Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species
Conditional promoters allowing both induction and silencing of gene expression are indispensable for basic and applied research. The xylP promoter (pxylP) from Penicillium chrysogenum was demonstrated to function in various mold species including Aspergillus fumigatus. pxylP allows high induction by...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-12-01
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| Series: | Metabolic Engineering Communications |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2214030122000232 |
| _version_ | 1828275558942769152 |
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| author | Annie Yap Irene Glarcher Matthias Misslinger Hubertus Haas |
| author_facet | Annie Yap Irene Glarcher Matthias Misslinger Hubertus Haas |
| author_sort | Annie Yap |
| collection | DOAJ |
| description | Conditional promoters allowing both induction and silencing of gene expression are indispensable for basic and applied research. The xylP promoter (pxylP) from Penicillium chrysogenum was demonstrated to function in various mold species including Aspergillus fumigatus. pxylP allows high induction by xylan or its degradation product xylose with low basal activity in the absence of an inducer. Here we structurally characterized and engineered pxylP in A. fumigatus to optimize its application. Mutational analysis demonstrated the importance of the putative TATA-box and a pyrimidine-rich region in the core promoter, both copies of a largely duplicated 91-bp sequence (91bpDS), as well as putative binding sites for the transcription factor XlnR and a GATA motif within the 91bpDS. In agreement, pxylP activity was found to depend on XlnR, while glucose repression appeared to be indirect. Truncation of the originally used 1643-bp promoter fragment to 725 bp largely preserved the promoter activity and the regulatory pattern. Integration of a third 91bpDS significantly increased promoter activity particularly under low inducer concentrations. Truncation of pxylP to 199 bp demonstrated that the upstream region including the 91bpDSs mediates not only inducer-dependent activation but also repression in the absence of inducer. Remarkably, the 1579-bp pxylP was found to act bi-bidirectionally with a similar regulatory pattern by driving expression of the upstream-located arabinofuranosidase gene. The latter opens the possibility of dual bidirectional use of pxylP. Comparison with a doxycycline-inducible TetOn system revealed a significantly higher dynamic range of pxylP. Taken together, this study identified functional elements of pxylP and opened new methodological opportunities for its application. |
| first_indexed | 2024-04-13T06:48:19Z |
| format | Article |
| id | doaj.art-5a2d2d26b94f422796751180987b24ad |
| institution | Directory Open Access Journal |
| issn | 2214-0301 |
| language | English |
| last_indexed | 2024-04-13T06:48:19Z |
| publishDate | 2022-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Metabolic Engineering Communications |
| spelling | doaj.art-5a2d2d26b94f422796751180987b24ad2022-12-22T02:57:29ZengElsevierMetabolic Engineering Communications2214-03012022-12-0115e00214Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal speciesAnnie Yap0Irene Glarcher1Matthias Misslinger2Hubertus Haas3Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, A6020, Innsbruck, Innrain 80-82, AustriaInstitute of Molecular Biology, Biocenter, Medical University of Innsbruck, A6020, Innsbruck, Innrain 80-82, AustriaInstitute of Molecular Biology, Biocenter, Medical University of Innsbruck, A6020, Innsbruck, Innrain 80-82, AustriaCorresponding author.; Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, A6020, Innsbruck, Innrain 80-82, AustriaConditional promoters allowing both induction and silencing of gene expression are indispensable for basic and applied research. The xylP promoter (pxylP) from Penicillium chrysogenum was demonstrated to function in various mold species including Aspergillus fumigatus. pxylP allows high induction by xylan or its degradation product xylose with low basal activity in the absence of an inducer. Here we structurally characterized and engineered pxylP in A. fumigatus to optimize its application. Mutational analysis demonstrated the importance of the putative TATA-box and a pyrimidine-rich region in the core promoter, both copies of a largely duplicated 91-bp sequence (91bpDS), as well as putative binding sites for the transcription factor XlnR and a GATA motif within the 91bpDS. In agreement, pxylP activity was found to depend on XlnR, while glucose repression appeared to be indirect. Truncation of the originally used 1643-bp promoter fragment to 725 bp largely preserved the promoter activity and the regulatory pattern. Integration of a third 91bpDS significantly increased promoter activity particularly under low inducer concentrations. Truncation of pxylP to 199 bp demonstrated that the upstream region including the 91bpDSs mediates not only inducer-dependent activation but also repression in the absence of inducer. Remarkably, the 1579-bp pxylP was found to act bi-bidirectionally with a similar regulatory pattern by driving expression of the upstream-located arabinofuranosidase gene. The latter opens the possibility of dual bidirectional use of pxylP. Comparison with a doxycycline-inducible TetOn system revealed a significantly higher dynamic range of pxylP. Taken together, this study identified functional elements of pxylP and opened new methodological opportunities for its application.http://www.sciencedirect.com/science/article/pii/S2214030122000232FungiAspergillusxylPXylosePromoterXlnR |
| spellingShingle | Annie Yap Irene Glarcher Matthias Misslinger Hubertus Haas Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species Metabolic Engineering Communications Fungi Aspergillus xylP Xylose Promoter XlnR |
| title | Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species |
| title_full | Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species |
| title_fullStr | Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species |
| title_full_unstemmed | Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species |
| title_short | Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species |
| title_sort | characterization and engineering of the xylose inducible xylp promoter for use in mold fungal species |
| topic | Fungi Aspergillus xylP Xylose Promoter XlnR |
| url | http://www.sciencedirect.com/science/article/pii/S2214030122000232 |
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