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Xpp1 regulates the expression of xylanases, but not of cellulases in Trichoderma reesei.

Derntl C, Rassinger A, Srebotnik E, Mach RL, Mach-Aigner AR - Biotechnol Biofuels (2015)

Bottom Line: Xpp1 expression was found to be up-regulated, additionally to d-glucose, by high d-xylose availability.These findings together with the observed xyn2 transcript levels during growth on xylan suggest that Xpp1 is the mediator of a feedback mechanism.Notably, Xpp1 has neither influence on the d-xylose metabolism nor on the expression of cellulases.

View Article: PubMed Central - PubMed

Affiliation: Department for Biotechnology and Microbiology, Institute of Chemical Engineering, TU Wien, Gumpendorfer Str. 1a, 1060 Vienna, Austria.

ABSTRACT

Background: The ascomycete Trichoderma reesei is industrially used for the production of cellulases. During the production process xylanases are co-secreted, which uses energy and nutrients. Cellulases and xylanases share the same main regulators, which makes a knowledge-based strain design difficult. However, previously a cis-element in the promoter of the main xylanase-encoding gene was identified as binding site for a putative repressor. Subsequently, three candidate repressors were identified in a pull-down approach. The expression of the most promising candidate, Xpp1 (Xylanase promoter-binding protein 1), was reported to be up-regulated on the repressing carbon source d-glucose and to bind the cis-element in vitro.

Results: In this study, Xpp1 was deleted and over-expressed in T. reesei. An in vivo DNA-footprint assay indicated that Xpp1 binds a palindromic sequence in the xyn2 promoter. Comparison of the deletion, the over-expression, and the parent strain demonstrated that Xpp1 regulates gene expression of xylanolytic enzymes at later cultivation stages. Xpp1 expression was found to be up-regulated, additionally to d-glucose, by high d-xylose availability. These findings together with the observed xyn2 transcript levels during growth on xylan suggest that Xpp1 is the mediator of a feedback mechanism. Notably, Xpp1 has neither influence on the d-xylose metabolism nor on the expression of cellulases.

Conclusions: Xpp1 as regulator acting on the expression of xylanases, but not cellulases, is a highly promising candidate for knowledge-based strain design to improve the cellulases-to-xylanases ratio during industrial cellulase production.

No MeSH data available.


Related in: MedlinePlus

Influence of Xpp1 on cellulase expression in T. reesei. T. reesei QM6aΔtmus53 (blue), the xpp1 deletion strain (green), and the xpp1 over-expression strain (yellow) were grown in MA medium containing 1% (w/v) CMC for 36, 48, 54, 60, 66, and 72 h. a Endo-cellulolytic activity was measured and normalized to the acquired biomass after 72 h. Relative transcript levels of cbh1 (b) and egl1 (c) were measured by qPCR using sar1 and act transcript levels for normalization and were referred to the reference sample (T. reesei QM6aΔtmus53, 36 h). Results are given as relative transcript ratios in logarithmic scale (Log). The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations.
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Fig7: Influence of Xpp1 on cellulase expression in T. reesei. T. reesei QM6aΔtmus53 (blue), the xpp1 deletion strain (green), and the xpp1 over-expression strain (yellow) were grown in MA medium containing 1% (w/v) CMC for 36, 48, 54, 60, 66, and 72 h. a Endo-cellulolytic activity was measured and normalized to the acquired biomass after 72 h. Relative transcript levels of cbh1 (b) and egl1 (c) were measured by qPCR using sar1 and act transcript levels for normalization and were referred to the reference sample (T. reesei QM6aΔtmus53, 36 h). Results are given as relative transcript ratios in logarithmic scale (Log). The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations.

Mentions: Owing to the fact that cellulolytic and xylanolytic enzymes are co-regulated in T. reesei, we were interested to know to what extent Xpp1 influences the expression of cellulases. The xpp1 deletion strain, the over-expression strain, and the parent strain were cultivated in minimal medium containing CMC for 72 h, and endo-cellulolytic activities of the resulting culture supernatants were assayed. We could not detect any differences on comparing the three strains (Fig. 7a). Further, transcript levels of the main cellulases, i.e., cbh1, cbh2, and egl1, were monitored throughout growth in CMC with qPCR assays. No differences between the deletion strain and the parent strain could be observed (Fig. 7b, c, data for cbh2 not shown).Fig. 7


Xpp1 regulates the expression of xylanases, but not of cellulases in Trichoderma reesei.

Derntl C, Rassinger A, Srebotnik E, Mach RL, Mach-Aigner AR - Biotechnol Biofuels (2015)

Influence of Xpp1 on cellulase expression in T. reesei. T. reesei QM6aΔtmus53 (blue), the xpp1 deletion strain (green), and the xpp1 over-expression strain (yellow) were grown in MA medium containing 1% (w/v) CMC for 36, 48, 54, 60, 66, and 72 h. a Endo-cellulolytic activity was measured and normalized to the acquired biomass after 72 h. Relative transcript levels of cbh1 (b) and egl1 (c) were measured by qPCR using sar1 and act transcript levels for normalization and were referred to the reference sample (T. reesei QM6aΔtmus53, 36 h). Results are given as relative transcript ratios in logarithmic scale (Log). The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4526299&req=5

Fig7: Influence of Xpp1 on cellulase expression in T. reesei. T. reesei QM6aΔtmus53 (blue), the xpp1 deletion strain (green), and the xpp1 over-expression strain (yellow) were grown in MA medium containing 1% (w/v) CMC for 36, 48, 54, 60, 66, and 72 h. a Endo-cellulolytic activity was measured and normalized to the acquired biomass after 72 h. Relative transcript levels of cbh1 (b) and egl1 (c) were measured by qPCR using sar1 and act transcript levels for normalization and were referred to the reference sample (T. reesei QM6aΔtmus53, 36 h). Results are given as relative transcript ratios in logarithmic scale (Log). The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations.
Mentions: Owing to the fact that cellulolytic and xylanolytic enzymes are co-regulated in T. reesei, we were interested to know to what extent Xpp1 influences the expression of cellulases. The xpp1 deletion strain, the over-expression strain, and the parent strain were cultivated in minimal medium containing CMC for 72 h, and endo-cellulolytic activities of the resulting culture supernatants were assayed. We could not detect any differences on comparing the three strains (Fig. 7a). Further, transcript levels of the main cellulases, i.e., cbh1, cbh2, and egl1, were monitored throughout growth in CMC with qPCR assays. No differences between the deletion strain and the parent strain could be observed (Fig. 7b, c, data for cbh2 not shown).Fig. 7

Bottom Line: Xpp1 expression was found to be up-regulated, additionally to d-glucose, by high d-xylose availability.These findings together with the observed xyn2 transcript levels during growth on xylan suggest that Xpp1 is the mediator of a feedback mechanism.Notably, Xpp1 has neither influence on the d-xylose metabolism nor on the expression of cellulases.

View Article: PubMed Central - PubMed

Affiliation: Department for Biotechnology and Microbiology, Institute of Chemical Engineering, TU Wien, Gumpendorfer Str. 1a, 1060 Vienna, Austria.

ABSTRACT

Background: The ascomycete Trichoderma reesei is industrially used for the production of cellulases. During the production process xylanases are co-secreted, which uses energy and nutrients. Cellulases and xylanases share the same main regulators, which makes a knowledge-based strain design difficult. However, previously a cis-element in the promoter of the main xylanase-encoding gene was identified as binding site for a putative repressor. Subsequently, three candidate repressors were identified in a pull-down approach. The expression of the most promising candidate, Xpp1 (Xylanase promoter-binding protein 1), was reported to be up-regulated on the repressing carbon source d-glucose and to bind the cis-element in vitro.

Results: In this study, Xpp1 was deleted and over-expressed in T. reesei. An in vivo DNA-footprint assay indicated that Xpp1 binds a palindromic sequence in the xyn2 promoter. Comparison of the deletion, the over-expression, and the parent strain demonstrated that Xpp1 regulates gene expression of xylanolytic enzymes at later cultivation stages. Xpp1 expression was found to be up-regulated, additionally to d-glucose, by high d-xylose availability. These findings together with the observed xyn2 transcript levels during growth on xylan suggest that Xpp1 is the mediator of a feedback mechanism. Notably, Xpp1 has neither influence on the d-xylose metabolism nor on the expression of cellulases.

Conclusions: Xpp1 as regulator acting on the expression of xylanases, but not cellulases, is a highly promising candidate for knowledge-based strain design to improve the cellulases-to-xylanases ratio during industrial cellulase production.

No MeSH data available.


Related in: MedlinePlus