Arentshorst Mark, Reijngoud Jos, van Tol Daan J C, Reid Ian D, Arendsen Yvonne, Pel Herman J, van Peij Noël N M E, Visser Jaap, Punt Peter J, Tsang Adrian, Ram Arthur F J
Microbial Sciences, Institute of Biology Leiden, Leiden University, Leiden, Netherlands.
Centre for Structural and Functional Genomics, Concordia University, Montreal, QC, Canada.
Front Fungal Biol. 2022 Nov 8;3:978845. doi: 10.3389/ffunb.2022.978845. eCollection 2022.
The feruloyl esterase B gene () is specifically induced by hydroxycinnamic acids (e.g. ferulic acid, caffeic acid and coumaric acid) but the transcriptional regulation network involved in induction and ferulic acid metabolism has only been partially addressed. To identify transcription factors involved in ferulic acid metabolism we constructed and screened a transcription factor knockout library of 239 strains for mutants unable to utilize ferulic acid as a carbon source. The transcription factor mutant, already known to be involved in fatty acid metabolism, could not utilize ferulic acid and other hydroxycinnamic acids. In addition to screening the transcription factor mutant collection, a forward genetic screen was performed to isolate mutants unable to express For this screen a and dual reporter strain was engineered. The rationale of the screen is that in this reporter strain ferulic acid induces (acetamidase) expression the promoter resulting in lethality on fluoro-acetamide. Conidia of this reporter strain were UV-mutagenized and plated on fluoro-acetamide medium in the presence of ferulic acid. Mutants unable to induce are expected to be fluoro-acetamide resistant and can be positively selected for. Using this screen, six fluoro-acetamide resistant mutants were obtained and phenotypically characterized. Three mutants had a phenotype identical to the mutant and sequencing the gene in these mutants indeed showed mutations in FarA which resulted in inability to growth on ferulic acid as well as on short and long chain fatty acids. The growth phenotype of the other three mutants was similar to the mutants in terms of the inability to grow on ferulic acid, but these mutants grew normally on short and long chain fatty acids. The genomes of these three mutants were sequenced and allelic mutations in one particular gene (NRRL3_09145) were found. The protein encoded by NRRL3_09145 shows similarity to the FarA and FarB transcription factors. However, whereas FarA and FarB contain both the Zn(II)Cys domain and a fungal-specific transcription factor domain, the protein encoded by NRRL3_09145 (FarD) lacks the canonical Zn(II)Cys domain and possesses only the fungal specific transcription factor domain.
阿魏酸酯酶B基因（）由羟基肉桂酸（如阿魏酸、咖啡酸和香豆酸）特异性诱导，但参与诱导和阿魏酸代谢的转录调控网络仅得到部分研究。为了鉴定参与阿魏酸代谢的转录因子，我们构建并筛选了239个菌株的转录因子敲除文库，以寻找不能利用阿魏酸作为碳源的突变体。已知参与脂肪酸代谢的转录因子突变体不能利用阿魏酸和其他羟基肉桂酸。除了筛选转录因子突变体文库外，还进行了正向遗传学筛选以分离不能表达的突变体。为此构建了一个和双报告菌株。该筛选的基本原理是，在这个报告菌株中，阿魏酸诱导（乙酰胺酶）表达，其启动子导致在氟乙酰胺上致死。该报告菌株的分生孢子经紫外线诱变后，在阿魏酸存在的情况下接种在氟乙酰胺培养基上。预期不能诱导的突变体对氟乙酰胺具有抗性，可以进行阳性选择。通过该筛选，获得了6个对氟乙酰胺具有抗性的突变体并对其进行了表型鉴定。3个突变体的表型与突变体相同，对这些突变体中的基因进行测序确实显示FarA发生了突变，导致其不能在阿魏酸以及短链和长链脂肪酸上生长。另外3个突变体的生长表型在不能在阿魏酸上生长方面与突变体相似，但这些突变体在短链和长链脂肪酸上能正常生长。对这3个突变体的基因组进行了测序，发现一个特定基因（NRRL3_09145）发生了等位基因突变。NRRL3_09145编码的蛋白质与FarA和FarB转录因子具有相似性。然而，FarA和FarB都包含Zn（II）Cys结构域和一个真菌特异性转录因子结构域，而NRRL3_09145（FarD）编码的蛋白质缺乏典型的Zn（II）Cys结构域，仅具有真菌特异性转录因子结构域。
