Engineering Light-Control in Biology

Abstract

Unraveling the transformative power of optogenetics in biology requires sophisticated engineering for the creation and optimization of light-regulatable proteins. In addition, diverse strategies have been used for the tuning of these light-sensitive regulators. This review highlights different protein engineering and synthetic biology approaches, which might aid in the development and optimization of novel optogenetic proteins (Opto-proteins). Focusing on non-neuronal optogenetics, chromophore availability, general strategies for creating light-controllable functions, modification of the photosensitive domains and their fusion to effector domains, as well as tuning concepts for Opto-proteins are discussed. Thus, this review shall not serve as an encyclopedic summary of light-sensitive regulators but aims at discussing important aspects for the engineering of light-controllable proteins through selected examples.

Keywords: bioengineering; light-control; optogenetics; photosensors; synthetic biology.

FIGURE 1

Chromophore availability in cells (A) Light sensing proteins contain either light-absorbing tryptophane conformations or bound small-molecule chromophores (B) Depending on the host organism and the photosensitive protein, the chromophore might be available in the cell through its native metabolism. Otherwise, availability of the chromophore can be realized through (C) external supplementation of the chromophore or its precursors, which requires their diffusion or active uptake into the cell, or by (D) transferring the genes necessary for its synthesis into the cells of interest.

FIGURE 2

Engineering strategies for light regulation (A) Transfer of light-sensing regulators and circuits from native host organisms into an organism of interest requires compatibility of the respective function and optimization for the new host (B) Change of the native sensing function of a protein to sensing of light through domain swapping (left), which was shown for the transformation of an osmoregulation bacterial two-component system (TCS) to a synthetic light-regulatable TCS (Levskaya et al., 2005). An intuitive way to categorize strategies for the creation of novel Opto-proteins is into intermolecular (C) and intramolecular light-control (D) (Baumschlager and Khammash 2021). Intermolecular light-control functions through proximity and distance of proteins. Thus, it usually comprises two or more interacting proteins, whose binding/distance can be regulated with light. Such regulation can be achieved through the reconstitution of inactive protein domains (e.g. split fragments in Opto-T7RNAP (Baumschlager et al., 2017); (C), left), the recruitment of an active protein to a location where it exerts function (e.g. membrane localization for control of phosphoinositide 3-kinase activity (Toettcher et al., 2011); (C), middle), or activation of a cellular function through clustering (e.g. activation of cell signaling with optoWnt (Rosenbloom et al., 2020); (C), right). In contrast, intramolecular control involves the development of a single protein chimera that comprises a light-responsive and an effector function. Regulation can be achieved allosterically in which light absorption leads to a structural change in the protein that changes the activity of the effector domain (e.g. enzymatic activity in yeast isocitrate dehydrogenase (Chen et al., 2021); (D), left), steric blocking/unblocking of the function of the effector (e.g. exposure of a transport signal in the light-inducible nuclear export system (LEXY) (Niopek et al., 2016); (D), middle), or steric regulation through occlusion (e.g. active site blocking of a protease (Zhou et al., 2012); (D), right).

FIGURE 3

Optimization of Opto-protein concentration (A–C) Potential scenarios encountered for light-induced and dark-state output/activity obtained with increasing Opto-protein concentrations. Optimal regulator expression is defined as the fold change of dark to light-induced reporter expression. In scenario one (A), dark and light-induced output increase proportionally, therefore the fold change is maintained for various regulator concentrations. Scenarios two (B) and three (C) show two examples in which the fold change is maximal at specific Opto-protein concentrations. Scenario two could be caused by the burden of the regulator expression which could lead to a reduction of the output, while in scenario three, a high regulator concentration might lead to increased dark state activation due to, for example, molecular crowding. (D) The concentration of the Opto-protein can also be adjusted throughout an experiment/biological process via a dual control scheme as shown for the Opto-T7RNAP, whose expression was controlled through the repressor rTetR (Baumschlager et al., 2020). rTetR binds tetO operator sequences of a corresponding promoter with anhydrotetracycline (aTc) bound, which represses transcription of the Opto-protein regulator. aTc can be inactivated with UVA light, leading to an induction of Opto-T7RNAP expression, which in turn can be activated with blue light. This regulation scheme was used to reduce the dark state activity of Opto-T7RNAP in the presence of aTc.