Exploring the Modularity of Thalassiosira oceanica Aureochrome1 Photoreceptor-Transcription Factor: Characterization and Component Engineering

The sensory transcription factors carry out gene expression in response to external stimuli like light. Further, they can be used in synthetic biological circuits to manipulate gene expression and thus can alter behavioral responses effectively. In this study, we characterize blue-light responsive photoreceptor transcription factor, Aureochrome (ToAureo1), from photosynthetic marine diatom Thalassiosira oceanica. In silico modeling and docking studies on C-terminal LOV (light-oxygen-voltage) and N-terminal bZIP (basic leucine zipper) domain (ToAubZL) revealed conserved 3D structures as well as interaction with Aureo-box containing substrate DNA. Modularity is an essential feature for synthetic biology inspired designs. Spectroscopy studies not only detected slow dark recovery kinetics, but also established the modular nature of both sensor and effector domains of ToAureo1. First, to check the compatibility of ToAureo1 bZIP with other non-Aureochrome LOV sensors; second, to explore the essentiality of heterogeneous linker connecting effector and sensor domains; and third, to generate temporal scaffolds for optogenetics, chimeric photoreceptor-transcription factor, AsTobZL was generated. Upon blue-light illumination, both the wildtype and synthetic constructs showed a four-fold increase in DNA binding activity. Formation of higher order structures in both cases could be important for transcription activation. Their dark recovery time, however, varied significantly by about ten-fold - further establishing modularity in LOV sensors. Our design further proves the non-essentiality of native ToAureo1 linker between LOV and bZIP in blue-light mediated DNA binding, thereby opening up newer options in optogenetics. Compatibility among different ToAureo-bZIP partners further strengthens their possible role in light-regulated transcription factor interaction networks through large-scale participation.