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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

Ptcu1-xyr1 displayed a full production of cellulases on non-inducing carbon sources. (A) SDS-PAGE analysis and pNPC hydrolytic activities of the extracellular proteins from QM9414 (left panel) and Ptcu1-xyr1 (right panel) strains grown on 1% (wt/vol) glucose without exogenous addition of CuSO4. (B) The same as in (A), but the strains were grown on 1% (wt/vol) glycerol. (C) Filter paper hydrolytic activity of the cellulases produced by Ptcu1-xyr1 on 1% (wt/vol) glycerol or those produced by QM9414 strain on 1% (wt/vol) Avicel or glycerol cultured with or without CuSO4. A significant difference (P < 0.05) was detected between the produced cellulases of Ptcu1-xyr1 on 1% (wt/vol) glycerol without CuSO4 and those of QM9414 on 1% (wt/vol) glycerol. (D) The extracellular pNPC hydrolytic activity of Ptcu1-xyr1 cultured on 1% (wt/vol) glycerol in the presence of the indicated concentrations of CuSO4. Equal amount of culture supernatant relative to biomass was measured for all the assays. Error bars are the SD from at least two biological replicates.
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Fig6: Ptcu1-xyr1 displayed a full production of cellulases on non-inducing carbon sources. (A) SDS-PAGE analysis and pNPC hydrolytic activities of the extracellular proteins from QM9414 (left panel) and Ptcu1-xyr1 (right panel) strains grown on 1% (wt/vol) glucose without exogenous addition of CuSO4. (B) The same as in (A), but the strains were grown on 1% (wt/vol) glycerol. (C) Filter paper hydrolytic activity of the cellulases produced by Ptcu1-xyr1 on 1% (wt/vol) glycerol or those produced by QM9414 strain on 1% (wt/vol) Avicel or glycerol cultured with or without CuSO4. A significant difference (P < 0.05) was detected between the produced cellulases of Ptcu1-xyr1 on 1% (wt/vol) glycerol without CuSO4 and those of QM9414 on 1% (wt/vol) glycerol. (D) The extracellular pNPC hydrolytic activity of Ptcu1-xyr1 cultured on 1% (wt/vol) glycerol in the presence of the indicated concentrations of CuSO4. Equal amount of culture supernatant relative to biomass was measured for all the assays. Error bars are the SD from at least two biological replicates.

Mentions: To further ask to what extent overexpression of xyr1 alone would effect on launching the production of cellulases under non-inducing conditions, we then determined the synthesis of cellulases by Ptcu1-xyr1 on various other carbon sources. As shown in Figure 6A,B, the expression of xyr1 in Ptcu1-xyr1 resulted in a constitutive expression of cellulases on either glucose or glycerol, whereas no cellulases could be detected for QM9414 under these conditions. Consistent with these results, significant extracellular pNPC and pNPG hydrolytic activities were detected for Ptcu1-xyr1 cultured without addition of copper, whereas hardly any such activities were observed when QM9414 was cultured under the same conditions (Figure 6A,B). Analysis of the hydrolytic activity toward filter paper revealed that significant activities were detected for cellulases produced by Ptcu1-xyr1 on glycerol over time, which was almost equivalent to cellulases produced by QM9414 induced with Avicel at 48 h (Figure 6C). Again, the cellulase production was inhibited with increasing amount of CuSO4 added to the culture, and BCS was able to partially recover the production after a complete inhibition was inflicted (Figure 6C,D). Altogether, the above results indicated that Ptcu1-xyr1-mediated expression of xyr1 is capable of achieving the full production of cellulases without induction.Figure 6


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)

Ptcu1-xyr1 displayed a full production of cellulases on non-inducing carbon sources. (A) SDS-PAGE analysis and pNPC hydrolytic activities of the extracellular proteins from QM9414 (left panel) and Ptcu1-xyr1 (right panel) strains grown on 1% (wt/vol) glucose without exogenous addition of CuSO4. (B) The same as in (A), but the strains were grown on 1% (wt/vol) glycerol. (C) Filter paper hydrolytic activity of the cellulases produced by Ptcu1-xyr1 on 1% (wt/vol) glycerol or those produced by QM9414 strain on 1% (wt/vol) Avicel or glycerol cultured with or without CuSO4. A significant difference (P < 0.05) was detected between the produced cellulases of Ptcu1-xyr1 on 1% (wt/vol) glycerol without CuSO4 and those of QM9414 on 1% (wt/vol) glycerol. (D) The extracellular pNPC hydrolytic activity of Ptcu1-xyr1 cultured on 1% (wt/vol) glycerol in the presence of the indicated concentrations of CuSO4. Equal amount of culture supernatant relative to biomass was measured for all the assays. Error bars are the SD from at least two biological replicates.
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Related In: Results  -  Collection

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Fig6: Ptcu1-xyr1 displayed a full production of cellulases on non-inducing carbon sources. (A) SDS-PAGE analysis and pNPC hydrolytic activities of the extracellular proteins from QM9414 (left panel) and Ptcu1-xyr1 (right panel) strains grown on 1% (wt/vol) glucose without exogenous addition of CuSO4. (B) The same as in (A), but the strains were grown on 1% (wt/vol) glycerol. (C) Filter paper hydrolytic activity of the cellulases produced by Ptcu1-xyr1 on 1% (wt/vol) glycerol or those produced by QM9414 strain on 1% (wt/vol) Avicel or glycerol cultured with or without CuSO4. A significant difference (P < 0.05) was detected between the produced cellulases of Ptcu1-xyr1 on 1% (wt/vol) glycerol without CuSO4 and those of QM9414 on 1% (wt/vol) glycerol. (D) The extracellular pNPC hydrolytic activity of Ptcu1-xyr1 cultured on 1% (wt/vol) glycerol in the presence of the indicated concentrations of CuSO4. Equal amount of culture supernatant relative to biomass was measured for all the assays. Error bars are the SD from at least two biological replicates.
Mentions: To further ask to what extent overexpression of xyr1 alone would effect on launching the production of cellulases under non-inducing conditions, we then determined the synthesis of cellulases by Ptcu1-xyr1 on various other carbon sources. As shown in Figure 6A,B, the expression of xyr1 in Ptcu1-xyr1 resulted in a constitutive expression of cellulases on either glucose or glycerol, whereas no cellulases could be detected for QM9414 under these conditions. Consistent with these results, significant extracellular pNPC and pNPG hydrolytic activities were detected for Ptcu1-xyr1 cultured without addition of copper, whereas hardly any such activities were observed when QM9414 was cultured under the same conditions (Figure 6A,B). Analysis of the hydrolytic activity toward filter paper revealed that significant activities were detected for cellulases produced by Ptcu1-xyr1 on glycerol over time, which was almost equivalent to cellulases produced by QM9414 induced with Avicel at 48 h (Figure 6C). Again, the cellulase production was inhibited with increasing amount of CuSO4 added to the culture, and BCS was able to partially recover the production after a complete inhibition was inflicted (Figure 6C,D). Altogether, the above results indicated that Ptcu1-xyr1-mediated expression of xyr1 is capable of achieving the full production of cellulases without induction.Figure 6

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