Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584, CT, Utrecht, The Netherlands.
Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke Street West, Montreal, Quebec, H4B 1R6, Canada.
Appl Microbiol Biotechnol. 2021 Jul;105(13):5553-5564. doi: 10.1007/s00253-021-11428-2. Epub 2021 Jul 8.
Aspergillus niger is a filamentous fungus well known for its ability to produce a wide variety of pectinolytic enzymes, which have many applications in the industry. The transcriptional activator GaaR is induced by 2-keto-3-deoxy-L-galactonate, a compound derived from D-galacturonic acid, and plays a major role in the regulation of pectinolytic genes. The requirement for inducer molecules can be a limiting factor for the production of enzymes. Therefore, the generation of chimeric transcription factors able to activate the expression of pectinolytic genes by using underutilized agricultural residues would be highly valuable for industrial applications. In this study, we used the CRISPR/Cas9 system to generate three chimeric GaaR-XlnR transcription factors expressed by the xlnR promoter by swapping the N-terminal region of the xylanolytic regulator XlnR to that of the GaaR in A. niger. As a test case, we constructed a PpgaX-hph reporter strain to evaluate the alteration of transcription factor specificity in the chimeric mutants. Our results showed that the chimeric GaaR-XlnR transcription factor was induced in the presence of D-xylose. Additionally, we generated a constitutively active GaaR-XlnR V756F version of the most efficient chimeric transcription factor to better assess its activity. Proteomics analysis confirmed the production of several pectinolytic enzymes by ΔgaaR mutants carrying the chimeric transcription factor. This correlates with the improved release of D-galacturonic acid from pectin by the GaaR-XlnR V756F mutant, as well as by the increased L-arabinose release from the pectin side chains by both chimeric mutants under inducing condition, which is required for efficient degradation of pectin. KEY POINTS: • Chimeric transcription factors were generated by on-site mutations using CRISPR/Cas9. • PpgaX-hph reporter strain allowed for the screening of functional GaaR-XlnR mutants. • Chimeric GaaR-XlnR induced pectinolytic activities in the presence of D-xylose.
黑曲霉是一种丝状真菌，以能够产生多种果胶酶而闻名，这些酶在工业中有许多应用。转录激活因子 GaaR 被 2-酮-3-脱氧-L-半乳糖酸诱导，该化合物来源于 D-半乳糖醛酸，在果胶酶基因的调控中起主要作用。诱导分子的需求可能是酶生产的限制因素。因此，产生能够利用未充分利用的农业残余物激活果胶酶基因表达的嵌合转录因子，对于工业应用将具有非常重要的价值。在这项研究中，我们使用 CRISPR/Cas9 系统通过交换木聚糖分解代谢调节剂 XlnR 的 N 端区域来产生三种由 xlnR 启动子表达的嵌合 GaaR-XlnR 转录因子黑曲霉中的 GaaR。作为一个测试案例，我们构建了 PpgaX-hph 报告菌株来评估嵌合突变体中转录因子特异性的改变。我们的结果表明，在存在 D-木糖的情况下，嵌合 GaaR-XlnR 转录因子被诱导。此外，我们生成了最有效的嵌合转录因子的组成型活性 GaaR-XlnR V756F 版本，以更好地评估其活性。蛋白质组学分析证实了携带嵌合转录因子的 ΔgaaR 突变体产生了几种果胶酶。这与 GaaR-XlnR V756F 突变体从果胶中更好地释放 D-半乳糖醛酸以及两种嵌合突变体在诱导条件下从果胶侧链中更好地释放 L-阿拉伯糖相关，这对于有效降解果胶是必需的。关键点：• 利用 CRISPR/Cas9 进行现场突变产生嵌合转录因子。• PpgaX-hph 报告菌株允许筛选功能性 GaaR-XlnR 突变体。• 嵌合 GaaR-XlnR 在 D-木糖存在下诱导果胶酶活性。
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