The Experts below are selected from a list of 211089 Experts worldwide ranked by ideXlab platform
Chaoguang Tian - One of the best experts on this subject based on the ideXlab platform.
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development of a genome editing crispr cas9 system in thermophilic fungal myceliophthora species and its application to hyper cellulase Production Strain engineering
Biotechnology for Biofuels, 2017Co-Authors: Qian Liu, Ranran Gao, Liangcai Lin, Wenliang Sun, Jingen Li, Junqi Zhao, Chaoguang TianAbstract:Over the past 3 years, the CRISPR/Cas9 system has revolutionized the field of genome engineering. However, its application has not yet been validated in thermophilic fungi. Myceliophthora thermophila, an important thermophilic biomass-degrading fungus, has attracted industrial interest for the Production of efficient thermostable enzymes. Genetic manipulation of Myceliophthora is crucial for metabolic engineering and to unravel the mechanism of lignocellulose deconstruction. The lack of a powerful, versatile genome-editing tool has impeded the broader exploitation of M. thermophila in biotechnology. In this study, a CRISPR/Cas9 system for efficient multiplexed genome engineering was successfully developed in the thermophilic species M. thermophila and M. heterothallica. This CRISPR/Cas9 system could efficiently mutate the imported amdS gene in the genome via NHEJ-mediated events. As a proof of principle, the genes of the cellulase Production pathway, including cre-1, res-1, gh1-1, and alp-1, were chosen as editing targets. Simultaneous multigene disruptions of up to four of these different loci were accomplished with neomycin selection marker integration via a single transformation using the CRISPR/Cas9 system. Using this genome-engineering tool, multiple Strains exhibiting pronounced hyper-cellulase Production were generated, in which the extracellular secreted protein and lignocellulase activities were significantly increased (up to 5- and 13-fold, respectively) compared with the parental Strain. A genome-wide engineering system for thermophilic fungi was established based on CRISPR/Cas9. Successful expansion of this system without modification to M. heterothallica indicates it has wide adaptability and flexibility for use in other Myceliophthora species. This system could greatly accelerate Strain engineering of thermophilic fungi for Production of industrial enzymes, such as cellulases as shown in this study and possibly bio-based fuels and chemicals in the future.
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MOESM1 of Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase Production Strain engineering
2017Co-Authors: Qian Liu, Ranran Gao, Liangcai Lin, Wenliang Sun, Junqi Zhao, Chaoguang TianAbstract:Additional file 1: Figure S1. Phenotype of WT and constitutive Cas9-expressing Strains. (A) Sodium dodecylsulfate-polyacrylamide gel electrophoresis of secreted protein of the WT and Cas9-OE after 4 days of culture on 2% Avicel. (B-E) Assays for protein concentration and xylanase, endoglucanase and exoglucanase activities of Cas9-OE and the WT in inducing medium with 2% Avicel after 3 and 4 days culture. (F) Mycelial dry weights of Cas9-OE and the WT from cultures on sucrose and Avicel. No significant difference (Tukey’s HSD, p > 0.5) was observed between Cas9-OE and the WT in the above assays
Qian Liu - One of the best experts on this subject based on the ideXlab platform.
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development of a genome editing crispr cas9 system in thermophilic fungal myceliophthora species and its application to hyper cellulase Production Strain engineering
Biotechnology for Biofuels, 2017Co-Authors: Qian Liu, Ranran Gao, Liangcai Lin, Wenliang Sun, Jingen Li, Junqi Zhao, Chaoguang TianAbstract:Over the past 3 years, the CRISPR/Cas9 system has revolutionized the field of genome engineering. However, its application has not yet been validated in thermophilic fungi. Myceliophthora thermophila, an important thermophilic biomass-degrading fungus, has attracted industrial interest for the Production of efficient thermostable enzymes. Genetic manipulation of Myceliophthora is crucial for metabolic engineering and to unravel the mechanism of lignocellulose deconstruction. The lack of a powerful, versatile genome-editing tool has impeded the broader exploitation of M. thermophila in biotechnology. In this study, a CRISPR/Cas9 system for efficient multiplexed genome engineering was successfully developed in the thermophilic species M. thermophila and M. heterothallica. This CRISPR/Cas9 system could efficiently mutate the imported amdS gene in the genome via NHEJ-mediated events. As a proof of principle, the genes of the cellulase Production pathway, including cre-1, res-1, gh1-1, and alp-1, were chosen as editing targets. Simultaneous multigene disruptions of up to four of these different loci were accomplished with neomycin selection marker integration via a single transformation using the CRISPR/Cas9 system. Using this genome-engineering tool, multiple Strains exhibiting pronounced hyper-cellulase Production were generated, in which the extracellular secreted protein and lignocellulase activities were significantly increased (up to 5- and 13-fold, respectively) compared with the parental Strain. A genome-wide engineering system for thermophilic fungi was established based on CRISPR/Cas9. Successful expansion of this system without modification to M. heterothallica indicates it has wide adaptability and flexibility for use in other Myceliophthora species. This system could greatly accelerate Strain engineering of thermophilic fungi for Production of industrial enzymes, such as cellulases as shown in this study and possibly bio-based fuels and chemicals in the future.
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MOESM1 of Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase Production Strain engineering
2017Co-Authors: Qian Liu, Ranran Gao, Liangcai Lin, Wenliang Sun, Junqi Zhao, Chaoguang TianAbstract:Additional file 1: Figure S1. Phenotype of WT and constitutive Cas9-expressing Strains. (A) Sodium dodecylsulfate-polyacrylamide gel electrophoresis of secreted protein of the WT and Cas9-OE after 4 days of culture on 2% Avicel. (B-E) Assays for protein concentration and xylanase, endoglucanase and exoglucanase activities of Cas9-OE and the WT in inducing medium with 2% Avicel after 3 and 4 days culture. (F) Mycelial dry weights of Cas9-OE and the WT from cultures on sucrose and Avicel. No significant difference (Tukey’s HSD, p > 0.5) was observed between Cas9-OE and the WT in the above assays
Vasileios Bampidis - One of the best experts on this subject based on the ideXlab platform.
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safety and efficacy of panavital feed d glyceric acid for chickens for fattening
EFSA Journal, 2020Co-Authors: Efsa Panel On Additives, Giovanna Azimonti, Vasileios Bampidis, Maria De Lourdes Bastos, Henrik Christensen, Birgit Dusemund, Maryline Kouba, Mojca Kos Durjava, Marta Lopezalonso, Secundino Lopez PuenteAbstract:Panavital feed is a preparation of d-glyceric acid intended to be used as a zootechnical additive in chickens for fattening. The active substance (d-glyceric acid) of the additive ■■■■■ by fermentation with Gluconobacter frateurii. The additive is intended to be used in chickens for fattening at a concentration from ■■■■■. The FEEDAP Panel notes that the data provided for the characterisation of the active substance are incomplete and that data on the characterisation of the formulated additive are not provided. Based on the information available, the FEEDAP Panel cannot assess the qualitative and quantitative composition of Panavital feed. Due to lack of data, the safety of the Production Strain claimed to be used to produce the active substance cannot be evaluated. Based on the data provided by the applicant, the FEEDAP Panel cannot conclude on the safety of Panavital feed for the target species, the consumer, the user and the environment and on the efficacy of Panavital feed when used as feed additive.
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Safety and efficacy of l-lysine monohydrochloride and l-lysine sulfate produced using Corynebacterium glutamicum CGMCC 7.266 for all animal species.
EFSA journal. European Food Safety Authority, 2020Co-Authors: Vasileios Bampidis, Giovanna Azimonti, Henrik Christensen, Birgit Dusemund, Maryline Kouba, Marta López-alonso, Maria De Lourdes Bastos, Mojca Kos Durjava, Secundino López PuenteAbstract:Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on l-lysine monohydrochloride and l-lysine sulfate produced using Corynebacterium glutamicum CGMCC 7.266 when used as a nutritional additive in feed and water for drinking for all animal species. The active substance is l-lysine and it is produced in two different forms (monohydrochloride or sulfate salts). None of those forms pose any safety concern associated with the genetic modification of the Production Strain. l-Lysine HCl and l-lysine sulfate produced by C. glutamicum CGMCC 7.266 are considered safe for the target species, for the consumer and for the environment. For both products, the FEEDAP Panel has concerns regarding the safety for the target species when the additives are administered via feed and water for drinking, simultaneously. In the absence of data, the FEEDAP Panel cannot conclude on the safety of both forms of the additive for the user. The products under assessment are considered efficacious sources of the amino acid l-lysine for all animal species. For these products to be as efficacious in ruminants as in non-ruminant species, they require protection against degradation in the rumen.
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Safety and efficacy of l-isoleucine produced by fermentation with Corynebacterium glutamicum KCCM 80189 for all animal species
'Wiley', 2020Co-Authors: Vasileios Bampidis, Giovanna Azimonti, Henrik Christensen, Birgit Dusemund, Maryline Kouba, Marta López-alonso, M.d.l. Bastos, Kos M. Durjava, Lopez S. Puente, F. MarconAbstract:Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of l-isoleucine produced by fermentation using Corynebacterium glutamicum KCCM 80189 when used as a nutritional additive in feed and water for drinking for all animal species. The Production Strain is not genetically modified. Viable cells of the Production Strain were not detected in the final additive. The additive does not give rise to any safety concern regarding the Production Strain. l-Isoleucine produced using C. glutamicum KCCM 80189 is considered safe for the target species, for the consumer and for the environment. l-Isoleucine produced by C. glutamicum KCCM 80189 is considered not toxic by inhalation, not irritant to skin or eyes and not a dermal sensitiser. Regarding the use in water, the FEEDAP Panel reiterates its concerns over the safety of l-isoleucine administered simultaneously via water for drinking and feed owing to the risk of nutritional imbalances and hygienic reasons. l-Isoleucine produced by C. glutamicum KCCM 80189 is considered as an efficacious source of the essential amino acid l-isoleucine for non-ruminant animal species. For the supplemental l-isoleucine to be as efficacious in ruminants as in non-ruminant species, it would require protection against degradation in the rumen
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Assessment of the application for renewal of the authorisation of Amaferm® (fermentation product of Aspergillus oryzae NRRL 458) as a feed additive for dairy cows
'Wiley', 2020Co-Authors: Vasileios Bampidis, Giovanna Azimonti, Birgit Dusemund, Marta López-alonso, M.d.l. Bastos, Kos M. Durjava, Lopez S. Puente, H. Christensen, M. Kouba, F. MarconAbstract:AMAFERM\uae is a fermentation product produced by Aspergillus oryzae NRRL 458, containing alpha-amylase and cellulase enzyme activities, authorised for use as a feed additive for cows. This scientific opinion concerns the renewal of the authorisation of this additive for its use in dairy cows. In its former opinion, the EFSA Panel on Additives and Product or Substances used in Animal Feed (FEEDAP), based on the data and knowledge available at that time, concluded that the additive is safe for cows, for the consumers and the environment. In that assessment, the Panel also concluded that the additive is non-irritant or a dermal sensitiser but should be considered a potential respiratory sensitiser. The applicant provided new information regarding the characterisation of the additive in terms of enzyme activities as well as information concerning the Production Strain. Regarding the enzyme activities in the fermentation product, weaknesses and limitations in the methods of analysis were noted. The information regarding the Production Strain did not permit to confirm its taxonomic classification, moreover uncertainty remains regarding the presence of viable cells/spores in the final product. Therefore, the FEEDAP Panel could not confirm the previously drawn conclusions regarding the safety of the Production Strain and consequently could not confirm the safety for the target species and consumers. There was no need for assessing the efficacy of the additive in the context of the renewal of the authorisation
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Safety and efficacy of l-tryptophan produced by fermentation using Escherichia coli CGMCC 7.267 for all animal species
'Wiley', 2020Co-Authors: Vasileios Bampidis, Giovanna Azimonti, Birgit Dusemund, Marta López-alonso, M.d.l. Bastos, Kos M. Durjava, Lopez S. Puente, H. Christensen, M. Kouba, F. MarconAbstract:Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on l-tryptophan produced by fermentation with a genetically modified Strain of Escherichia coli CGMCC 7.267 when used as a nutritional additive in feed and water for drinking for all animal species and categories. The Production Strain E. coli CGMCC 7.267 is safe for the Production of l-tryptophan. No viable cells or DNA of the Production Strain were detected in the additive under assessment. The use of l-tryptophan produced using E. coli CGMCC 7.267 in supplementing feed to compensate for tryptophan deficiency in feedingstuffs is safe for non-ruminant target species. However, excess doses would create amino acid imbalances with negative consequences on animal performance. The use of unprotected l-tryptophan in feed poses safety concerns for ruminants. The use of l-tryptophan produced by fermentation with E. coli CGMCC 7.267 in animal nutrition is considered safe for the consumers and for the environment. The endotoxin activity in the product and its dusting potential indicate an inhalation risk for the user. In the absence of data, the FEEDAP Panel cannot conclude on the potential of the additive to be irritant to skin and eyes or to be a skin sensitiser. The additive l-tryptophan produced using E. coli CGMCC 7.267 is regarded as an effective source of the amino acid l-tryptophan. In order to be as efficacious in ruminants as in non-ruminants, it should be protected from ruminal degradation
F. Marcon - One of the best experts on this subject based on the ideXlab platform.
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Safety and efficacy of l-isoleucine produced by fermentation with Corynebacterium glutamicum KCCM 80189 for all animal species
'Wiley', 2020Co-Authors: Vasileios Bampidis, Giovanna Azimonti, Henrik Christensen, Birgit Dusemund, Maryline Kouba, Marta López-alonso, M.d.l. Bastos, Kos M. Durjava, Lopez S. Puente, F. MarconAbstract:Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of l-isoleucine produced by fermentation using Corynebacterium glutamicum KCCM 80189 when used as a nutritional additive in feed and water for drinking for all animal species. The Production Strain is not genetically modified. Viable cells of the Production Strain were not detected in the final additive. The additive does not give rise to any safety concern regarding the Production Strain. l-Isoleucine produced using C. glutamicum KCCM 80189 is considered safe for the target species, for the consumer and for the environment. l-Isoleucine produced by C. glutamicum KCCM 80189 is considered not toxic by inhalation, not irritant to skin or eyes and not a dermal sensitiser. Regarding the use in water, the FEEDAP Panel reiterates its concerns over the safety of l-isoleucine administered simultaneously via water for drinking and feed owing to the risk of nutritional imbalances and hygienic reasons. l-Isoleucine produced by C. glutamicum KCCM 80189 is considered as an efficacious source of the essential amino acid l-isoleucine for non-ruminant animal species. For the supplemental l-isoleucine to be as efficacious in ruminants as in non-ruminant species, it would require protection against degradation in the rumen
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Assessment of the application for renewal of the authorisation of Amaferm® (fermentation product of Aspergillus oryzae NRRL 458) as a feed additive for dairy cows
'Wiley', 2020Co-Authors: Vasileios Bampidis, Giovanna Azimonti, Birgit Dusemund, Marta López-alonso, M.d.l. Bastos, Kos M. Durjava, Lopez S. Puente, H. Christensen, M. Kouba, F. MarconAbstract:AMAFERM\uae is a fermentation product produced by Aspergillus oryzae NRRL 458, containing alpha-amylase and cellulase enzyme activities, authorised for use as a feed additive for cows. This scientific opinion concerns the renewal of the authorisation of this additive for its use in dairy cows. In its former opinion, the EFSA Panel on Additives and Product or Substances used in Animal Feed (FEEDAP), based on the data and knowledge available at that time, concluded that the additive is safe for cows, for the consumers and the environment. In that assessment, the Panel also concluded that the additive is non-irritant or a dermal sensitiser but should be considered a potential respiratory sensitiser. The applicant provided new information regarding the characterisation of the additive in terms of enzyme activities as well as information concerning the Production Strain. Regarding the enzyme activities in the fermentation product, weaknesses and limitations in the methods of analysis were noted. The information regarding the Production Strain did not permit to confirm its taxonomic classification, moreover uncertainty remains regarding the presence of viable cells/spores in the final product. Therefore, the FEEDAP Panel could not confirm the previously drawn conclusions regarding the safety of the Production Strain and consequently could not confirm the safety for the target species and consumers. There was no need for assessing the efficacy of the additive in the context of the renewal of the authorisation
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Safety and efficacy of l-tryptophan produced by fermentation using Escherichia coli CGMCC 7.267 for all animal species
'Wiley', 2020Co-Authors: Vasileios Bampidis, Giovanna Azimonti, Birgit Dusemund, Marta López-alonso, M.d.l. Bastos, Kos M. Durjava, Lopez S. Puente, H. Christensen, M. Kouba, F. MarconAbstract:Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on l-tryptophan produced by fermentation with a genetically modified Strain of Escherichia coli CGMCC 7.267 when used as a nutritional additive in feed and water for drinking for all animal species and categories. The Production Strain E. coli CGMCC 7.267 is safe for the Production of l-tryptophan. No viable cells or DNA of the Production Strain were detected in the additive under assessment. The use of l-tryptophan produced using E. coli CGMCC 7.267 in supplementing feed to compensate for tryptophan deficiency in feedingstuffs is safe for non-ruminant target species. However, excess doses would create amino acid imbalances with negative consequences on animal performance. The use of unprotected l-tryptophan in feed poses safety concerns for ruminants. The use of l-tryptophan produced by fermentation with E. coli CGMCC 7.267 in animal nutrition is considered safe for the consumers and for the environment. The endotoxin activity in the product and its dusting potential indicate an inhalation risk for the user. In the absence of data, the FEEDAP Panel cannot conclude on the potential of the additive to be irritant to skin and eyes or to be a skin sensitiser. The additive l-tryptophan produced using E. coli CGMCC 7.267 is regarded as an effective source of the amino acid l-tryptophan. In order to be as efficacious in ruminants as in non-ruminants, it should be protected from ruminal degradation
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Safety and efficacy of l-tryptophan produced by fermentation with Escherichia coli KCCM 80135 for all animal species
'Wiley', 2019Co-Authors: Vasileios Bampidis, Birgit Dusemund, Marta López-alonso, Kos M. Durjava, Lopez S. Puente, F. Marcon, H. Christensen, M. Kouba, M. De Lourdes Bastos, B. MayoAbstract:Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on l-tryptophan produced by fermentation using Escherichia coli KCCM 80135 when used as a nutritional additive in feed and water for drinking for all animal species. The Production Strain and its recombinant DNA were not detected in the additive. l-Tryptophan produced by fermentation with E. coli KCCM 80135 does not raise any safety concern with regard to the genetic modification of the Production Strain. The use of l-tryptophan produced using E. coli KCCM 80135 in supplementing feed to compensate for tryptophan deficiency in feedingstuffs is safe for non-ruminant target species. Using unprotected forms of tryptophan in ruminants can be a risk. The use of l-tryptophan produced by fermentation using E. coli KCCM 80135 in animal nutrition presents no safety concerns to consumers of animal products. l-Tryptophan produced by E. coli KCCM 80135 is not toxic by inhalation. The additive is not an irritant to skin and eyes, and it is not a skin sensitiser. The additive under assessment is considered safe for the environment. It is regarded as an effective source of the amino acid l-tryptophan for all non-ruminant species. If the additive l-tryptophan is intended for use in ruminants, it should be protected from ruminal degradation
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Safety and efficacy of l‐tryptophan produced by fermentation with Escherichia coli CGMCC 7.248 for all animal species
'Wiley', 2019Co-Authors: Vasileios Bampidis, Giovanna Azimonti, Birgit Dusemund, M.d.l. Bastos, Kos M. Durjava, Lopez S. Puente, H. Christensen, M. Kouba, Marta López‐alonso, F. MarconAbstract:Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on L-tryptophan produced by fermentation with a genetically modified Strain of Escherichia coli CGMCC 7.248 when used as a nutritional additive in feed and water for drinking for all animal species and categories. The Production Strain E. coli CGMCC 7.248 and its recombinant DNA were not detected in the final product. The product L-tryptophan, manufactured by fermentation with E. coli CGMCC 7.248, does not give rise to any safety concern with regard to the genetic modification of the Production Strain. L-Tryptophan produced by E. coli CGMCC 7.248 is safe for non-ruminant target species. The use of unprotected L-tryptophan in ruminant feed should be avoided. L-Tryptophan produced by fermentation by E. coli CGMCC 7.248 is safe for the consumer. The level of endotoxins present in the product and its dusting potential indicate an inhalation risk for the user. L-Tryptophan produced by E. coli CGMCC 7.248 is not a skin or eye irritant but it is a dermal sensitiser. The use of L-tryptophan produced by E. coli CGMCC 7.248 in animal nutrition does not pose a risk to the environment. The product under assessment is regarded as an effective source of the amino acid L-tryptophan for all non-ruminant species. For the supplemental L-tryptophan to be as efficacious in ruminants as in non-ruminant species, it requires protection against degradation in the rumen
Junqi Zhao - One of the best experts on this subject based on the ideXlab platform.
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development of a genome editing crispr cas9 system in thermophilic fungal myceliophthora species and its application to hyper cellulase Production Strain engineering
Biotechnology for Biofuels, 2017Co-Authors: Qian Liu, Ranran Gao, Liangcai Lin, Wenliang Sun, Jingen Li, Junqi Zhao, Chaoguang TianAbstract:Over the past 3 years, the CRISPR/Cas9 system has revolutionized the field of genome engineering. However, its application has not yet been validated in thermophilic fungi. Myceliophthora thermophila, an important thermophilic biomass-degrading fungus, has attracted industrial interest for the Production of efficient thermostable enzymes. Genetic manipulation of Myceliophthora is crucial for metabolic engineering and to unravel the mechanism of lignocellulose deconstruction. The lack of a powerful, versatile genome-editing tool has impeded the broader exploitation of M. thermophila in biotechnology. In this study, a CRISPR/Cas9 system for efficient multiplexed genome engineering was successfully developed in the thermophilic species M. thermophila and M. heterothallica. This CRISPR/Cas9 system could efficiently mutate the imported amdS gene in the genome via NHEJ-mediated events. As a proof of principle, the genes of the cellulase Production pathway, including cre-1, res-1, gh1-1, and alp-1, were chosen as editing targets. Simultaneous multigene disruptions of up to four of these different loci were accomplished with neomycin selection marker integration via a single transformation using the CRISPR/Cas9 system. Using this genome-engineering tool, multiple Strains exhibiting pronounced hyper-cellulase Production were generated, in which the extracellular secreted protein and lignocellulase activities were significantly increased (up to 5- and 13-fold, respectively) compared with the parental Strain. A genome-wide engineering system for thermophilic fungi was established based on CRISPR/Cas9. Successful expansion of this system without modification to M. heterothallica indicates it has wide adaptability and flexibility for use in other Myceliophthora species. This system could greatly accelerate Strain engineering of thermophilic fungi for Production of industrial enzymes, such as cellulases as shown in this study and possibly bio-based fuels and chemicals in the future.
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MOESM1 of Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase Production Strain engineering
2017Co-Authors: Qian Liu, Ranran Gao, Liangcai Lin, Wenliang Sun, Junqi Zhao, Chaoguang TianAbstract:Additional file 1: Figure S1. Phenotype of WT and constitutive Cas9-expressing Strains. (A) Sodium dodecylsulfate-polyacrylamide gel electrophoresis of secreted protein of the WT and Cas9-OE after 4 days of culture on 2% Avicel. (B-E) Assays for protein concentration and xylanase, endoglucanase and exoglucanase activities of Cas9-OE and the WT in inducing medium with 2% Avicel after 3 and 4 days culture. (F) Mycelial dry weights of Cas9-OE and the WT from cultures on sucrose and Avicel. No significant difference (Tukey’s HSD, p > 0.5) was observed between Cas9-OE and the WT in the above assays
