Influence of cis Element Arrangement on Promoter Strength in Trichoderma reesei

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.