Limits...
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 xylose reductase expression in T. reesei. T. reesei QM6aΔtmus53 (blue) and the xpp1 deletion strain (green) were grown in MA medium containing 1% (w/v) xylan. Samples were taken after 18, 30, 36, 48, and 72 h growth. a Xylose reductase activity in cell-free extracts was measured in vitro and normalized to the total protein concentration of the cell-free extracts. b Transcript levels of the xyl1 gene were measured by qPCR using sar1 and act transcript levels for normalization and were referred to the reference sample (T. reesei QM6aΔtmus53, 18 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
getmorefigures.php?uid=PMC4526299&req=5

Fig6: Influence of Xpp1 on xylose reductase expression in T. reesei. T. reesei QM6aΔtmus53 (blue) and the xpp1 deletion strain (green) were grown in MA medium containing 1% (w/v) xylan. Samples were taken after 18, 30, 36, 48, and 72 h growth. a Xylose reductase activity in cell-free extracts was measured in vitro and normalized to the total protein concentration of the cell-free extracts. b Transcript levels of the xyl1 gene were measured by qPCR using sar1 and act transcript levels for normalization and were referred to the reference sample (T. reesei QM6aΔtmus53, 18 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: Degradation of xylan results in the release of d-xylose, which is taken up by T. reesei and metabolized via the pentose phosphate way. The initial reaction is the reduction of d-xylose to xylitol, catalyzed by xylose reductase (EC1.1.1.307). We measured xylose reductase activity of the xpp1 deletion strain and its parent strain after 72 h growth on xylan. We could not detect any difference between the two strains (Fig. 6a). Further, the transcript levels of xyl1, encoding the xylose reductase, were monitored in the two strains throughout growth on xylan. Samples were taken periodically and xyl1 transcript levels were measured with a qPCR assay. Matching the obtained enzymatic activities, no differences of xyl1 transcript levels could be observed between the two strains (Fig. 6b). Xpp1 regulates the gene expression of the extracellular xylanolytic enzymes, but not of the intracellular xylose reductase. We presume that the xylose reductase can be used as an indicator for the whole downstream d-xylose metabolism in this context.Fig. 6


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 xylose reductase expression in T. reesei. T. reesei QM6aΔtmus53 (blue) and the xpp1 deletion strain (green) were grown in MA medium containing 1% (w/v) xylan. Samples were taken after 18, 30, 36, 48, and 72 h growth. a Xylose reductase activity in cell-free extracts was measured in vitro and normalized to the total protein concentration of the cell-free extracts. b Transcript levels of the xyl1 gene were measured by qPCR using sar1 and act transcript levels for normalization and were referred to the reference sample (T. reesei QM6aΔtmus53, 18 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

Fig6: Influence of Xpp1 on xylose reductase expression in T. reesei. T. reesei QM6aΔtmus53 (blue) and the xpp1 deletion strain (green) were grown in MA medium containing 1% (w/v) xylan. Samples were taken after 18, 30, 36, 48, and 72 h growth. a Xylose reductase activity in cell-free extracts was measured in vitro and normalized to the total protein concentration of the cell-free extracts. b Transcript levels of the xyl1 gene were measured by qPCR using sar1 and act transcript levels for normalization and were referred to the reference sample (T. reesei QM6aΔtmus53, 18 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: Degradation of xylan results in the release of d-xylose, which is taken up by T. reesei and metabolized via the pentose phosphate way. The initial reaction is the reduction of d-xylose to xylitol, catalyzed by xylose reductase (EC1.1.1.307). We measured xylose reductase activity of the xpp1 deletion strain and its parent strain after 72 h growth on xylan. We could not detect any difference between the two strains (Fig. 6a). Further, the transcript levels of xyl1, encoding the xylose reductase, were monitored in the two strains throughout growth on xylan. Samples were taken periodically and xyl1 transcript levels were measured with a qPCR assay. Matching the obtained enzymatic activities, no differences of xyl1 transcript levels could be observed between the two strains (Fig. 6b). Xpp1 regulates the gene expression of the extracellular xylanolytic enzymes, but not of the intracellular xylose reductase. We presume that the xylose reductase can be used as an indicator for the whole downstream d-xylose metabolism in this context.Fig. 6

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