Optogenetics refers to the use of light for the control of gene expression. This is achieved via light sensing domains, which are coupled to regulatory proteins that control gene expression. Starting in 2002 with channel rhodopsins the field has undergone rapid development and since then, rhodopsins, BLUF domains and phytochromes have now all been exploited for this purpose. Optogenetics has become an indispensible tool for neuroscientists to study neural networks and brain function. It is clear that optogenetics also has the potential to revolutionise the field of synthetic biology by allowing light-dependent control of gene expression in biosynthetic pathways. The phytochromes have been the subject of detailed photochemical and structural studies. Although phytochromes have proven to be an attractive target for new applications in the field of optogenetics, their use has been limited due to their narrow wavelength sensitivity. However, several atypical phytochromes that exhibit broad spectral properties have been discovered recently. Here, we have expressed and purified two of these atypical phytochromes, one from cyanobacteria and one from algae. Several biophysical techniques, including time-resolved visible and infrared spectroscopy, cryo-trapping and time-resolved X-Ray scattering, were used to understand the photocycles and characterise the mechanism of photoreactions of these atypical phytochromes in more detail. These findings are discussed in relation to the photocycles of the well-studied typical red/far-red phytochromes. In order to exploit the broad spectral diversity, we used these atypical phytochromes to engineer an optogenetic toolkit that allows light-induced modulation of gene expression. The modular nature of phytochromes makes them an ideal target for coupling different photosensory core modules with selected effector domains in a "mix and match" manner. We demonstrated that this approach can be used to make functional chimaeras, which showed both in-vitro and in-vivo activity. This work was done in parallel with refactoring of a published optogenetic system onto the linalool biosynthesis pathway, which will eventually be used as a benchmark to test the chimaeric systems against. Ultimately, this work has set the foundations to allow an optogenetic toolkit to be established for the light-dependent control of biosynthetic pathways for biotechnology applications.
- Time Resolved Spectroscopy