Programmable Synthetic Upstream Activating Sequence Library for Fine-Tuning Gene Expression Levels in Saccharomyces cerevisiae

• DOI: 10.1021/acssynbio.1c00511
• Publication Date: 2022-02-23T17:45:09Z
• Authors: Shiyun Li; Lizhou Ma; Wenxuan Fu; Ruifang Su; Yunying Zhao; Yu Deng
• Keywords: 0303 health sciences; 03 medical and health sciences; Transcription, Genetic; Gene Expression Regulation, Fungal; Gene Expression; Saccharomyces cerevisiae; Promoter Regions, Genetic; Gene Library

A wide dynamic range of promoters is necessary for fine-tuning transcription levels. However, weak intensity and narrow dynamic range limit transcriptional regulation via constitutive promoters. The upstream activation sequence (UAS) located upstream of the core promoter is a crucial region that could obviously enhance promoter strength. Herein, we created a random mutagenesis library consisting of 330 different variants based on the UAS of the TDH3 promoter with an ∼37-fold dynamic range by error-prone polymerase chain reaction (PCR) and obtained strong intensity mutant UAS, which was ∼12-fold greater than the wild-type UASTDH3. Analysis of the mutant library revealed 15 strength-enhancing sites and their corresponding bases of the UASTDH3 regions, which provided the impetus for a synthetic library. The resulting 32 768 mutant UAS library was constructed by permutation and combination of the bases of the 15 enhancing sites. To characterize the library, a strength prediction model was built by correlating DNA structural features and UAS strength, which provided a model between UAS sequence and intensity. Following characterization, the UAS library was applied to precisely regulate gene expression in the production of β-carotene, proving that the UAS library would be a useful tool for gene tuning in metabolic engineering. In summary, we designed, constructed, and characterized a UAS library that facilitated precise tuning of transcription levels of target proteins.