Random Mutagenesis for the Generation of Repertoire of Aureochrome-based Optogenetic Scaffolds

Engineering of the natural light-regulated transcription factors for the purposeful generation of synthetic photo-switches is a widely adopted strategy in synthetic biology and optogenetics. Aureochrome is one such potential, naturally occurring photoreceptor-cum-transcription factor that can be used for the development of synthetic optogenetic tools, especially meant for light-regulated gene expression. It is however well-known that different biological events demand different duration of signaling state lifetime and diverse affinity towards different DNA substrates. Therefore, in this study, we produce multiple variants of aureochromes via random mutagenesis - mimicking single generation directed evolution. After screening the single generation variants for in-frame transcripts, we sort them on the basis of altered photocycle kinetics as well as DNA-binding affinity. The variants that exhibited reversible photochemistry and light-regulated DNA-binding ability similar/comparable to that of the wild-type despite incurring mutations, are characterized and discussed in this manuscript. These can later be subjected to successive rounds of random mutations to get an eventual superior variety with enhanced functionality. Even the apparently unsuccessful ones, which depicted drastic alteration of photochemical/DNA-binding properties, helped us to identify amino acid residues - lesser known to be indispensable for a certain biological activity. The process of diversification and selection via random mutagenesis not only explains the functional significance of the different amino acids in aureochromes but also generates a repertoire of appropriate aureochrome variants that may be used in optogenetics.