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

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.