Curated Optogenetic Publication Database

Abstract: Optogenetics has emerged as a powerful tool for regulating cellular processes due to its noninvasive nature and precise spatiotemporal control. Far-red light (FRL) has increasingly been used in the optogenetic control of mammalian cells due to its low toxicity and high tissue penetration. However, robust and orthogonal FRL sensors are lacking in bacteria. Here, we established an orthogonal FRL sensor in Escherichia coli with a maximum dynamic range exceeding 230-fold based on the RfpA-RfpC-RfpB (RfpABC) signaling system that regulates the far-red light photoacclimation (FaRLiP) in cyanobacteria. We identified a conserved DNA motif in the promoter sequences of the Chl f synthase gene and other genes in the FaRLiP gene clusters, termed the far-red light-regulatory (FLR) motif, which enables the light-responsive activation of gene expression through its interaction with RfpB. Based on the FLR motif, we simplified the FLR-containing promoters and characterized their activation abilities and dynamic ranges, which can be utilized in different synthetic biology scenarios. Additionally, one or two FLR motifs are present at other loci within the FaRLiP gene cluster, providing further FRL-inducible promoter resources. The FRL sensor exhibits effective activation and suppression under low-intensity FRL and white light, respectively, and remains functional in darkness. In conclusion, this study advances the understanding of the regulatory mechanisms of FaRLiP in cyanobacteria and provides robust and orthogonal FRL sensors for synthetic biology applications.

Abstract: Class III adenylyl cyclases generate the ubiquitous second messenger cAMP from ATP often in response to environmental or cellular cues. During evolution, soluble adenylyl-cyclase catalytic domains have been repeatedly juxtaposed with signal-input domains to place cAMP synthesis under the control of a wide variety of these environmental and endogenous signals. Adenylyl cyclases with light-sensing domains have proliferated in photosynthetic species depending on light as an energy source, yet are also widespread in non-photosynthetic species. Among such naturally occurring light sensors, several flavin-based photoactivated adenylyl cyclases (PACs) have been adopted as optogenetic tools to manipulate cellular processes with blue light. In this report, we report the discovery of a cyanobacteriochrome-based photoswitchable adenylyl cyclase (cPAC) from the cyanobacterium Microcoleussp. PCC 7113. Unlike flavin-dependent PACs, which must thermally decay to be deactivated, cPAC exhibited a bistable photocycle whose adenylyl cyclase could be reversibly activated and inactivated by blue and green light, respectively. Through domain exchange experiments, we also document the ability to extend the wavelength-sensing specificity of cPAC into the near IR. In summary, our work has uncovered a cyanobacteriochrome-based adenylyl cyclase that holds great potential for design of bistable photoswitchable adenylyl cyclases to fine-tune cAMP-regulated processes in cells. tissues, and whole organisms with light across the visible spectrum and into near IR.