deletion of gene trFKBP12, there is no impact of rapamycin on cellulase production, indicating that trFKBP12 mediates the observed temporary inhibition effect of rapamycin. Conclusion Our study shows for the first time that only high-concentration rapamycin induced a transient impact on T. reesei RUT-C30 at its early cultivation stage, demonstrating T. reesei RUT-C30 is highly resistant to rapamycin, probably due to that trTOR and its related signaling pathways were not that sensitive to rapamycin. This temporary influence of rapamycin was facilitated by gene trFKBP12. These findings add to our knowledge on the roles of rapamycin and the TOR signaling pathways play in T. reesei.

scholarly journals Glutamine involvement in nitrogen regulation of cellulase production in fungi

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Ai-Ping Pang ◽  
Funing Zhang ◽  
Xin Hu ◽  
Yongsheng Luo ◽  
Haiyan Wang ◽  
...  
Keyword(s):  
Cell Growth ◽  
Rational Design ◽  
Cellulase Production ◽  
Nitrogen Regulation ◽  
Transcriptional Level ◽  
Regulation Mechanism ◽  
Mrna Levels ◽  
Inhibition Effect ◽  
The Impact ◽  
Rut C30

Abstract Background Cellulase synthesized by fungi can environment-friendly and sustainably degrades cellulose to fermentable sugars for producing cellulosic biofuels, biobased medicine and fine chemicals. Great efforts have been made to study the regulation mechanism of cellulase biosynthesis in fungi with the focus on the carbon sources, while little attention has been paid to the impact and regulation mechanism of nitrogen sources on cellulase production. Results Glutamine displayed the strongest inhibition effect on cellulase biosynthesis in Trichoderma reesei, followed by yeast extract, urea, tryptone, ammonium sulfate and l-glutamate. Cellulase production, cell growth and sporulation in T. reesei RUT-C30 grown on cellulose were all inhibited with the addition of glutamine (a preferred nitrogen source) with no change for mycelium morphology. This inhibition effect was attributed to both l-glutamine itself and the nitrogen excess induced by its presence. In agreement with the reduced cellulase production, the mRNA levels of 44 genes related to the cellulase production were decreased severely in the presence of glutamine. The transcriptional levels of genes involved in other nitrogen transport, ribosomal biogenesis and glutamine biosynthesis were decreased notably by glutamine, while the expression of genes relevant to glutamate biosynthesis, amino acid catabolism, and glutamine catabolism were increased noticeably. Moreover, the transcriptional level of cellulose signaling related proteins ooc1 and ooc2, and the cellular receptor of rapamycin trFKBP12 was increased remarkably, whose deletion exacerbated the cellulase depression influence of glutamine. Conclusion Glutamine may well be the metabolite effector in nitrogen repression of cellulase synthesis, like the role of glucose plays in carbon catabolite repression. Glutamine under excess nitrogen condition repressed cellulase biosynthesis significantly as well as cell growth and sporulation in T. reesei RUT-C30. More importantly, the presence of glutamine notably impacted the transport and metabolism of nitrogen. Genes ooc1, ooc2, and trFKBP12 are associated with the cellulase repression impact of glutamine. These findings advance our understanding of nitrogen regulation of cellulase production in filamentous fungi, which would aid in the rational design of strains and fermentation strategies for cellulase production in industry.

scholarly journals Cellular effects of glycine and trehalose air-polishing powders on human gingival fibroblasts in vitro

2021 ◽  
Author(s):  
Jens Weusmann ◽  
James Deschner ◽  
Jean-Claude Imber ◽  
Anna Damanaki ◽  
Natalia D. P. Leguizamón ◽  
...  
Keyword(s):  
Wound Healing ◽  
Cell Function ◽  
Nuclear Translocation ◽  
In Vitro Study ◽  
Human Gingival Fibroblasts ◽  
Mrna Levels ◽  
Gingival Fibroblasts ◽  
Air Polishing ◽  
Wide Range

Abstract Objectives Air-polishing has been used in the treatment of periodontitis and gingivitis for years. The introduction of low-abrasive powders has enabled the use of air-polishing devices for subgingival therapy. Within the last decade, a wide range of different low-abrasive powders for subgingival use has been established. In this study, the effects of a glycine powder and a trehalose powder on human gingival fibroblasts (HGF) were investigated. Methods HGF were derived from three systemically and periodontally healthy donors. After 24 h and 48 h of incubation time, mRNA levels, and after 48 h, protein levels of TNFα, IL-8, CCL2, and VEGF were determined. In addition, NF-κB p65 nuclear translocation and in vitro wound healing were assessed. Statistical analysis was performed by ANOVA and post hoc Dunnett’s and Tukey’s tests (p < 0.05). Results Glycine powder significantly increased the expression of proinflammatory genes and showed exploitation of the NF-κB pathway, albeit trehalose powder hardly interfered with cell function and did not trigger the NF-κB pathway. In contrast to trehalose, glycine showed a significant inhibitory effect on the in vitro wound healing rate. Conclusion Subgingivally applicable powders for air-polishing devices can regulate cell viability and proliferation as well as cytokine expression. Our in vitro study suggests that the above powders may influence HGF via direct cell effects. Trehalose appears to be relatively inert compared to glycine powder.

scholarly journals Strain improvement ofPhaffia rhodozymaby protoplast fusion

1992 ◽  
Vol 93 (3) ◽  
pp. 221-226 ◽  
Author(s):  
Soon Bai Chun ◽  
Jong Eon Chin ◽  
Suk Bai ◽  
Gil-Hwan An
Keyword(s):  
Protoplast Fusion ◽  
Strain Improvement

scholarly journals Wild type Bacillus subtilis treated with ethyl methyl sulphonate produced catabolite insensitive mutants with improved cellulase production

2021 ◽  
Author(s):  
Oladipo Olaniyi
Keyword(s):  
Bacillus Subtilis ◽  
Carbon Source ◽  
Enzyme Production ◽  
Cellulase Activity ◽  
Cellulase Production ◽  
Minimal Salt Medium ◽  
Salt Medium ◽  
Production Medium ◽  
Wild Type ◽  
Ethyl Methyl

Abstract The goal of this present investigation was to mutagenize Bacillus subtilis with Ethyl Methyl Sulphonate (EMS), screen the mutants for cellulase production and evaluate the influence of different glucose concentrations on their cellulase production potentials. The wild type B. subtilis was treated with 20, 40, 60 and 80 µl of EMS and the mutants generated were screened for cellulase production in minimal salt medium containing carboxylmethylcellulose (CMC) as the carbon source. Quantitatively, cellulase activity and protein contents were determined by dinitrosalicylic acid and Lowry methods respectively. Seven mutants were developed from each of the EMS concentration bringing the total to twenty-eight from all the concentrations. Approximately 14 and 57% of the mutants developed from 40 and 60µl of EMS had higher cellulase activities than the wild type, while none of the mutants developed from 20 and 80 µl of EMS had better activities than the wild type. The supplementation of 0.2, 0.5, 1.0 and 1.5% glucose in enzyme production medium caused approximately 100, 14, 29 and 14% cellulase repression respectively in the mutants developed from 60µl EMS. Mutants MSSS02 and MSSS05 were considered as catabolite insensitive mutants because their cellulase production were enhanced in comparison to wild type.

scholarly journals Response surface methodology-artificial neural network based optimization and strain improvement of cellulase production by Streptomyces sp.

2020 ◽  
Vol 36 (4) ◽  
Author(s):  
Ega Soujanya Lakshmi ◽  
Manda Rama Narasinga Rao ◽  
Muddada Sudhamani
Keyword(s):  
Neural Network ◽  
Artificial Neural Network ◽  
Response Surface Methodology ◽  
Response Surface ◽  
Wild Strain ◽  
Cellulase Activity ◽  
Cellulase Production ◽  
Optimal Conditions ◽  
Zone Formation ◽  
Artificial Neural

ABSTRACT Thirty seven different colonies were isolated from decomposing logs of textile industries. From among these, a thermotolerant, grampositive, filamentous soil bacteria Streptomyces durhamensis vs15 was selected and screened for cellulase production. The strain showed clear zone formation on CMC agar plate after Gram’s iodine staining.  Streptomyces durhamensis vs15 was further confirmed for cellulase production by estimating the reducing sugars through dinitrosalicylic acid (DNS) method. The activity was enhanced by sequential mutagenesis using three mutagens of ultraviolet irradiation (UV), N methyl-N’-nitro-N-nitrosoguanidine (NTG) and Ethyl methane sulphonate (EMS). After mutagenesis, the cellulase activity of GC23 (mutant) was improved to 1.86 fold compared to the wild strain (vs15). Optimal conditions for the production of cellulase by the GC 23 strain were evaluated using Response Surface Methodology (RSM) and Artificial Neural Network (ANN). Effect of pH, temperature, duration of incubation, , and substrate concentration on cellulase production were evaluated. Optimal conditions for the production of cellulase enzyme using Carboxy Methyl Cellulase as a substrate are 55 oC of temperature, pH of 5.0 and incubation for 40 h. The cellulase activity of the mutant Streptomyces durhamensis GC23 was further optimised to 2 fold of the activity of the wild type by RSM and ANN.  

Strain improvement of newly isolated Lactobacillus acidophilus MS1 for enhanced bacteriocin production

2017 ◽  
Vol 43 (3) ◽  
pp. 323-332 ◽  
Author(s):  
Mahwish Salman ◽  
Shazia Anwer Bukhari ◽  
Muhammad Shahid ◽  
Tanzila Sahar ◽  
Shazia Naheed
Keyword(s):  
Survival Rate ◽  
Ethidium Bromide ◽  
Lactobacillus Acidophilus ◽  
Strain Improvement ◽  
Bacteriocin Production ◽  
Chemical Mutagenesis ◽  
16S Rdna Sequence Analysis ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
The Stability

Abstract Background: Lactic acid bacteria (LAB) are considered as GRAS (generally recognized as safe) and being used extensively as bio-preservatives. Bacteriocins, the metabolites of LAB, belong to a diverse family of naturally synthesized antimicrobial peptides. Objective: Strain improvement for enhanced bacteriocin production by physical and chemical mutagenesis. Methods: The bacterial strain was identified by 16S rDNA sequence analysis and improved by ultraviolet and ethidium bromide mutation. The resultant bacteriocin was biochemically characterized, purified and analyzed for mass determination. Results: Among mutants of identified Lactobacillus acidophilus MS1, the UV6 (ultraviolet mutant) revealed 3400 AU bacteriocin activity with 42% survival rate and EB5 (ethidium bromide mutant) exhibited 4020 AU with 28% survival rate. Bacteriocin of 6.5 kDa was purified by cation exchange and gel exclusion chromatography. It was found to be thermally stable at 100°C for 30 min and maintained the stability up to 121°C. The activity was monitored in a wide range of pH (4–9). Conclusion: Being resistant to several biochemical parameters, the bacteriocins have an effective incorporation in food, forage and pharmacy. There is a need to engage more efforts to explore novel bacteriocins and multifarious applications.

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