Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.
Dutch DNA Biotech, Hugo R Kruytgebouw 4-Noord, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
Appl Microbiol Biotechnol. 2019 May;103(10):4125-4136. doi: 10.1007/s00253-019-09777-0. Epub 2019 Apr 8.
The AraR transcription factor of Aspergillus niger encodes a Zn(II)Cys transcription factor required for the induction of genes encoding arabinolytic enzymes. One of the target genes of AraR is abfA, encoding an arabinofuranosidase. The expression of abfA as well as other L-arabinose-induced genes in A. niger requires the presence of L-arabinose or its derivative L-arabitol as an inducer to activate AraR-dependant gene expression. In this study, mutants were isolated that express L-arabinose-induced genes independently of the presence of an inducer under derepressing conditions. To obtain these mutants, a reporter strain was constructed in a ΔcreA background containing the L-arabinose-responsive promoter (PabfA) fused to the acetamidase (amdS) gene. Spores of the ΔcreA PabfA-amdS reporter strain were UV-mutagenized and mutants were obtained by their ability to grow on acetamide without the presence of inducer. From a total of 164 mutants, 15 mutants were identified to contain transacting mutations resulting in high arabinofuranosidase activity in the medium after growth under non-inducing conditions. Sequencing of the araR gene of the 15 constitutive mutants revealed that 14 mutants carried a mutation in AraR. Some mutations were found more than once and in total nine different point mutations were identified in AraR. The AraR point mutation was reintroduced into a parental strain and confirmed that this point mutation leads to inducer-independent expression of AraR target genes. The inducer independent of L-arabinose-induced genes in the AraR mutant was found to be sensitive to carbon catabolite repression, indicating that the CreA-mediated carbon catabolite repression is dominant over the AraR mutant allele. These mutations in AraR provide new opportunities to improve arabinase production in industrial fungal strains.
黑曲霉的 AraR 转录因子编码一个 Zn(II)Cys 转录因子，该转录因子是诱导编码阿拉伯糖分解酶基因所必需的。AraR 的一个靶标基因是 abfA，它编码阿拉伯呋喃糖苷酶。在黑曲霉中，abfA 以及其他 L-阿拉伯糖诱导的基因的表达需要 L-阿拉伯糖或其衍生物 L-阿拉伯醇作为诱导物来激活 AraR 依赖的基因表达。在这项研究中，分离到了在去阻遏条件下独立于诱导物存在表达 L-阿拉伯糖诱导基因的突变体。为了获得这些突变体，在含有 L-阿拉伯糖响应启动子（PabfA）与乙酰氨基葡萄糖苷酶（amdS）基因融合的ΔcreA 背景中构建了报告菌株。ΔcreA PabfA-amdS 报告菌株的孢子经紫外线诱变，通过在没有诱导物的情况下利用乙酰胺生长的能力获得突变体。在总共 164 个突变体中，有 15 个突变体被鉴定为含有反式作用突变，导致在非诱导条件下生长后培养基中阿拉伯呋喃糖苷酶活性升高。对 15 个组成型突变体的 araR 基因进行测序发现，14 个突变体携带 AraR 突变。一些突变不止出现一次，总共在 AraR 中鉴定出 9 个不同的点突变。将 AraR 点突变重新引入亲本菌株，并证实该点突变导致 AraR 靶标基因的诱导物非依赖性表达。在 AraR 突变体中，L-阿拉伯糖诱导基因的非诱导依赖被发现对碳源分解代谢物阻遏敏感，这表明 CreA 介导的碳源分解代谢物阻遏对 AraR 突变等位基因是显性的。这些 AraR 突变为在工业真菌菌株中提高阿拉伯酶的生产提供了新的机会。
