Optogenetic Downregulation of Protein Levels with an Ultrasensitive Switch

Abstract

Optogenetic control of protein activity is a versatile technique to gain control over cellular processes, for example, for biomedical and biotechnological applications. Among other techniques, the regulation of protein abundance by controlling either transcription or protein stability found common use as this controls the activity of any type of target protein. Here, we report modules of an improved variant of the photosensitive degron module and a light-sensitive transcription factor, which we compared to doxycyclinedependent transcriptional control. Given their modularity the combined control of synthesis and stability of a given target protein resulted in the synergistic down regulation of its abundance by light. This combined module exhibits very high switching ratios, profound downregulation of protein abundance at low lightfluxes, and fast protein depletion kinetics. Overall, this synergistic optogenetic multistep control (SOMCo) module is easy to implement and results in a regulation of protein abundance superior to each individual component.

Degradation of integral membrane proteins modified with the photosensitive degron module requires the cytosolic endoplasmic reticulum–associated degradation pathway

“…Also, for RFP-psd, we observed a slower degradation in uba1 ts cells than in the control cells (Figure 10C). Next, we followed RFP-psd degradation by fluorescence measurements (Hasenjäger et al , 2019). We performed a cycloheximide chase assay in control cells, as well as in ubc6Δ , ubc7Δ , ubc6Δ ubc7Δ , and doa10Δ mutant cells.…”

“…The software Fiji was used for image processing and fluorescence quantification (Schindelin et al , 2012). Flow cytometer measurements were performed as described by Hasenjäger et al (2019). Briefly, yeast cells in logarithmic growth phase were treated with sodium azide (10 mM final concentration) and transferred to a 96-well plate.…”

Dual Systems for Enhancing Control of Protein Activity through Induced Dimerization Approaches

“…[60] Taking a similar approach in yeast, Hasenjager et al used a different light-sensitive degron consisting of the degron cODC1 caged by AsLOV2. [64] The authors also generated a new light-disrupted transcriptional system using AsLOV2 and Zdk to control association of a DNA-binding domain and VP16 activation domain (VP16) in dark, and trigger dissociation in light. To VP16, they added the light-induced degron element, to enable degradation of this component with light application.…”

The Promise of Optogenetics for Bioproduction: Dynamic Control Strategies and Scale-Up Instruments

“…The widely used yeast Pichia pastoris has not, to date, been adapted to optogenetic control, but we expect this to be achieved within a few years. Moreover, widely used synthetic biology systems have also been adapted for optogenetics: photo-inducible CRE recombinases [36] and photosensitive degrons [37] could be used to complement the current optogenetic systems used for bioproduction.…”