Glutamine involvement in nitrogen regulation of cellulase production in fungi

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.

Download Full-text

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

Biotechnology for Biofuels ◽

10.1186/s13068-021-01926-w ◽

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.

Download Full-text

High-dose rapamycin excerts a temporary impact on T. reesei through gene trFKBP12

10.21203/rs.3.rs-110351/v1 ◽

2020 ◽

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 ◽

High Concentration ◽

Tor Signaling ◽

Inhibition Effect ◽

Temporary Inhibition

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 TOR (Target of Rapamycin) 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 remains 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 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 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 at its early cultivation stage, demonstrating T. reesei 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.

Download Full-text

Glucose-lactose mixture feeds in industry-like conditions: a gene regulatory network analysis on the hyperproducing Trichoderma reesei strain Rut-C30

10.21203/rs.3.rs-29020/v2 ◽

2020 ◽

Author(s):

Aurélie Pirayre ◽

Laurent Duval ◽

Corinne Blugeon ◽

Cyril Firmo ◽

Sandrine Perrin ◽

...

Keyword(s):

Transcription Factors ◽

Gene Regulatory Network ◽

Trichoderma Reesei ◽

Regulatory Network ◽

Cellulase Production ◽

Biofuel Production ◽

Lactose Concentration ◽

Gene Regulatory ◽

The Impact ◽

Rut C30

Abstract Background: The degradation of cellulose and hemicellulose molecules into simpler sugars such as glucose is part of the second generation biofuel production process. Hydrolysis of lignocellulosic substrates is usually performed by enzymes produced and secreted by the fungus Trichoderma reesei . Studies identifying transcription factors involved in the regulation of cellulase production have been conducted but no overview of the whole regulation network is available. A transcriptomic approach with mixtures of glucose and lactose, used as a substrate for cellulase induction, was used to help us decipher missing parts in the network of T. reesei Rut-C30.Results: Experimental results on the Rut-C30 hyperproducing strain confirmed the impact of sugar mixtures on the enzymatic cocktail composition. The transcriptomic study shows a temporal regulation of the main transcription factors and a lactose concentration impact on the transcriptional profile. A gene regulatory network built using BRANE Cut software reveals three sub-networks related to iq a positive correlation between lactose concentration and cellulase production, iiq a particular dependence of the lactose onto the β-glucosidase regulation and iiiq a negative regulation of the development process and growth.Conclusions: This work is the first investigating a transcriptomic study regarding the effects of pure and mixed carbon sources in a fed-batch mode. Our study expose a co-orchestration of xyr1 , clr2 and ace3 for cellulase and hemicellulase induction and production, a fine regulation of the β-glucosidase and a decrease of growth in favor of cellulase production. These conclusions provide us with potential targets for further genetic engineering leading to bettercellulase-producing strains in industry-like conditions.

Download Full-text

Glucose-lactose mixture feeds in industry-like conditions: a gene regulatory network analysis on the hyperproducing Trichoderma reesei strain Rut-C30

BMC Genomics ◽

10.1186/s12864-020-07281-8 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Aurélie Pirayre ◽

Laurent Duval ◽

Corinne Blugeon ◽

Cyril Firmo ◽

Sandrine Perrin ◽

...

Keyword(s):

Transcription Factors ◽

Gene Regulatory Network ◽

Trichoderma Reesei ◽

Regulatory Network ◽

Cellulase Production ◽

Biofuel Production ◽

Lactose Concentration ◽

Gene Regulatory ◽

The Impact ◽

Rut C30

Abstract Background The degradation of cellulose and hemicellulose molecules into simpler sugars such as glucose is part of the second generation biofuel production process. Hydrolysis of lignocellulosic substrates is usually performed by enzymes produced and secreted by the fungus Trichoderma reesei. Studies identifying transcription factors involved in the regulation of cellulase production have been conducted but no overview of the whole regulation network is available. A transcriptomic approach with mixtures of glucose and lactose, used as a substrate for cellulase induction, was used to help us decipher missing parts in the network of T. reesei Rut-C30. Results Experimental results on the Rut-C30 hyperproducing strain confirmed the impact of sugar mixtures on the enzymatic cocktail composition. The transcriptomic study shows a temporal regulation of the main transcription factors and a lactose concentration impact on the transcriptional profile. A gene regulatory network built using BRANE Cut software reveals three sub-networks related to i) a positive correlation between lactose concentration and cellulase production, ii) a particular dependence of the lactose onto the β-glucosidase regulation and iii) a negative regulation of the development process and growth. Conclusions This work is the first investigating a transcriptomic study regarding the effects of pure and mixed carbon sources in a fed-batch mode. Our study expose a co-orchestration of xyr1, clr2 and ace3 for cellulase and hemicellulase induction and production, a fine regulation of the β-glucosidase and a decrease of growth in favor of cellulase production. These conclusions provide us with potential targets for further genetic engineering leading to better cellulase-producing strains in industry-like conditions.

Download Full-text

Glucose-lactose mixture feeds in industry-like conditions: a gene regulatory network analysis on the hyperproducing Trichoderma reesei strain Rut-C30

10.21203/rs.3.rs-29020/v4 ◽

2020 ◽

Author(s):

Aurélie Pirayre ◽

Laurent Duval ◽

Corinne Blugeon ◽

Cyril Firmo ◽

Sandrine Perrin ◽

...

Keyword(s):

Transcription Factors ◽

Gene Regulatory Network ◽

Trichoderma Reesei ◽

Regulatory Network ◽

Cellulase Production ◽

Biofuel Production ◽

Lactose Concentration ◽

Gene Regulatory ◽

The Impact ◽

Rut C30

Abstract Background: The degradation of cellulose and hemicellulose molecules into simpler sugars such as glucose is part of the second generation biofuel production process. Hydrolysis of lignocellulosic substrates is usually performed by enzymes produced and secreted by the fungus Trichoderma reesei. Studies identifying transcription factors involved in the regulation of cellulase production have been conducted but no overview of the whole regulation network is available. A transcriptomic approach with mixtures of glucose and lactose, used as a substrate for cellulase induction, was used to help us decipher missing parts in the network of T. reesei Rut-C30. Results: Experimental results on the Rut-C30 hyperproducing strain confirmed the impact of sugar mixtures on the enzymatic cocktail composition. The transcriptomic study shows a temporal regulation of the main transcription factors and a lactose concentration impact on the transcriptional profile. A gene regulatory network built using BRANE Cut software reveals three sub-networks related to i) a positive correlation between lactose concentration and cellulase production, ii) a particular dependence of the lactose onto the β-glucosidase regulation and iii) a negative regulation of the development process and growth. Conclusions: This work is the first investigating a transcriptomic study regarding the effects of pure and mixed carbon sources in a fed-batch mode. Our study expose a co-orchestration of xyr1, clr2 and ace3 for cellulase and hemicellulase induction and production, a fine regulation of the β-glucosidase and a decrease of growth in favor of cellulase production. These conclusions provide us with potential targets for further genetic engineering leading to better cellulase-producing strains in industry-like conditions.

Download Full-text

Glucose-lactose mixture feeds in industry-like conditions: a gene regulatory network analysis on the hyperproducing Trichoderma reesei strain Rut-C30

10.21203/rs.3.rs-29020/v3 ◽

2020 ◽

Author(s):

Aurélie Pirayre ◽

Laurent Duval ◽

Corinne Blugeon ◽

Cyril Firmo ◽

Sandrine Perrin ◽

...

Keyword(s):

Transcription Factors ◽

Gene Regulatory Network ◽

Trichoderma Reesei ◽

Regulatory Network ◽

Cellulase Production ◽

Biofuel Production ◽

Lactose Concentration ◽

Gene Regulatory ◽

The Impact ◽

Rut C30

Abstract Background: The degradation of cellulose and hemicellulose molecules into simpler sugars such as glucose is part of the second generation biofuel production process. Hydrolysis of lignocellulosic substrates is usually performed by enzymes produced and secreted by the fungus Trichoderma reesei. Studies identifying transcription factors involved in the regulation of cellulase production have been conducted but no overview of the whole regulation network is available. A transcriptomic approach with mixtures of glucose and lactose, used as a substrate for cellulase induction, was used to help us decipher missing parts in the network of T. reesei Rut-C30. Results: Experimental results on the Rut-C30 hyperproducing strain confirmed the impact of sugar mixtures on the enzymatic cocktail composition. The transcriptomic study shows a temporal regulation of the main transcription factors and a lactose concentration impact on the transcriptional profile. A gene regulatory network built using BRANE Cut software reveals three sub-networks related to i) a positive correlation between lactose concentration and cellulase production, ii) a particular dependence of the lactose onto the β-glucosidase regulation and iii) a negative regulation of the development process and growth. Conclusions: This work is the first investigating a transcriptomic study regarding the effects of pure and mixed carbon sources in a fed-batch mode. Our study expose a co-orchestration of xyr1, clr2 and ace3 for cellulase and hemicellulase induction and production, a fine regulation of the β-glucosidase and a decrease of growth in favor of cellulase production. These conclusions provide us with potential targets for further genetic engineering leading to better cellulase-producing strains in industry-like conditions.

Download Full-text

Eukarion-134 Attenuates Endoplasmic Reticulum Stress-Induced Mitochondrial Dysfunction in Human Skeletal Muscle Cells

Antioxidants ◽

10.3390/antiox9080710 ◽

2020 ◽

Vol 9 (8) ◽

pp. 710

Author(s):

Anastasia Thoma ◽

Max Lyon ◽

Nasser Al-Shanti ◽

Gareth A. Nye ◽

Robert G. Cooper ◽

...

Keyword(s):

Skeletal Muscle ◽

Endoplasmic Reticulum ◽

Er Stress ◽

Mitochondrial Dysfunction ◽

Muscle Weakness ◽

Ros Generation ◽

Transcriptional Level ◽

Mrna Levels ◽

Human Skeletal Muscle Cells ◽

The Impact

Maladaptive endoplasmic reticulum (ER) stress is associated with modified reactive oxygen species (ROS) generation and mitochondrial abnormalities; and is postulated as a potential mechanism involved in muscle weakness in myositis, an acquired autoimmune neuromuscular disease. This study investigates the impact of ROS generation in an in vitro model of ER stress in skeletal muscle, using the ER stress inducer tunicamycin (24 h) in the presence or absence of a superoxide dismutase/catalase mimetic Eukarion (EUK)-134. Tunicamycin induced maladaptive ER stress, which was mitigated by EUK-134 at the transcriptional level. ER stress promoted mitochondrial dysfunction, described by substantial loss of mitochondrial membrane potential, as well as a reduction in respiratory control ratio, reserve capacity, phosphorylating respiration, and coupling efficiency, which was ameliorated by EUK-134. Tunicamycin induced ROS-mediated biogenesis and fusion of mitochondria, which, however, had high propensity of fragmentation, accompanied by upregulated mRNA levels of fission-related markers. Increased cellular ROS generation was observed under ER stress that was prevented by EUK-134, even though no changes in mitochondrial superoxide were noticeable. These findings suggest that targeting ROS generation using EUK-134 can amend aspects of ER stress-induced changes in mitochondrial dynamics and function, and therefore, in instances of chronic ER stress, such as in myositis, quenching ROS generation may be a promising therapy for muscle weakness and dysfunction.

Download Full-text

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

Canadian Journal of Microbiology ◽

10.1139/cjm-2020-0438 ◽

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.

Download Full-text

Analysis of the Impact of Three Phthalates on the Freshwater Gastropod Physella Acuta at the Transcriptional Level

10.21203/rs.3.rs-154303/v1 ◽

2021 ◽

Author(s):

Marina Prieto-Amador ◽

Patricia Caballero ◽

Jose-Luis Martinez-Guitarte

Keyword(s):

Stress Responses ◽

Molecular Level ◽

Transcriptional Level ◽

Mrna Levels ◽

Plastic Pollution ◽

Physella Acuta ◽

Butyl Phthalate ◽

General Response ◽

Almost All ◽

The Impact

Abstract Plastic pollution is one of the leading environmental problems. Phthalates are widely used plastic additives released into the environment. Although the phthalates' effects have been extensively studied on vertebrates, there is a gap in knowledge on their effects on invertebrates. This work analyzes the impact of three phthalates, diethyl phthalate (DEP), benzyl butyl phthalate (BBP), and bis-(2-ethylhexyl) phthalate (DEHP), on the gastropod Physella acuta at the molecular level to establish the putative pathways involved in its response to them. By real-time PCR, we obtained the expression profile of thirty genes in one-week exposed animals at 0.1, 10, and 1000 µg/L. The genes cover the DNA repairing mechanism, detoxification mechanisms, apoptosis, oxidative and stress responses, immunity, energy reserves, and lipid transport. The results show that while DEP and DEHP did not cause alteration of the mRNA levels, BBP modulates almost all the genes tested. It can be concluded that the impact of BBP is extensive at the molecular level. However, it cannot be dismissed that the increase in transcriptional activity is a general response due to this compound’s well-known role as an endocrine disruptor. Additional research is needed to elucidate the differences observed in the impact of these compounds on this gastropod.

Download Full-text

Analysis of the impact of three phthalates on the freshwater gastropod Physella acuta at the transcriptional level

Scientific Reports ◽

10.1038/s41598-021-90934-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marina Prieto-Amador ◽

Patricia Caballero ◽

José-Luis Martínez-Guitarte

Keyword(s):

Stress Responses ◽

Molecular Level ◽

Transcriptional Level ◽

Mrna Levels ◽

Plastic Pollution ◽

Physella Acuta ◽

Butyl Phthalate ◽

General Response ◽

Almost All ◽

The Impact

AbstractPlastic pollution is one of the leading environmental problems. Phthalates are widely used plastic additives released into the environment. Although the effects of phthalates on vertebrates have been extensively studied, there is a knowledge gap regarding their effects on invertebrates. This work analyzes the impact of three phthalates, diethyl phthalate (DEP), benzyl butyl phthalate (BBP), and bis-(2-ethylhexyl) phthalate (DEHP), on the gastropod Physella acuta at the molecular level to establish the putative pathways involved in its response to them. By real-time PCR, we obtained the expression profile of 30 genes in animals exposed for 1 week to 0.1, 10, and 1000 μg/L of each phthalate. The genes cover DNA repair, detoxification, apoptosis, oxidative and stress responses, immunity, energy reserves, and lipid transport. The results show that while DEP and DEHP did not alter the mRNA levels, BBP modulated almost all the analyzed genes. It can be concluded that the impact of BBP is extensive at the molecular level. However, it cannot be dismissed that the increase in transcriptional activity is a general response due to this compound’s well-known role as an endocrine disruptor. Additional research is needed to elucidate the differences observed in the impact of these compounds on the gastropod P. acuta.

Download Full-text