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

In vivo footprinting analysis of the Xpp1-binding region within the xyn2 promoter. T. reesei QM6aΔtmus53 and the xpp1 deletion strain were pre-cultured on glycerol and thereafter transferred to MA medium containing 50 mM d-glucose. DNA was methylated in vivo with DMS after 3 h incubation. Analysis of data was performed using ivFAST [22]. Significant differences in methylation intensities of individual purine nucleotides between the two strains are represented by squares (white, no difference; light gray, difference with ratios of more than 1.1 and less than 1.3; dark gray, differences with ratios of more than 1.3 and less 1.5; black, differences with ratios of more than 1.5). The sequence of the coding (upper lane) and non-coding (lower lane) strand from positions −241 to −220 of the xyn2 promoter is given. Bold letters indicate the previously described AGAA-boxes, underlined letters the palindromic sequence, and italic letters an atypical Xyr1-binding site [20].
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: In vivo footprinting analysis of the Xpp1-binding region within the xyn2 promoter. T. reesei QM6aΔtmus53 and the xpp1 deletion strain were pre-cultured on glycerol and thereafter transferred to MA medium containing 50 mM d-glucose. DNA was methylated in vivo with DMS after 3 h incubation. Analysis of data was performed using ivFAST [22]. Significant differences in methylation intensities of individual purine nucleotides between the two strains are represented by squares (white, no difference; light gray, difference with ratios of more than 1.1 and less than 1.3; dark gray, differences with ratios of more than 1.3 and less 1.5; black, differences with ratios of more than 1.5). The sequence of the coding (upper lane) and non-coding (lower lane) strand from positions −241 to −220 of the xyn2 promoter is given. Bold letters indicate the previously described AGAA-boxes, underlined letters the palindromic sequence, and italic letters an atypical Xyr1-binding site [20].

Mentions: As mentioned, Xpp1 was identified by a pull-down assay using a probe that contained the AGAA-boxes of the xyn2 promoter [23]. To test whether this motif is bound in vivo by Xpp1, we performed a dimethyl sulfate (DMS)-induced in vivo footprint comparing the xpp1 deletion strain and its parent strain. In a replacement experiment, both strains were pre-grown on glycerol and then transferred to minimal medium containing 50 mM d-glucose. We could observe drastic differences between the two strains comparing the accessibility of the part of the xyn2 promoter that contains the AGAA-boxes (Fig. 1). Two nucleotides in the downstream AGAA-box were strongly hypermethylated in the xpp1 deletion strain, but only one nucleotide of the upstream AGAA-box. Interestingly, three nucleotides adjacent to the downstream AGAA-box were also hypermethylated. These three nucleotides are part of a palindromic sequence (5′-TCTAGA-3′) that overlaps with the downstream AGAA-box (Fig. 1).Fig. 1


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)

In vivo footprinting analysis of the Xpp1-binding region within the xyn2 promoter. T. reesei QM6aΔtmus53 and the xpp1 deletion strain were pre-cultured on glycerol and thereafter transferred to MA medium containing 50 mM d-glucose. DNA was methylated in vivo with DMS after 3 h incubation. Analysis of data was performed using ivFAST [22]. Significant differences in methylation intensities of individual purine nucleotides between the two strains are represented by squares (white, no difference; light gray, difference with ratios of more than 1.1 and less than 1.3; dark gray, differences with ratios of more than 1.3 and less 1.5; black, differences with ratios of more than 1.5). The sequence of the coding (upper lane) and non-coding (lower lane) strand from positions −241 to −220 of the xyn2 promoter is given. Bold letters indicate the previously described AGAA-boxes, underlined letters the palindromic sequence, and italic letters an atypical Xyr1-binding site [20].
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: In vivo footprinting analysis of the Xpp1-binding region within the xyn2 promoter. T. reesei QM6aΔtmus53 and the xpp1 deletion strain were pre-cultured on glycerol and thereafter transferred to MA medium containing 50 mM d-glucose. DNA was methylated in vivo with DMS after 3 h incubation. Analysis of data was performed using ivFAST [22]. Significant differences in methylation intensities of individual purine nucleotides between the two strains are represented by squares (white, no difference; light gray, difference with ratios of more than 1.1 and less than 1.3; dark gray, differences with ratios of more than 1.3 and less 1.5; black, differences with ratios of more than 1.5). The sequence of the coding (upper lane) and non-coding (lower lane) strand from positions −241 to −220 of the xyn2 promoter is given. Bold letters indicate the previously described AGAA-boxes, underlined letters the palindromic sequence, and italic letters an atypical Xyr1-binding site [20].
Mentions: As mentioned, Xpp1 was identified by a pull-down assay using a probe that contained the AGAA-boxes of the xyn2 promoter [23]. To test whether this motif is bound in vivo by Xpp1, we performed a dimethyl sulfate (DMS)-induced in vivo footprint comparing the xpp1 deletion strain and its parent strain. In a replacement experiment, both strains were pre-grown on glycerol and then transferred to minimal medium containing 50 mM d-glucose. We could observe drastic differences between the two strains comparing the accessibility of the part of the xyn2 promoter that contains the AGAA-boxes (Fig. 1). Two nucleotides in the downstream AGAA-box were strongly hypermethylated in the xpp1 deletion strain, but only one nucleotide of the upstream AGAA-box. Interestingly, three nucleotides adjacent to the downstream AGAA-box were also hypermethylated. These three nucleotides are part of a palindromic sequence (5′-TCTAGA-3′) that overlaps with the downstream AGAA-box (Fig. 1).Fig. 1

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