Curated Optogenetic Publication Database

Abstract: Precise temporal and spatial control of gene expression greatly benefits the study of specific cellular circuits and activities. Compared to chemical inducers, light-dependent control of gene expression by optogenetics achieves a higher spatial and temporal resolution. Beyond basic research, this could also prove decisive for manufacturing difficult-to-express proteins in pharmaceutical bioproduction. However, current optogenetic gene-expression systems limit this application in mammalian cells, as expression levels and the degree of induction upon light stimulation are insufficient. To overcome this limitation, we designed a photoswitch by fusing the blue light-activated light-oxygen-voltage receptor EL222 from Erythrobacter litoralis to the three transcriptional activator domains VP64, p65, and Rta in tandem. The resultant photoswitch, dubbed DEL-VPR, allows up to a 570-fold induction of target gene expression by blue light, thereby achieving expression levels of strong constitutive promoters. Here, we used DEL-VPR to enable light-induced expression of complex monoclonal and bispecific antibodies with reduced byproduct expression and increased yield of functional protein complexes. Our approach offers temporally controlled yet strong gene expression and applies to academic and industrial settings.

Abstract: Sensory photoreceptors abound in nature and enable organisms to adapt behavior, development, and physiology to environmental light. In optogenetics, photoreceptors allow spatiotemporally precise, reversible, and non-invasive control by light of cellular processes. Notwithstanding the development of numerous optogenetic circuits, an unmet demand exists for efficient systems sensitive to red light, given its superior penetration of biological tissue. Bacteriophytochrome photoreceptors sense the ratio of red and far-red light to regulate the activity of enzymatic effector modules. The recombination of bacteriophytochrome photosensor modules with cyclase effectors underlies photoactivated adenylyl cyclases (PAC) that catalyze the synthesis of the ubiquitous second messenger 3', 5'-cyclic adenosine monophosphate (cAMP). Via homologous exchanges of the photosensor unit, we devised novel PACs, with the variant DmPAC exhibiting 40-fold activation of cyclase activity under red light, thus surpassing previous red-light-responsive PACs. Modifications of the PHY tongue modulated the responses to red and far-red light. Exchanges of the cyclase effector offer an avenue to further enhancing PACs but require optimization of the linker to the photosensor. DmPAC and a derivative for 3', 5'-cyclic guanosine monophosphate allow the manipulation of cyclic-nucleotide-dependent processes in mammalian cells by red light. Taken together, we advance the optogenetic control of second-messenger signaling and provide insight into the signaling and design of bacteriophytochrome receptors.