6.5 Conclusions 121been applied to mammalian synthetic gene circuits [132]. Repression by steric
hindrance can also be obtained by using a CRISPR‐derived system. A complex
consisting of sgRNA and dCas9 competes with the transcription activator of the
target promoter, as it targets the same TFBS. The sgRNA–dCas9 complex pre-
vents transcription initiation by blocking the access of the TFBS [117]. However,
neither the tetR nor other bacterial repressor domains nor the CRISPR‐based
systems show a sufficiently tight repression of the basal level to be useful in a
broad setting.
The basal level of heterologous repression systems can be further reduced by
fusing a DNA‐binding domain to a component of the eukaryotic transcriptional
repressor complex, like Tup1 or Cyc8. LexA, when fused either to Tup1 or
to Cyc8, mediates repression of hybrid promoters containing LexA TFBSs
[133, 134]. The CRISPR‐derived system has been used in a similar strategy.
dCas9 was fused to a mammalian repressor that recruits a yeast histone deacety-
lase. This repressor was targeted to the TEF1 promoter by designing a specific
sgRNA [117]. As a drawback, these systems slow down the transcriptional induc-
tion kinetics and affect the expression levels of (endogenous) genes located in
close proximity.
6.5 Conclusions
In this chapter, we highlighted some examples of both regulated and constitutive
natural yeast promoters. The characterization of these sequences allowed for the
identification of structural and functional features that are exploited to build
synthetic promoters and heterologous transcription factors. Examples of the
application of these promoters in synthetic biology have been reviewed in [75, 91,
135–137].
Today, in the context of implementation of novel functions in cells, the
construction of robust promoters is crucial [99]. Recent efforts to transform
biotechnology and synthetic biology into more engineering‐like disciplines
also motivate the construction of synthetic promoters. In fact, their implemen-
tation is an essential step for the abstraction and standardization of concepts
like promoter structure and transcription initiation [138]. In this perspective,
modularization and orthogonality are the aspects that need to be further
developed.
Modularization enables the implementation of new promoters and transcrip-
tion factors by combining well‐characterized and structurally independent mod-
ules. Orthogonal systems do not depend on and influence the endogenous
metabolism. This ensures a robust behavior and the possibility to reuse the sys-
tem in different environments and contexts. Today, zinc finger, TALE, and
CRISPR‐based technologies allow the design of artificial transcription factors
that recognize unique sequences [99, 100, 139]. Additionally, the CRISPR‐based
toolkits available now allow for simple construction of strains containing con-
structs with the different mechanisms discussed in this review [140]. Together,
modularization and orthogonality ensure versatility and the possibility to easily
construct new systems with improved or new functionalities.