Modulating transcription through development of semi-synthetic yeast core promoters


Autoři: Thomas Decoene aff001;  Sofie L. De Maeseneire aff002;  Marjan De Mey aff001
Působiště autorů: Centre for Synthetic Biology (CSB), Ghent University, Ghent, Belgium aff001;  Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Ghent University, Coupure links, Ghent, Belgium aff002
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224476

Souhrn

Altering gene expression regulation by promoter engineering is a very effective way to fine-tune heterologous pathways in eukaryotic hosts. Typically, pathway building approaches in yeast still use a limited set of long, native promoters. With the today’s introduction of longer and more complex pathways, an expansion of this synthetic biology toolbox is necessary. In this study we elucidated the core promoter structure of the well-characterized yeast TEF1 promoter and determined the minimal length needed for sufficient protein expression. Furthermore, this minimal core promoter sequence was used for the creation of a promoter library covering different expression strengths. This resulted in a group of short, 69 bp promoters with an 8.0-fold expression range. One exemplar had a two and four times higher expression compared to the native CYC1 and ADH1 promoter, respectively. Additionally, as it was described that the protein expression range could be broadened by upstream activating sequences (UASs), we integrated earlier described single and multiple short, synthetic UASs in front of the strongest yeast core promoter. This approach resulted to further variation in protein expression and an overall promoter library spanning a 20-fold activity range and covering a length from 69 bp to maximally 129 bp. Furthermore, the robustness of this library was assessed on three alternative carbon sources besides glucose. As such, the suitability of short yeast core promoters for metabolic engineering applications on different media, either in an individual context or combined with UAS elements, was demonstrated.

Klíčová slova:

DNA transcription – Engineering and technology – Gene expression – Protein expression – Saccharomyces cerevisiae – Transcriptional control – Yeast – TATA box


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