6.4 Synthetic Yeast Promoters 117An alternative strategy for obtaining promoter libraries is the substitution of
the non‐consensus sequences of the promoter with random sequences. Non‐
consensus sequences usually do not play a direct role in transcription initiation
regulation; that is, they do not bind to specific proteins. However, those sequences
might modulate the process indirectly, for example, by keeping the optimal dis-
tance between functional sequences [91], by influencing the local DNA helical
parameters [95], or by modulating the efficiency of nucleosome formation and
clearance [43, 96]. Therefore, promoter variants containing modifications in
non‐consensus sequences will differ from each other by small changes in
strength. The modification strategy is based on the synthesis of libraries of
oligonucleotides encoding the promoter sequence. In each oligonucleotide the
consensus sequences are separated by degenerate stretches of nucleotides of
variable length [88]. This approach was used to modify the profilin (PFY1)
promoter. The library obtained spanned a range of activities from 11% to 100%
relative to the starting promoter [89].
6.4.2 Synthetic Hybrid Promoters
Synthetic hybrid promoters combine DNA sequences originally belonging to
different promoters, but retain the typical bipartite structure of natural promot-
ers [91] (Figure 6.2).
The choice of the core promoter has effects on the overall performance of the
hybrid promoter, as it controls the efficiency of the PIC assembly [23] and the
identification of the TSS(s) [26]. Frequently, synthetic hybrid promoters contain
the native core promoter of inducible genes, for example, LEU2 or CYC1
[3, 90, 97]. Strong synthetic core promoters have been isolated from DNA librar-
ies where the TATA element and the TSS consensus sequence were separated by
a randomized spacer of 30 nucleotides. An additional stretch of 30 nucleotides
placed between the TATA element and the upstream TFBSs improves the core
promoter robustness by possibly avoiding steric hindrances between the tran-
scription factors, TBP, and other general transcription factors that bind to the
TFBSs or the core promoter [98].
The main advantage of the synthetic hybrid promoter approach is the possi-
bility of using any DNA sequence targeted by a protein as an upstream element.
Endogenous TFBSs link the synthetic promoter to a regulatory pathway. For
example, by placing the UAS of GAL1–10 in front of the TDH3 promoter, a
hybrid promoter that is active in glucose and further stimulated by galactose
was obtained [90]. Heterologous TFBSs either belong originally to other
species or are artificial sequences. They enable the implementation of orthog-
onal transcription systems that are independent of metabolism [99]. In fact,
heterologous sequences are not recognized by any yeast transcription factor,
unless they are homologous or, by chance, similar to endogenous sequences
[34, 97, 100]. Promoters containing combinations of TFBSs are sensitive to
several stimuli [90].
Modification of the binding affinity between the transcription factor and its
cognate TFBS results in promoter strength modulation [99, 101]. Alternatively,
the strength of the promoter can be tuned by increasing the number of TFBS