A novel transcriptional repressor specifically regulates xylanase gene 1 in Trichoderma reesei.
A novel transcriptional repressor specifically regulates xylanase gene 1 in Trichoderma reesei.
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DOI：
10.1186/s13068-023-02417-w
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发表时间：
2023-10-27
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著名的工业真菌里氏木霉（Trichoderma reesei）具有分泌大量纤维素酶和木聚糖酶的优异能力。纤维素酶和木聚糖酶基因的诱导表达在转录水平上受到严格控制。然而，相对于纤维素酶基因复杂调控机制的深入研究，木聚糖酶基因调控机制的研究相对有限，阻碍了木聚糖酶产量的进一步提高。reesei通过合理的应变工程。目的：筛选木聚糖酶基因表达调控因子。reesei，酵母单杂交筛选基于两个主要的胞外木聚糖酶基因xyn 1和xyn 2的启动子进行。一个假定的转录因子命名为XTR 1显示显着的结合能力的xyn 1启动子，但不是xyn 2的，被成功地分离。xtr 1的缺失显著提高了xyn 1的转录水平，但对xyn 2的转录水平仅产生了轻微的促进作用。去除XTR 1后，木聚糖酶活性提高了约50%，但纤维素酶活性几乎不受影响。融合绿色荧光蛋白的XTR 1的亚细胞定位分析证明XTR 1是核蛋白。进一步的分析揭示了XTR 1的精确结合位点和xyn 1启动子内结合的关键核苷酸。此外，竞争性EMSA表明，XTR 1竞争与xyn 1启动子结合的基本反式激活因子XYR 1。XTR 1是一种新的特异性调控木聚糖酶基因表达的转录抑制因子。该因子的分离和功能鉴定不仅有助于进一步了解木聚糖酶基因的调控网络，而且为促进木聚糖酶在木霉中的生物合成提供了重要线索。reesei。在线版本包含补充材料，可通过10.1186/s13068-023-02417-w获得。
The well-known industrial fungus Trichoderma reesei has an excellent capability of secreting a large amount of cellulases and xylanases. The induced expression of cellulase and xylanase genes is tightly controlled at the transcriptional level. However, compared to the intensive studies on the intricate regulatory mechanism of cellulase genes, efforts to understand how xylanase genes are regulated are relatively limited, which impedes the further improvement of xylanase production by T. reesei via rational strain engineering. To identify transcription factors involved in regulating xylanase gene expression in T. reesei, yeast one-hybrid screen was performed based on the promoters of two major extracellular xylanase genes xyn1 and xyn2. A putative transcription factor named XTR1 showing significant binding capability to the xyn1 promoter but not that of xyn2, was successfully isolated. Deletion of xtr1 significantly increased the transcriptional level of xyn1, but only exerted a minor promoting effect on that of xyn2. The xylanase activity was increased by ~ 50% with XTR1 elimination but the cellulase activity was hardly affected. Subcellular localization analysis of XTR1 fused to a green fluorescence protein demonstrated that XTR1 is a nuclear protein. Further analyses revealed the precise binding site of XTR1 and nucleotides critical for the binding within the xyn1 promoter. Moreover, competitive EMSAs indicated that XTR1 competes with the essential transactivator XYR1 for binding to the xyn1 promoter. XTR1 represents a new transcriptional repressor specific for controlling xylanase gene expression. Isolation and functional characterization of this new factor not only contribute to further understanding the stringent regulatory network of xylanase genes, but also provide important clues for boosting xylanase biosynthesis in T. reesei. The online version contains supplementary material available at 10.1186/s13068-023-02417-w.
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