mation of a photo-adduct disrupts the interaction between LOV and HTH, leading to the
protein homodimerization and light-dependent
gene expression (Zoltowski et al. 2013). Considering these EL222 features, a one-component
gene expression system was developed and
tested in different mammalian cell lines,
obtaining a wide range of luciferase expression
levels in response to blue light stimulation.
Moreover, the system showed fast kinetics of
activation/deactivation and low basal activity in
the dark (Motta-Mena et al. 2014). Notably, this
approach was recently implemented in S. cerevisiae. In this work, the EL222 optogenetic system was used to directly control the expression
of a gfp reporter gene under the synthetic C120
promoter. The circuit, named OptoEXP, was
evaluated upon different light treatments, displaying fluorescence levels that are similar to
those obtained with an adh1 constitutive promoter (Zhao et al. 2018). On the other hand, the
OptoINVRT circuit allows light-dependent
gene repression. In this case, a constitutively
activated GAL4 transcription factor allowed
the constant expression of the fluorescent
reporter controlled by gal1 promoter in darkness by, while the transcription levels were
switched off by the light-dependent production
of the GAL80 repressor (Zhao et al. 2018).
Thereby, depending on the gene controlled by
the synthetic EL222 system, it was possible to
activate or repress different genes in response
to light using OptoEXP or OptoINVRT circuits,
respectively. In this way, the biosynthesis of a
metabolite of interest could be inhibited during
the growth phase, whereas it could be produced
(at will) when enough cell biomass has been
accumulated in a yeast fermentation process.
Using these optogenetic metabolic valves, it
was possible to obtain high yields of isobutanol
and 2-methyl-1-butanol production after a
light/dark transition by controlling an essential
mitochondrial pathway (Fig. 12.3a). This work
is a remarkable case of control of multiple
genes and represents one of the first examples
of successful applications of optogenetics to
metabolic engineering of molecules of interest
in yeast (Zhao et al. 2018). Other blue light
sensing domains will be covered in the upcoming section.
C. Beyond Red and Blue Light
The so far mentioned optogenetic systems
respond to wavelengths that are part of classic
white light (~400–700 nm). However, the sunlight also includes ultraviolet light (UV, ~100–
400 nm) and near-infrared light (NIR, >700 nm).
Since UV causes DNA damage and generation of
reactive oxygen species (ROS), sessile organisms
such as plants had to develop tolerance and
acclimation mechanisms (Yin and Ulm 2017;
Jenkins 2017). In A. thaliana, the photoreceptor
UV-B resistance 8 (UVR8) forms inactive homodimers in darkness conditions by an interaction
between charged amino acids. In response to
UV-B light (~280–315 nm), a conformational
change breaks these intermolecular interactions
and UVR8 behaves as a monomer (Christie et al.
2012). In this state, the photoreceptor can interact with the constitutive photomorphogenic 1
(COP1) protein (Fig. 12.2a), triggering the UVprotection signaling pathways (Oravecz et al.
2006; Favory et al. 2009). Interestingly, UVR8
does not require a chromophore, and its photoperception is based on the intrinsic amino acids
of the protein, where tryptophan (Trp233 and
Trp285) residues are responsible for UV-B light
sensing (Rizzini et al. 2011). Although optogenetic systems based on UVR8/COP1 interaction
have been implemented, they have been less
exploited due to the cellular damages caused by
high-energy UV radiation. On the other hand, it
has been reported that certain bacterial species
have the capacity to respond to NIR. For
instance, the BphP1 protein from Rhodopseudomonas palustris can perceive NIR, and upon light
stimulation interacts with the PpsR2 protein
(Fig. 12.2a, b) (Kojadinovic et al. 2008). In comparison with shorter wavelengths, NIR causes
less phototoxicity at constant exposures and is
more effective to penetrate into mammalian tissues (Weissleder and Ntziachristos 2003). Moreover, this interaction does not require a
chromophore of bilin nature, such as PCB, utilizing instead biliverdin as cofactor. In that context,
an optoinducible system based on BphP1/PpsR2
interaction was implemented in mammalian cell
lines, allowing the control of cell signaling, gene
expression and subcellular protein localization
(Kaberniuk et al. 2016).
304
V. Rojas et al.
protein homodimerization and light-dependent
gene expression (Zoltowski et al. 2013). Considering these EL222 features, a one-component
gene expression system was developed and
tested in different mammalian cell lines,
obtaining a wide range of luciferase expression
levels in response to blue light stimulation.
Moreover, the system showed fast kinetics of
activation/deactivation and low basal activity in
the dark (Motta-Mena et al. 2014). Notably, this
approach was recently implemented in S. cerevisiae. In this work, the EL222 optogenetic system was used to directly control the expression
of a gfp reporter gene under the synthetic C120
promoter. The circuit, named OptoEXP, was
evaluated upon different light treatments, displaying fluorescence levels that are similar to
those obtained with an adh1 constitutive promoter (Zhao et al. 2018). On the other hand, the
OptoINVRT circuit allows light-dependent
gene repression. In this case, a constitutively
activated GAL4 transcription factor allowed
the constant expression of the fluorescent
reporter controlled by gal1 promoter in darkness by, while the transcription levels were
switched off by the light-dependent production
of the GAL80 repressor (Zhao et al. 2018).
Thereby, depending on the gene controlled by
the synthetic EL222 system, it was possible to
activate or repress different genes in response
to light using OptoEXP or OptoINVRT circuits,
respectively. In this way, the biosynthesis of a
metabolite of interest could be inhibited during
the growth phase, whereas it could be produced
(at will) when enough cell biomass has been
accumulated in a yeast fermentation process.
Using these optogenetic metabolic valves, it
was possible to obtain high yields of isobutanol
and 2-methyl-1-butanol production after a
light/dark transition by controlling an essential
mitochondrial pathway (Fig. 12.3a). This work
is a remarkable case of control of multiple
genes and represents one of the first examples
of successful applications of optogenetics to
metabolic engineering of molecules of interest
in yeast (Zhao et al. 2018). Other blue light
sensing domains will be covered in the upcoming section.
C. Beyond Red and Blue Light
The so far mentioned optogenetic systems
respond to wavelengths that are part of classic
white light (~400–700 nm). However, the sunlight also includes ultraviolet light (UV, ~100–
400 nm) and near-infrared light (NIR, >700 nm).
Since UV causes DNA damage and generation of
reactive oxygen species (ROS), sessile organisms
such as plants had to develop tolerance and
acclimation mechanisms (Yin and Ulm 2017;
Jenkins 2017). In A. thaliana, the photoreceptor
UV-B resistance 8 (UVR8) forms inactive homodimers in darkness conditions by an interaction
between charged amino acids. In response to
UV-B light (~280–315 nm), a conformational
change breaks these intermolecular interactions
and UVR8 behaves as a monomer (Christie et al.
2012). In this state, the photoreceptor can interact with the constitutive photomorphogenic 1
(COP1) protein (Fig. 12.2a), triggering the UVprotection signaling pathways (Oravecz et al.
2006; Favory et al. 2009). Interestingly, UVR8
does not require a chromophore, and its photoperception is based on the intrinsic amino acids
of the protein, where tryptophan (Trp233 and
Trp285) residues are responsible for UV-B light
sensing (Rizzini et al. 2011). Although optogenetic systems based on UVR8/COP1 interaction
have been implemented, they have been less
exploited due to the cellular damages caused by
high-energy UV radiation. On the other hand, it
has been reported that certain bacterial species
have the capacity to respond to NIR. For
instance, the BphP1 protein from Rhodopseudomonas palustris can perceive NIR, and upon light
stimulation interacts with the PpsR2 protein
(Fig. 12.2a, b) (Kojadinovic et al. 2008). In comparison with shorter wavelengths, NIR causes
less phototoxicity at constant exposures and is
more effective to penetrate into mammalian tissues (Weissleder and Ntziachristos 2003). Moreover, this interaction does not require a
chromophore of bilin nature, such as PCB, utilizing instead biliverdin as cofactor. In that context,
an optoinducible system based on BphP1/PpsR2
interaction was implemented in mammalian cell
lines, allowing the control of cell signaling, gene
expression and subcellular protein localization
(Kaberniuk et al. 2016).
304
V. Rojas et al.