Kiesenhofer Daniel P, Mach Robert L, Mach-Aigner Astrid R
Research Area Biochemical Technology, Institute of Chemical, Environmental and Biological Engineering, TU Wien, Vienna, Austria.
Research Area Biochemical Technology, Institute of Chemical, Environmental and Biological Engineering, TU Wien, Vienna, Austria
Appl Environ Microbiol. 2017 Dec 15;84(1). doi: 10.1128/AEM.01742-17. Print 2018 Jan 1.
can produce up to 100 g/liter of extracellular proteins. The major and industrially relevant products are cellobiohydrolase I (CBHI) and the hemicellulase XYNI. The genes encoding both enzymes are transcriptionally activated by the regulatory protein Xyr1. The first 850 nucleotides of the promoter contain 14 Xyr1-binding sites (XBS), and 8 XBS are present in the promoter. Some of these XBS are arranged in tandem and others as inverted repeats. One such element, an inverted repeat, plays a crucial role in the inducibility of the promoter. We investigated the impact of the properties of such elements by shuffling them by insertion, exchange, deletion, and rearrangement of elements in both the and promoter. A promoter-reporter assay using the gene allowed us to measure changes in the promoter strength and inducibility. Most strikingly, we found that an inverted repeat of XBS causes an important increase in promoter strength and allows induction by xylan or wheat straw. Furthermore, evidence is provided that the distances of elements to the transcription start site have important influence on promoter activity. Our results suggest that the arrangement and distances of elements have large impacts on the strength of the promoter, whereas the sheer number of XBS has only secondary importance. Ultimately, the biotechnologically important promoter can be improved by element rearrangement. In the present study, we demonstrate that the arrangement of elements has a major impact on promoter strength and inducibility. We discovered an influence on promoter activity by the distances of elements to the transcription start site. Furthermore, we found that the configuration of elements has a greater effect on promoter strength than does the sheer number of transactivator binding sites present in the promoter. Altogether, the arrangement of elements is an important factor that should not be overlooked when enhancement of gene expression is desired.
可产生高达100克/升的细胞外蛋白质。主要的且与工业相关的产物是纤维二糖水解酶I（CBHI）和半纤维素酶XYNI。编码这两种酶的基因由调节蛋白Xyr1转录激活。该启动子的前850个核苷酸包含14个Xyr1结合位点（XBS），且在该启动子中存在8个XBS。其中一些XBS串联排列，其他的则为反向重复序列。这样一个元件，即反向重复序列，在该启动子的诱导性中起关键作用。我们通过在该启动子和另一启动子中对元件进行插入、交换、缺失和重排来改组它们，从而研究这些元件特性的影响。使用该基因的启动子 - 报告基因测定使我们能够测量启动子强度和诱导性的变化。最引人注目的是，我们发现XBS的反向重复序列会导致该启动子强度显著增加，并允许木聚糖或麦秸诱导。此外，有证据表明元件到转录起始位点的距离对启动子活性有重要影响。我们的结果表明，元件的排列和距离对该启动子的强度有很大影响，而XBS的数量仅具有次要重要性。最终，通过元件重排可以改进具有生物技术重要性的该启动子。在本研究中，我们证明元件的排列对启动子强度和诱导性有重大影响。我们发现元件到转录起始位点
