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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 xyn1, bxl1, and bxl2 in T. reesei. T. 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 growth. Transcript levels of xyn1 (a), bxl1 (b), and bxl2 (c) were measured by qPCR using sar1 and act transcript levels for normalization and referred to the reference sample (T. reesei QM6aΔtmus53, 24 h for xyn1 and bxl1; xpp1 deletion strain, 24 h for bxl2). 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. nd not detected.
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Fig5: Influence of Xpp1 on transcript levels of xyn1, bxl1, and bxl2 in T. reesei. T. 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 growth. Transcript levels of xyn1 (a), bxl1 (b), and bxl2 (c) were measured by qPCR using sar1 and act transcript levels for normalization and referred to the reference sample (T. reesei QM6aΔtmus53, 24 h for xyn1 and bxl1; xpp1 deletion strain, 24 h for bxl2). 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. nd not detected.

Mentions: Since further enzymes besides XYNII contribute to the xylanolytic activities of T. reesei, we investigated if Xpp1 also controls their expression. First, we analyzed xyn1 transcript levels, which turned out to be higher in the xpp1 deletion strain compared to the parent strain (Fig. 5a), similar to xyn2 (compare Fig. 4b). Since we detected increased β-xylosidase activity in the supernatant of the xpp1 deletion strain (compare Fig. 3b), we measured the bxl1 transcript levels. Surprisingly, we detected equal levels in both strains (Fig. 5b), which contradicts the measured enzyme activity. However, the protein ID 58450 (on http://genome.jgi-psf.org/Trire2/Trire2.home.html) is annotated as a putative β-xylosidase [25]. Consequently, we analyzed the transcript levels of the gene encoding for this candidate β-xylosidase (in the following termed bxl2). They were indeed higher in the xpp1 deletion strain compared to its parental strain at later cultivation stages (Fig. 5c).Fig. 5


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 xyn1, bxl1, and bxl2 in T. reesei. T. 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 growth. Transcript levels of xyn1 (a), bxl1 (b), and bxl2 (c) were measured by qPCR using sar1 and act transcript levels for normalization and referred to the reference sample (T. reesei QM6aΔtmus53, 24 h for xyn1 and bxl1; xpp1 deletion strain, 24 h for bxl2). 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. nd not detected.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Influence of Xpp1 on transcript levels of xyn1, bxl1, and bxl2 in T. reesei. T. 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 growth. Transcript levels of xyn1 (a), bxl1 (b), and bxl2 (c) were measured by qPCR using sar1 and act transcript levels for normalization and referred to the reference sample (T. reesei QM6aΔtmus53, 24 h for xyn1 and bxl1; xpp1 deletion strain, 24 h for bxl2). 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. nd not detected.
Mentions: Since further enzymes besides XYNII contribute to the xylanolytic activities of T. reesei, we investigated if Xpp1 also controls their expression. First, we analyzed xyn1 transcript levels, which turned out to be higher in the xpp1 deletion strain compared to the parent strain (Fig. 5a), similar to xyn2 (compare Fig. 4b). Since we detected increased β-xylosidase activity in the supernatant of the xpp1 deletion strain (compare Fig. 3b), we measured the bxl1 transcript levels. Surprisingly, we detected equal levels in both strains (Fig. 5b), which contradicts the measured enzyme activity. However, the protein ID 58450 (on http://genome.jgi-psf.org/Trire2/Trire2.home.html) is annotated as a putative β-xylosidase [25]. Consequently, we analyzed the transcript levels of the gene encoding for this candidate β-xylosidase (in the following termed bxl2). They were indeed higher in the xpp1 deletion strain compared to its parental strain at later cultivation stages (Fig. 5c).Fig. 5

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