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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 recovered cellulase gene expression on induction by 1% lactose. (A) SDS-PAGE and Western blot analysis of the secreted proteins in the culture supernatant of QM9414 (A) and Ptcu1-xyr1(B) strains grown on 1% (wt/vol) lactose with (lower panel) or without (upper panel) addition of CuSO4. (C) pNPC and pNPG hydrolytic activities of the culture filtrates from QM9414 and Ptcu1-xyr1 strains induced with 1% (wt/vol) lactose and cultured in the presence or absence of CuSO4. A significant difference (P < 0.05) was detected for the culture filtrates of Ptcu1-xyr1 after 72 h without copper addition compared with those of QM9414. Error bars are the SD from at least two biological replicates.
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Fig5: Ptcu1-xyr1 recovered cellulase gene expression on induction by 1% lactose. (A) SDS-PAGE and Western blot analysis of the secreted proteins in the culture supernatant of QM9414 (A) and Ptcu1-xyr1(B) strains grown on 1% (wt/vol) lactose with (lower panel) or without (upper panel) addition of CuSO4. (C) pNPC and pNPG hydrolytic activities of the culture filtrates from QM9414 and Ptcu1-xyr1 strains induced with 1% (wt/vol) lactose and cultured in the presence or absence of CuSO4. A significant difference (P < 0.05) was detected for the culture filtrates of Ptcu1-xyr1 after 72 h without copper addition compared with those of QM9414. Error bars are the SD from at least two biological replicates.

Mentions: XYR1 has been established as the major regulator for the cellulolytic response to inducing substrate including Avicel or lactose in T. reesei [4,5,15]. Strongly increased basal expression of xyr1 has thus been found to correlate with the hyperproduction of cellulases though that still entails the presence of inducers [16,17]. To probe the effect of copper-controlled expression of xyr1 on cellulase production, we constructed a recombinant strain expressing xyr1 under the control of Ptcu1. After transformation of the Δxyr1 strain, several independent transformants were isolated based on their recovered ability to form hydrolytic halos on Avicel plate (data not shown). One transformant with the largest halo (Ptcu1-xyr1) was chosen for further analysis. To first test whether the Ptcu1-driven expression of xyr1 could recover the cellulolytic phenotype of the Δxyr1 strain, the proteins induced by 1% Avicel in the culture filtrate were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Whereas cellulases were efficiently induced in the strain QM9414 regardless of the addition of copper or not (Figure 4A), comparable levels and patterns of induced cellulases were only observed for Ptcu1-xyr1 without addition of copper (Figure 4B), indicating that the induced production of cellulases in the Δxyr1 strain was absolutely dependent on the copper-controlled expression of xyr1. Analysis of the extracellular pNPC and p-nitrophenyl-β-D-glucopyranoside (pNPG) hydrolytic activities demonstrated that, in accordance with the SDS-PAGE results, the hydrolytic activities as displayed by the culture supernatant of Ptcu1-xyr1 without addition of CuSO4 were overall comparable to those of QM9414 (Figure 4C). Hardly any hydrolytic activities were detected for Ptcu1-xyr1 in the presence of 10 μΜ CuSO4. Efficient production of cellulases by Ptcu1-xyr1 was also observed upon incubation with lactose, and the detected hydrolytic activities of the culture filtrate were significantly higher than those of QM9414 under the same culture conditions after 72 h of induction (Figure 5).Figure 4


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 recovered cellulase gene expression on induction by 1% lactose. (A) SDS-PAGE and Western blot analysis of the secreted proteins in the culture supernatant of QM9414 (A) and Ptcu1-xyr1(B) strains grown on 1% (wt/vol) lactose with (lower panel) or without (upper panel) addition of CuSO4. (C) pNPC and pNPG hydrolytic activities of the culture filtrates from QM9414 and Ptcu1-xyr1 strains induced with 1% (wt/vol) lactose and cultured in the presence or absence of CuSO4. A significant difference (P < 0.05) was detected for the culture filtrates of Ptcu1-xyr1 after 72 h without copper addition compared with those of QM9414. Error bars are the SD from at least two biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4413991&req=5

Fig5: Ptcu1-xyr1 recovered cellulase gene expression on induction by 1% lactose. (A) SDS-PAGE and Western blot analysis of the secreted proteins in the culture supernatant of QM9414 (A) and Ptcu1-xyr1(B) strains grown on 1% (wt/vol) lactose with (lower panel) or without (upper panel) addition of CuSO4. (C) pNPC and pNPG hydrolytic activities of the culture filtrates from QM9414 and Ptcu1-xyr1 strains induced with 1% (wt/vol) lactose and cultured in the presence or absence of CuSO4. A significant difference (P < 0.05) was detected for the culture filtrates of Ptcu1-xyr1 after 72 h without copper addition compared with those of QM9414. Error bars are the SD from at least two biological replicates.
Mentions: XYR1 has been established as the major regulator for the cellulolytic response to inducing substrate including Avicel or lactose in T. reesei [4,5,15]. Strongly increased basal expression of xyr1 has thus been found to correlate with the hyperproduction of cellulases though that still entails the presence of inducers [16,17]. To probe the effect of copper-controlled expression of xyr1 on cellulase production, we constructed a recombinant strain expressing xyr1 under the control of Ptcu1. After transformation of the Δxyr1 strain, several independent transformants were isolated based on their recovered ability to form hydrolytic halos on Avicel plate (data not shown). One transformant with the largest halo (Ptcu1-xyr1) was chosen for further analysis. To first test whether the Ptcu1-driven expression of xyr1 could recover the cellulolytic phenotype of the Δxyr1 strain, the proteins induced by 1% Avicel in the culture filtrate were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Whereas cellulases were efficiently induced in the strain QM9414 regardless of the addition of copper or not (Figure 4A), comparable levels and patterns of induced cellulases were only observed for Ptcu1-xyr1 without addition of copper (Figure 4B), indicating that the induced production of cellulases in the Δxyr1 strain was absolutely dependent on the copper-controlled expression of xyr1. Analysis of the extracellular pNPC and p-nitrophenyl-β-D-glucopyranoside (pNPG) hydrolytic activities demonstrated that, in accordance with the SDS-PAGE results, the hydrolytic activities as displayed by the culture supernatant of Ptcu1-xyr1 without addition of CuSO4 were overall comparable to those of QM9414 (Figure 4C). Hardly any hydrolytic activities were detected for Ptcu1-xyr1 in the presence of 10 μΜ CuSO4. Efficient production of cellulases by Ptcu1-xyr1 was also observed upon incubation with lactose, and the detected hydrolytic activities of the culture filtrate were significantly higher than those of QM9414 under the same culture conditions after 72 h of induction (Figure 5).Figure 4

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