Entwicklung und Charakterisierung von Flavoprotein-basierten optogenetischen Modulen in Saccharomyces cerevisiae

In the last decade, the field of optogenetics gained more and more attention in science. Several tools were developed, starting with light controlled ion-channels and devices to control the protein level, up to the control of physiological processes in living cells. These tools are based on photoreceptors, which are light-sensitive proteins for distinct wavelengths. A protein family frequently used in optogenetics are LOV domains. LOV domains are blue-light photoreceptors and have a small size of under 20 kDa. Cryptochromes are another class of blue-light photoreceptors, which are recently getting more attention in the field of optogenetics. In this thesis an optogenetics tool was developed based on the aureochrome 1a LOV domain form Phaeodactylum tricornutum (AuLOV). Therefore, different mutants of the AuLOV domain were characterized in vitro, a new dimer interface of the AuLOV domain was identified by crystallization experiments and was characterized by bioinformatics. In order to create an optogenetic tool, AuLOV was tested in the photosensitive degron module (psd) and a darkness destabilization tag (AuLOVV254M/V349W-cODC1CA) was developed. This tag was fused to the photoactive adenylate cyclase bPAC to create AuPAC. AuPAC is a synergistic, photoactive adenylate cyclase, which is able to control cellular functions in S. cerevisiae. Furthermore, the soluble in vivo construct mCherry-P2A-AuLOV-cODC1 was expressed and characterized. Another photoreceptor, the „animal-like Cryptochrome“ from C. reinhardtii (CraCry), was characterized in the psd-module under different light conditions in S. cerevisiae. Based on the different redox forms of the chromophore, CraCry is additionally hypothesized to set as red-light sensitive photoreceptor. CraCry has the potential for different optogenetic applications.