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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 transcript levels of xyn2 in T. reesei.aT. reesei QM6aΔtmus53 (blue bars) and the xpp1 deletion strain (green bars) were pre-cultured on glycerol and thereafter transferred to MA media without carbon source (NC), or containing 50 mM d-glucose (G), or 66 mM d-xylose (XO) and incubated for 3 h. Transcript levels refer to the reference sample (QM6aΔtmus53, NC). bT. reesei QM6aΔtmus53 (blue squares) and the xpp1 deletion strain (green squares) were grown in MA medium containing 1% (w/v) xylan. Samples were taken after 18, 24, 30, 36, 48, and 72 h of cultivation. Transcript levels of xyn2 were measured by qPCR using sar1 and act transcript levels for normalization and referred to the reference sample (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.
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Fig4: Influence of Xpp1 on transcript levels of xyn2 in T. reesei.aT. reesei QM6aΔtmus53 (blue bars) and the xpp1 deletion strain (green bars) were pre-cultured on glycerol and thereafter transferred to MA media without carbon source (NC), or containing 50 mM d-glucose (G), or 66 mM d-xylose (XO) and incubated for 3 h. Transcript levels refer to the reference sample (QM6aΔtmus53, NC). bT. reesei QM6aΔtmus53 (blue squares) and the xpp1 deletion strain (green squares) were grown in MA medium containing 1% (w/v) xylan. Samples were taken after 18, 24, 30, 36, 48, and 72 h of cultivation. Transcript levels of xyn2 were measured by qPCR using sar1 and act transcript levels for normalization and referred to the reference sample (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: We aimed to test whether Xpp1 does in fact act as a transcription factor on the regulation of gene expression of xyn2 directly. In a first experiment, the xpp1 deletion strain and its parent strain were subjected to a replacement experiment using 50 mM d-glucose or 66 mM d-xylose. The transcript levels of xyn2 were quantified by a qPCR assay. Surprisingly, we could not observe higher xyn2 transcript levels in the deletion strain on both tested carbon sources after 3 h compared to the parent strain (Fig. 4a). Xpp1 does not seem to have an effect on the inducibility of xyn2 gene expression, which conflicts with the elevated xylanolytic activity of the xpp1 deletion strain observed, e.g., after 72 h growth on xylan. Therefore, we monitored xyn2 transcript levels in the two strains throughout growth on xylan. We could detect higher xyn2 transcript levels in the deletion strain compared to its parent strain after 30, 36, 48, and 72 h (Fig. 4b). Only the early (24 h) xyn2 transcript level is lower in the deletion strain. These findings match the observed xylanolytic activities. Deletion and parent strain had the same xylanolytic activities after 48 h (Fig. 3a). During growth xyn2 transcript levels drop in the parent strain, whereas they stay at a high level in the deletion strain (Fig. 4b), which results in a higher xylanolytic activity of the deletion strain at later cultivation stages (Fig. 3a).Fig. 4


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 transcript levels of xyn2 in T. reesei.aT. reesei QM6aΔtmus53 (blue bars) and the xpp1 deletion strain (green bars) were pre-cultured on glycerol and thereafter transferred to MA media without carbon source (NC), or containing 50 mM d-glucose (G), or 66 mM d-xylose (XO) and incubated for 3 h. Transcript levels refer to the reference sample (QM6aΔtmus53, NC). bT. reesei QM6aΔtmus53 (blue squares) and the xpp1 deletion strain (green squares) were grown in MA medium containing 1% (w/v) xylan. Samples were taken after 18, 24, 30, 36, 48, and 72 h of cultivation. Transcript levels of xyn2 were measured by qPCR using sar1 and act transcript levels for normalization and referred to the reference sample (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.
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Related In: Results  -  Collection

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Fig4: Influence of Xpp1 on transcript levels of xyn2 in T. reesei.aT. reesei QM6aΔtmus53 (blue bars) and the xpp1 deletion strain (green bars) were pre-cultured on glycerol and thereafter transferred to MA media without carbon source (NC), or containing 50 mM d-glucose (G), or 66 mM d-xylose (XO) and incubated for 3 h. Transcript levels refer to the reference sample (QM6aΔtmus53, NC). bT. reesei QM6aΔtmus53 (blue squares) and the xpp1 deletion strain (green squares) were grown in MA medium containing 1% (w/v) xylan. Samples were taken after 18, 24, 30, 36, 48, and 72 h of cultivation. Transcript levels of xyn2 were measured by qPCR using sar1 and act transcript levels for normalization and referred to the reference sample (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: We aimed to test whether Xpp1 does in fact act as a transcription factor on the regulation of gene expression of xyn2 directly. In a first experiment, the xpp1 deletion strain and its parent strain were subjected to a replacement experiment using 50 mM d-glucose or 66 mM d-xylose. The transcript levels of xyn2 were quantified by a qPCR assay. Surprisingly, we could not observe higher xyn2 transcript levels in the deletion strain on both tested carbon sources after 3 h compared to the parent strain (Fig. 4a). Xpp1 does not seem to have an effect on the inducibility of xyn2 gene expression, which conflicts with the elevated xylanolytic activity of the xpp1 deletion strain observed, e.g., after 72 h growth on xylan. Therefore, we monitored xyn2 transcript levels in the two strains throughout growth on xylan. We could detect higher xyn2 transcript levels in the deletion strain compared to its parent strain after 30, 36, 48, and 72 h (Fig. 4b). Only the early (24 h) xyn2 transcript level is lower in the deletion strain. These findings match the observed xylanolytic activities. Deletion and parent strain had the same xylanolytic activities after 48 h (Fig. 3a). During growth xyn2 transcript levels drop in the parent strain, whereas they stay at a high level in the deletion strain (Fig. 4b), which results in a higher xylanolytic activity of the deletion strain at later cultivation stages (Fig. 3a).Fig. 4

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