1 research outputs found
Synthetic Core Promoters as Universal Parts for Fine-Tuning Expression in Different Yeast Species
Synthetic
biology and metabolic engineering experiments frequently
require the fine-tuning of gene expression to balance and optimize
protein levels of regulators or metabolic enzymes. A key concept of
synthetic biology is the development of modular parts that can be
used in different contexts. Here, we have applied a computational
multifactor design approach to generate <i>de novo</i> synthetic
core promoters and 5′ untranslated regions (UTRs) for yeast
cells. In contrast to upstream <i>cis</i>-regulatory modules
(CRMs), core promoters are typically not subject to specific regulation,
making them ideal engineering targets for gene expression fine-tuning.
112 synthetic core promoter sequences were designed on the basis of
the sequence/function relationship of natural core promoters, nucleosome
occupancy and the presence of short motifs. The synthetic core promoters
were fused to the <i>Pichia pastoris AOX1</i> CRM, and the
resulting activity spanned more than a 200-fold range (0.3% to 70.6%
of the wild type <i>AOX1</i> level). The top-ten synthetic
core promoters with highest activity were fused to six additional
CRMs (three in <i>P. pastoris</i> and three in <i>Saccharomyces cerevisiae</i>). Inducible CRM constructs showed
significantly higher activity than constitutive CRMs, reaching up
to 176% of natural core promoters. Comparing the activity of the same
synthetic core promoters fused to different CRMs revealed high correlations
only for CRMs within the same organism. These data suggest that modularity
is maintained to some extent but only within the same organism. Due
to the conserved role of eukaryotic core promoters, this rational
design concept may be transferred to other organisms as a generic
engineering tool