CRY2 oligomerization achieved high-affinity
clustering of these proteins (Bugaj et al. 2015).
In the same context, “CYR2olig” is a point
mutant protein (E490G) that shows increased
levels of light-mediated clustering (Taslimi
et al. 2014). Using a similar logic, the “LARIAT”
system was developed to inactivate proteins by
its clustering upon CRY2/CIB1 interaction.
Thus, the fusion of CIB1 to a multimeric protein
and CRY2 to the protein of interest permitted
the sequestration of this protein in response to
light (Lee et al. 2014).
An alternative set of blue light photoreceptors are the ones containing light-oxygen-voltage (LOV) domains (Pudasaini et al. 2015).
These domains belong to the Per-Arnt-Sim
(PAS) protein family, which are found in organisms across different kingdoms (Glantz et al.
2016). In general, LOV-containing proteins
bind flavin derivatives (FMN or FAD) as the
chromophore. In response to blue light, a thiol
photo-adduct between the flavin chromophore
and a conserved cysteine (Cys) residue in the
LOV domain is formed, generating a conformational change in the protein (Pudasaini et al.
2015; Glantz et al. 2016). This cysteinyl-flavin
interaction is spontaneously hydrolyzed in
darkness, however, the time for photo-adduct
reversion to basal inactive state depending on
the photoreceptors being analyzed (Pudasaini
et al. 2015; Glantz et al. 2016). For instance,
Flavin-binding kelch domain F-box protein
(FKF1) from A. thaliana is a LOV-containing
protein that binds its natural partner GIGANTEA (GI) in response to blue light (Fig. 12.2a,
b). This light-induced association occurs in a
few minutes and is stable for several hours
(Sawa et al. 2007). In this way, optogenetic
systems based on FKF1/GI interaction have
been developed for light-controlled gene
expression and subcellular protein localization
(Fig. 12.2b, c) (Yazawa et al. 2009), but its slow
photocycle limits temporal resolution. The blue
light photoreceptor AsLOV2 from the plant
Avena sativa overcomes the photocycle limits,
showing fast kinetics between inactive and
active states (Swartz et al. 2001). The LOV2
domain of this photoreceptor is flanked by
two helixes (A’a and Ja) that are unfolded by
blue light stimulation and return to the basal
condition in a few seconds each (Fig. 12.2e)
(Harper et al. 2003). In this way, AsLOV2
light-dependent unfolding allows implementing one-component optogenetic systems by
caging. Caging approaches allow the temporal
hindrance of binding/active sites of a specific
protein, depending on how this protein is fused
to AsLOV2. Thus, the caging interaction can be
modified by blue light, exposing the functional
domain of a protein and providing fine control
at the post-translational level. The AsLOV2
domain was also used to implement an exciting
approach aimed to diminish the variability of
gene expression levels. By fusing this LOV
domain to different components of the Tet system, gene circuits based on negative feedback
allowed the reduction of transcriptional noise
in human cell lines (Guinn and Bala ´zsi 2019). In
addition, the AsLOV2 domain has been adapted
to implement two-component systems
mediated by light-dependent interaction. For
instance, the “TULIP” system exploits the caging mechanism in a fusion protein between
AsLOV2 and a specific peptide where, in
response to blue light, this epitope is exposed
and interacts with a synthetic PSD95-Dlg1-zo1
(PDZ) domain (Strickland et al. 2012). Similarly, the phototropin 1 (PHOT1) from A. thaliana has a LOV domain (AtLOV2) that shares
the basic mechanism of photoperception with
AsLOV2 (Christie et al. 1999). In that context,
Renicke et al. (2013) utilized a degradation
sequence sterically blocked by the Ja-helix of
the AtLOV2, which in turn was linked to a
specific protein of interest. Thus, upon blue
light stimulation, the displacement of Ja-helix
exposed the degradation signal, changing the
stability of relevant proteins in the yeast cell
cycle (Renicke et al. 2013). On the other hand,
LOV-containing photoreceptors can also be
found in bacteria. For instance, the transcription factor EL222 from Erythrobacter litoralis
has been also used to implement synthetic
light-inducible systems (Fig. 12.2a, b). Besides
a helix-turn-helix (HTH) DBD, this protein
contains a LOV module that allows transcriptional activation upon blue light stimulation
(Zoltowski et al. 2013). In the dark, both
domains interact and a critical dimerization
sequence is blocked, avoiding the binding of
EL222 to specific promoter regions of the
genome. Under blue light conditions, the for12 Exploiting Fungal Photobiology as a Source of Novel Bio-blocks for Optogenetic Systems
303
clustering of these proteins (Bugaj et al. 2015).
In the same context, “CYR2olig” is a point
mutant protein (E490G) that shows increased
levels of light-mediated clustering (Taslimi
et al. 2014). Using a similar logic, the “LARIAT”
system was developed to inactivate proteins by
its clustering upon CRY2/CIB1 interaction.
Thus, the fusion of CIB1 to a multimeric protein
and CRY2 to the protein of interest permitted
the sequestration of this protein in response to
light (Lee et al. 2014).
An alternative set of blue light photoreceptors are the ones containing light-oxygen-voltage (LOV) domains (Pudasaini et al. 2015).
These domains belong to the Per-Arnt-Sim
(PAS) protein family, which are found in organisms across different kingdoms (Glantz et al.
2016). In general, LOV-containing proteins
bind flavin derivatives (FMN or FAD) as the
chromophore. In response to blue light, a thiol
photo-adduct between the flavin chromophore
and a conserved cysteine (Cys) residue in the
LOV domain is formed, generating a conformational change in the protein (Pudasaini et al.
2015; Glantz et al. 2016). This cysteinyl-flavin
interaction is spontaneously hydrolyzed in
darkness, however, the time for photo-adduct
reversion to basal inactive state depending on
the photoreceptors being analyzed (Pudasaini
et al. 2015; Glantz et al. 2016). For instance,
Flavin-binding kelch domain F-box protein
(FKF1) from A. thaliana is a LOV-containing
protein that binds its natural partner GIGANTEA (GI) in response to blue light (Fig. 12.2a,
b). This light-induced association occurs in a
few minutes and is stable for several hours
(Sawa et al. 2007). In this way, optogenetic
systems based on FKF1/GI interaction have
been developed for light-controlled gene
expression and subcellular protein localization
(Fig. 12.2b, c) (Yazawa et al. 2009), but its slow
photocycle limits temporal resolution. The blue
light photoreceptor AsLOV2 from the plant
Avena sativa overcomes the photocycle limits,
showing fast kinetics between inactive and
active states (Swartz et al. 2001). The LOV2
domain of this photoreceptor is flanked by
two helixes (A’a and Ja) that are unfolded by
blue light stimulation and return to the basal
condition in a few seconds each (Fig. 12.2e)
(Harper et al. 2003). In this way, AsLOV2
light-dependent unfolding allows implementing one-component optogenetic systems by
caging. Caging approaches allow the temporal
hindrance of binding/active sites of a specific
protein, depending on how this protein is fused
to AsLOV2. Thus, the caging interaction can be
modified by blue light, exposing the functional
domain of a protein and providing fine control
at the post-translational level. The AsLOV2
domain was also used to implement an exciting
approach aimed to diminish the variability of
gene expression levels. By fusing this LOV
domain to different components of the Tet system, gene circuits based on negative feedback
allowed the reduction of transcriptional noise
in human cell lines (Guinn and Bala ´zsi 2019). In
addition, the AsLOV2 domain has been adapted
to implement two-component systems
mediated by light-dependent interaction. For
instance, the “TULIP” system exploits the caging mechanism in a fusion protein between
AsLOV2 and a specific peptide where, in
response to blue light, this epitope is exposed
and interacts with a synthetic PSD95-Dlg1-zo1
(PDZ) domain (Strickland et al. 2012). Similarly, the phototropin 1 (PHOT1) from A. thaliana has a LOV domain (AtLOV2) that shares
the basic mechanism of photoperception with
AsLOV2 (Christie et al. 1999). In that context,
Renicke et al. (2013) utilized a degradation
sequence sterically blocked by the Ja-helix of
the AtLOV2, which in turn was linked to a
specific protein of interest. Thus, upon blue
light stimulation, the displacement of Ja-helix
exposed the degradation signal, changing the
stability of relevant proteins in the yeast cell
cycle (Renicke et al. 2013). On the other hand,
LOV-containing photoreceptors can also be
found in bacteria. For instance, the transcription factor EL222 from Erythrobacter litoralis
has been also used to implement synthetic
light-inducible systems (Fig. 12.2a, b). Besides
a helix-turn-helix (HTH) DBD, this protein
contains a LOV module that allows transcriptional activation upon blue light stimulation
(Zoltowski et al. 2013). In the dark, both
domains interact and a critical dimerization
sequence is blocked, avoiding the binding of
EL222 to specific promoter regions of the
genome. Under blue light conditions, the for12 Exploiting Fungal Photobiology as a Source of Novel Bio-blocks for Optogenetic Systems
303