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Characterization of a copper responsive promoter and its mediated overexpression of the xylanase regulator 1 results in an induction-independent production of cellulases in Trichoderma reesei.

Lv X, Zheng F, Li C, Zhang W, Chen G, Liu W - Biotechnol Biofuels (2015)

Bottom Line: Whereas excess copper repressed the expression of tcu1 from T. reesei, eliminating copper addition in the medium resulted in a high-level transcription of tcu1.Expression of T. reesei tcu1 gene was tightly controlled by copper availability, and a homologous protein expression system was developed based on this promoter.Deregulation of XYR1 (xylanase regulator 1) mediated by the tcu1 promoter not only overcame the carbon catabolite repression of cellulases but also resulted in their full expression even on the non-inducing carbon sources.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, No.27 Shanda South Road, Jinan, 250100 Shandong People's Republic of China.

ABSTRACT

Background: Trichoderma reesei represents an important workhorse for industrial production of cellulases as well as other proteins. The large-scale production is usually performed in a substrate-inducing manner achieved by a fine-tuned cooperation of a suite of transcription factors. Their production and subsequent analysis are, however, often either difficult to manipulate or complicated by the concomitant production of other inducible proteins. Alternatives to control gene expression independent of the nutritional state are thus preferred in some cases to facilitate not only biochemical studies of proteins but also genetic engineering of the producer.

Results: We identified a copper transporter encoding gene tcu1 (jgi:Trire2:52315) in T. reesei, the transcription of which was highly responsive to copper availability. Whereas excess copper repressed the expression of tcu1 from T. reesei, eliminating copper addition in the medium resulted in a high-level transcription of tcu1. The usefulness of the system was further illustrated by the high-level expression of specific cellulases driven by the tcu1 promoter in T. reesei when cultivated on D-glucose or glycerol as the sole carbon source. A recombinant T. reesei strain, which overexpressed the main transcription activator of hydrolases (xylanase regulator 1) under the control of tcu1 promoter, was found to be relieved from the carbon catabolite repression and thus displayed a constitutive cellulase expression. Moreover, the amount and activities of cellulases produced by this strain on glycerol or glucose fully recapitulated those of the parental strain produced on Avicel.

Conclusion: Expression of T. reesei tcu1 gene was tightly controlled by copper availability, and a homologous protein expression system was developed based on this promoter. Deregulation of XYR1 (xylanase regulator 1) mediated by the tcu1 promoter not only overcame the carbon catabolite repression of cellulases but also resulted in their full expression even on the non-inducing carbon sources.

No MeSH data available.


Related in: MedlinePlus

Homologous expression of CEL7A and CEL7B mediated by Ptcu1. SDS-PAGE analysis of the extracellular proteins of Ptcu1-cel7b grown on 1% (wt/vol) glucose (A) or glycerol (B) with (lower panel) or without (upper panel) addition of CuSO4. The extracellular CMC and pNPC hydrolytic activities of Ptcu1-cel7b and its parental strain Δcel7a grown on 1% (wt/vol) glucose (C) or glycerol (D). Error bars are the SD from two biological replicates. (E) qRT-PCR analysis of the cel7b mRNA of Ptcu1-cel7b cultured with 1% (wt/vol) glucose or 1% (wt/vol) glycerol in the presence or absence of 10 μM CuSO4. A significant difference (P < 0.05) existed for the transcription of cel7b with and without 10 μΜ Cu2+. Error bars are the SD from three biological replicates. (F) The extracellular pNPC hydrolytic activities of Ptcu1-cel7b in the presence of different concentrations of copper sulfate. (G) SDS-PAGE and Western blot analysis of the extracellular production of CEL7A by Ptcu1-cel7a grown on 1% (wt/vol) glucose with (right panel) or without (left panel) addition of CuSO4. Equal amount of culture supernatant relative to biomass was measured for all the assays.
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Fig3: Homologous expression of CEL7A and CEL7B mediated by Ptcu1. SDS-PAGE analysis of the extracellular proteins of Ptcu1-cel7b grown on 1% (wt/vol) glucose (A) or glycerol (B) with (lower panel) or without (upper panel) addition of CuSO4. The extracellular CMC and pNPC hydrolytic activities of Ptcu1-cel7b and its parental strain Δcel7a grown on 1% (wt/vol) glucose (C) or glycerol (D). Error bars are the SD from two biological replicates. (E) qRT-PCR analysis of the cel7b mRNA of Ptcu1-cel7b cultured with 1% (wt/vol) glucose or 1% (wt/vol) glycerol in the presence or absence of 10 μM CuSO4. A significant difference (P < 0.05) existed for the transcription of cel7b with and without 10 μΜ Cu2+. Error bars are the SD from three biological replicates. (F) The extracellular pNPC hydrolytic activities of Ptcu1-cel7b in the presence of different concentrations of copper sulfate. (G) SDS-PAGE and Western blot analysis of the extracellular production of CEL7A by Ptcu1-cel7a grown on 1% (wt/vol) glucose with (right panel) or without (left panel) addition of CuSO4. Equal amount of culture supernatant relative to biomass was measured for all the assays.

Mentions: To further test the potential of developing a Ptcu1-based expression system in T. reesei, two cellulase genes including cellobiohydrolase encoding gene (cel7a) and endocellulase encoding gene (cel7b) were chosen for testing the expression. The respective expression plasmids were transformed into a Δcel7a strain to avoid the potential contamination of endogenous CEL7A, and the extracellular protein production as well as the hydrolytic activity was followed during growth on 1% D-glucose or 1% glycerol. The growth rate was similar with or without CuSO4 (data not shown). As shown in Figure 3A,B,G, a significant amount of CEL7B and CEL7A was detected in the culture supernatant of transformants Ptcu1-cel7b and Ptcu1-cel7a. The produced CEL7B and CEL7A was not due to an endogenous expression since no corresponding protein bands could be detected in the parental Δcel7a strain on glucose or glycerol regardless of the presence or absence of copper (data not shown). Moreover, these proteins were absent from the culture supernatant of the respective transformants when 10 μM CuSO4 was included, suggesting the expression was specifically responsive to the copper. Further examination of the cel7b transcripts by quantitative real-time PCR (qRT-PCR) revealed that, while the expression of the endogenous cel7b was below a detectable level, a significant induced transcription was observed for cel7b in the Ptcu1-cel7b strain in the absence of copper, which was dramatically decreased by 10 μM copper (Figure 3E). The produced CEL7A was verified by Western blot (Figure 3G). Analysis of the hydrolytic activities verified that the produced CEL7B displayed significant carboxymethylcellulose (CMC) and p-nitrophenol-D-cellbioside (pNPC) hydrolytic activities which were strictly dependent on copper availability (Figure 3C,D,F), whereas hardly any pNPC hydrolytic activity was detected from the culture supernatant of Ptcu1-cel7a (data not shown), indicating that CEL7A produced under this condition may not adopt a correct conformation. Thus, the T. reesei tcu1 promoter can be used for copper-controlled expression of both homologous and heterologous proteins.Figure 3


Characterization of a copper responsive promoter and its mediated overexpression of the xylanase regulator 1 results in an induction-independent production of cellulases in Trichoderma reesei.

Lv X, Zheng F, Li C, Zhang W, Chen G, Liu W - Biotechnol Biofuels (2015)

Homologous expression of CEL7A and CEL7B mediated by Ptcu1. SDS-PAGE analysis of the extracellular proteins of Ptcu1-cel7b grown on 1% (wt/vol) glucose (A) or glycerol (B) with (lower panel) or without (upper panel) addition of CuSO4. The extracellular CMC and pNPC hydrolytic activities of Ptcu1-cel7b and its parental strain Δcel7a grown on 1% (wt/vol) glucose (C) or glycerol (D). Error bars are the SD from two biological replicates. (E) qRT-PCR analysis of the cel7b mRNA of Ptcu1-cel7b cultured with 1% (wt/vol) glucose or 1% (wt/vol) glycerol in the presence or absence of 10 μM CuSO4. A significant difference (P < 0.05) existed for the transcription of cel7b with and without 10 μΜ Cu2+. Error bars are the SD from three biological replicates. (F) The extracellular pNPC hydrolytic activities of Ptcu1-cel7b in the presence of different concentrations of copper sulfate. (G) SDS-PAGE and Western blot analysis of the extracellular production of CEL7A by Ptcu1-cel7a grown on 1% (wt/vol) glucose with (right panel) or without (left panel) addition of CuSO4. Equal amount of culture supernatant relative to biomass was measured for all the assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Fig3: Homologous expression of CEL7A and CEL7B mediated by Ptcu1. SDS-PAGE analysis of the extracellular proteins of Ptcu1-cel7b grown on 1% (wt/vol) glucose (A) or glycerol (B) with (lower panel) or without (upper panel) addition of CuSO4. The extracellular CMC and pNPC hydrolytic activities of Ptcu1-cel7b and its parental strain Δcel7a grown on 1% (wt/vol) glucose (C) or glycerol (D). Error bars are the SD from two biological replicates. (E) qRT-PCR analysis of the cel7b mRNA of Ptcu1-cel7b cultured with 1% (wt/vol) glucose or 1% (wt/vol) glycerol in the presence or absence of 10 μM CuSO4. A significant difference (P < 0.05) existed for the transcription of cel7b with and without 10 μΜ Cu2+. Error bars are the SD from three biological replicates. (F) The extracellular pNPC hydrolytic activities of Ptcu1-cel7b in the presence of different concentrations of copper sulfate. (G) SDS-PAGE and Western blot analysis of the extracellular production of CEL7A by Ptcu1-cel7a grown on 1% (wt/vol) glucose with (right panel) or without (left panel) addition of CuSO4. Equal amount of culture supernatant relative to biomass was measured for all the assays.
Mentions: To further test the potential of developing a Ptcu1-based expression system in T. reesei, two cellulase genes including cellobiohydrolase encoding gene (cel7a) and endocellulase encoding gene (cel7b) were chosen for testing the expression. The respective expression plasmids were transformed into a Δcel7a strain to avoid the potential contamination of endogenous CEL7A, and the extracellular protein production as well as the hydrolytic activity was followed during growth on 1% D-glucose or 1% glycerol. The growth rate was similar with or without CuSO4 (data not shown). As shown in Figure 3A,B,G, a significant amount of CEL7B and CEL7A was detected in the culture supernatant of transformants Ptcu1-cel7b and Ptcu1-cel7a. The produced CEL7B and CEL7A was not due to an endogenous expression since no corresponding protein bands could be detected in the parental Δcel7a strain on glucose or glycerol regardless of the presence or absence of copper (data not shown). Moreover, these proteins were absent from the culture supernatant of the respective transformants when 10 μM CuSO4 was included, suggesting the expression was specifically responsive to the copper. Further examination of the cel7b transcripts by quantitative real-time PCR (qRT-PCR) revealed that, while the expression of the endogenous cel7b was below a detectable level, a significant induced transcription was observed for cel7b in the Ptcu1-cel7b strain in the absence of copper, which was dramatically decreased by 10 μM copper (Figure 3E). The produced CEL7A was verified by Western blot (Figure 3G). Analysis of the hydrolytic activities verified that the produced CEL7B displayed significant carboxymethylcellulose (CMC) and p-nitrophenol-D-cellbioside (pNPC) hydrolytic activities which were strictly dependent on copper availability (Figure 3C,D,F), whereas hardly any pNPC hydrolytic activity was detected from the culture supernatant of Ptcu1-cel7a (data not shown), indicating that CEL7A produced under this condition may not adopt a correct conformation. Thus, the T. reesei tcu1 promoter can be used for copper-controlled expression of both homologous and heterologous proteins.Figure 3

Bottom Line: Whereas excess copper repressed the expression of tcu1 from T. reesei, eliminating copper addition in the medium resulted in a high-level transcription of tcu1.Expression of T. reesei tcu1 gene was tightly controlled by copper availability, and a homologous protein expression system was developed based on this promoter.Deregulation of XYR1 (xylanase regulator 1) mediated by the tcu1 promoter not only overcame the carbon catabolite repression of cellulases but also resulted in their full expression even on the non-inducing carbon sources.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, No.27 Shanda South Road, Jinan, 250100 Shandong People's Republic of China.

ABSTRACT

Background: Trichoderma reesei represents an important workhorse for industrial production of cellulases as well as other proteins. The large-scale production is usually performed in a substrate-inducing manner achieved by a fine-tuned cooperation of a suite of transcription factors. Their production and subsequent analysis are, however, often either difficult to manipulate or complicated by the concomitant production of other inducible proteins. Alternatives to control gene expression independent of the nutritional state are thus preferred in some cases to facilitate not only biochemical studies of proteins but also genetic engineering of the producer.

Results: We identified a copper transporter encoding gene tcu1 (jgi:Trire2:52315) in T. reesei, the transcription of which was highly responsive to copper availability. Whereas excess copper repressed the expression of tcu1 from T. reesei, eliminating copper addition in the medium resulted in a high-level transcription of tcu1. The usefulness of the system was further illustrated by the high-level expression of specific cellulases driven by the tcu1 promoter in T. reesei when cultivated on D-glucose or glycerol as the sole carbon source. A recombinant T. reesei strain, which overexpressed the main transcription activator of hydrolases (xylanase regulator 1) under the control of tcu1 promoter, was found to be relieved from the carbon catabolite repression and thus displayed a constitutive cellulase expression. Moreover, the amount and activities of cellulases produced by this strain on glycerol or glucose fully recapitulated those of the parental strain produced on Avicel.

Conclusion: Expression of T. reesei tcu1 gene was tightly controlled by copper availability, and a homologous protein expression system was developed based on this promoter. Deregulation of XYR1 (xylanase regulator 1) mediated by the tcu1 promoter not only overcame the carbon catabolite repression of cellulases but also resulted in their full expression even on the non-inducing carbon sources.

No MeSH data available.


Related in: MedlinePlus