WC-1 is a blue light photoreceptor containing a
LOV domain (He et al. 2002; Froehlich et al.
2002), which under blue light illumination
passes to an active form allowing interaction
with another WC-1 protein through a LOVLOV interaction, affecting the WCC stoichiometry (Cheng et al. 2003). Thus, the sequence
specificity of WCC changes and a photoactivated multimer binds to specific elements
found in promoters of several light-activated
genes (He and Liu 2005; Chen et al. 2009). One
of these genes is vvd, and, therefore, after prolonged illumination, VVD is accumulated and
becomes light-activated, breaking the WCCWCC interactions by competitive LOV-LOV
binding between VVD and WC-1. This latter
interaction leads to the attenuation of lightinducible transcription in N. crassa in a process
known as photoadaptation (Chen et al. 2010;
Malzahn et al. 2010; Hunt et al. 2010; Gin et al.
2013). Thereby, the natural WC-1/VVD interaction is an interesting combination of fungal
photoreceptors to generate a novel lightinducible approach. Recently, our group used
this feature from N. crassa photobiology to
implement an optogenetic system in yeast
that we termed “FUN-LOV” (Fig. 12.2a). We
showed that “FUN-LOV” allows high expression of luc upon constant light illumination,
showing almost null residual activity in the
dark (Salinas et al. 2018). Thus, “FUN-LOV”
yielded high levels of the reporter under constant white or blue lights, achieving superior
results over those obtained with the classical
galactose induction strategy. Importantly, we
achieved induction levels of ~1300-fold when
comparing light versus dark conditions, and
even over 2500-fold when analyzing luciferase
levels from extracts of cells grown in the light
versus dark. Moreover, the “FUN-LOV” system
showed a very dynamic on/off gene expression
pattern in response to light/dark cycles, reaching strong induction during lights on and quick
decrease of the signal as lights were turned off.
We also tested if longer exposure to light pulses
would increase expression of the reporter gene,
observing a rise in the signal as the duration of
exposure increases. On the other hand, despite
that a doubling in light intensity (from 20 to
40 mmol m
À2 s
À1 ) did not produce an increase
in luciferase levels (Salinas et al. 2018), we have
observed a slower induction kinetic and diminished maximal levels in low light intensity
such as 1 mmol m
À2 s
À1 (Delgado, unpublished
results). These results confirm that “FUN-LOV”
not only allows reaching high levels of expression and displaying low background signals but
that it is also tunable by modifying the intensity
and the duration of the light stimulus. We also
assessed the feasibility of the “FUN-LOV” system to control the expression of a heterologous
protein such as limonene synthase from Cannabis sativa, observing a strong production of the
enzyme in constant light conditions by western
blot assays (Salinas et al. 2018). Importantly, as
proof of principle, we evaluated the performance of this synthetic optogenetic switch to
induce a biotechnologically relevant phenotype
such as flocculation (Fig. 12.3c). Depending on
the genetic target of “FUN-LOV,” we controlled
the formation of yeast cell aggregates by light or
darkness, demonstrating the versatility of our
system (Salinas et al. 2018).
B. Optogenetic Approaches Directly
Implemented in Filamentous Fungi
In past years, different groups have attempted
to harness the power of light to control gene
expression directly in filamentous fungi, which
are already capable of sensing light. An example
of this was utilizing the promoter of the vvd
gene from N. crassa, to control the expression
of autologous and heterologous proteins in this
organism. The expression of vvd is quite high,
reaching augmented expression within minutes
of turning on the lights, while levels are
extremely low in constant darkness (Cesbron
et al. 2013). Thus, the vvd promoter was set to
control gfp reporter gene expression and transformed ectopically both in wild-type (WT) and
Dvvd backgrounds, the latter to eliminate
photoadaptation (for continuous high expression in constant light). As expected, the system
revealed tight control as GFP levels were almost
undetectable when the fungus was kept in constant darkness, whereas within 1 h of light
exposure, GFP was high and its levels increased
linearly for almost 2 days in constant light.
308
V. Rojas et al.
LOV domain (He et al. 2002; Froehlich et al.
2002), which under blue light illumination
passes to an active form allowing interaction
with another WC-1 protein through a LOVLOV interaction, affecting the WCC stoichiometry (Cheng et al. 2003). Thus, the sequence
specificity of WCC changes and a photoactivated multimer binds to specific elements
found in promoters of several light-activated
genes (He and Liu 2005; Chen et al. 2009). One
of these genes is vvd, and, therefore, after prolonged illumination, VVD is accumulated and
becomes light-activated, breaking the WCCWCC interactions by competitive LOV-LOV
binding between VVD and WC-1. This latter
interaction leads to the attenuation of lightinducible transcription in N. crassa in a process
known as photoadaptation (Chen et al. 2010;
Malzahn et al. 2010; Hunt et al. 2010; Gin et al.
2013). Thereby, the natural WC-1/VVD interaction is an interesting combination of fungal
photoreceptors to generate a novel lightinducible approach. Recently, our group used
this feature from N. crassa photobiology to
implement an optogenetic system in yeast
that we termed “FUN-LOV” (Fig. 12.2a). We
showed that “FUN-LOV” allows high expression of luc upon constant light illumination,
showing almost null residual activity in the
dark (Salinas et al. 2018). Thus, “FUN-LOV”
yielded high levels of the reporter under constant white or blue lights, achieving superior
results over those obtained with the classical
galactose induction strategy. Importantly, we
achieved induction levels of ~1300-fold when
comparing light versus dark conditions, and
even over 2500-fold when analyzing luciferase
levels from extracts of cells grown in the light
versus dark. Moreover, the “FUN-LOV” system
showed a very dynamic on/off gene expression
pattern in response to light/dark cycles, reaching strong induction during lights on and quick
decrease of the signal as lights were turned off.
We also tested if longer exposure to light pulses
would increase expression of the reporter gene,
observing a rise in the signal as the duration of
exposure increases. On the other hand, despite
that a doubling in light intensity (from 20 to
40 mmol m
À2 s
À1 ) did not produce an increase
in luciferase levels (Salinas et al. 2018), we have
observed a slower induction kinetic and diminished maximal levels in low light intensity
such as 1 mmol m
À2 s
À1 (Delgado, unpublished
results). These results confirm that “FUN-LOV”
not only allows reaching high levels of expression and displaying low background signals but
that it is also tunable by modifying the intensity
and the duration of the light stimulus. We also
assessed the feasibility of the “FUN-LOV” system to control the expression of a heterologous
protein such as limonene synthase from Cannabis sativa, observing a strong production of the
enzyme in constant light conditions by western
blot assays (Salinas et al. 2018). Importantly, as
proof of principle, we evaluated the performance of this synthetic optogenetic switch to
induce a biotechnologically relevant phenotype
such as flocculation (Fig. 12.3c). Depending on
the genetic target of “FUN-LOV,” we controlled
the formation of yeast cell aggregates by light or
darkness, demonstrating the versatility of our
system (Salinas et al. 2018).
B. Optogenetic Approaches Directly
Implemented in Filamentous Fungi
In past years, different groups have attempted
to harness the power of light to control gene
expression directly in filamentous fungi, which
are already capable of sensing light. An example
of this was utilizing the promoter of the vvd
gene from N. crassa, to control the expression
of autologous and heterologous proteins in this
organism. The expression of vvd is quite high,
reaching augmented expression within minutes
of turning on the lights, while levels are
extremely low in constant darkness (Cesbron
et al. 2013). Thus, the vvd promoter was set to
control gfp reporter gene expression and transformed ectopically both in wild-type (WT) and
Dvvd backgrounds, the latter to eliminate
photoadaptation (for continuous high expression in constant light). As expected, the system
revealed tight control as GFP levels were almost
undetectable when the fungus was kept in constant darkness, whereas within 1 h of light
exposure, GFP was high and its levels increased
linearly for almost 2 days in constant light.
308
V. Rojas et al.