Curated Optogenetic Publication Database

Abstract: No organism is an island: organisms of varying taxonomic complexity, including genetic variants of a single species, can coexist in particular niches, cooperating for survival while simultaneously competing for environmental resources. In recent years, synthetic biology strategies have witnessed a surge of efforts focused on creating artificial microbial communities to tackle pressing questions about the complexity of natural systems and the interactions that underpin them. These engineered ecosystems depend on the number and nature of their members, allowing complex cell communication designs to recreate and create diverse interactions of interest. Due to its experimental simplicity, the budding yeast Saccharomyces cerevisiae has been harnessed to establish a mixture of varied cell populations with the potential to explore synthetic ecology, metabolic bioprocessing, biosensing, and pattern formation. Indeed, engineered yeast communities enable advanced molecule detection dynamics and logic operations. Here, we present a concise overview of the state-of-the-art, highlighting examples that exploit optogenetics to manipulate, through light stimulation, key yeast phenotypes at the community level, with unprecedented spatial and temporal regulation. Hence, we envision a bright future where the application of optogenetic approaches in synthetic communities (optoecology) illuminates the intricate dynamics of complex ecosystems and drives innovations in metabolic engineering strategies.

Abstract: Collective cell migration and tissue morphogenesis play a variety of important roles in the development of many species. Tissue morphogenesis often generates mechanical forces that alter cell shapes and arrangements, resembling collective cell migration-like behaviors. Genetic methods have been widely used to study collective cell migration and its like behavior, advancing our understanding of these processes during development. However, a growing body of research shows that collective cell migration during development is not a simple behavior but is often combined with other cellular and tissue processes. In addition, different surrounding environments can also influence migrating cells, further complicating collective cell migration during development. Due to the complexity of developmental processes and tissues, traditional genetic approaches often encounter challenges and limitations. Thus, some methods with spatiotemporal control become urgent in dissecting collective cell migration and tissue morphogenesis during development. Optogenetics is a method that combines optics and genetics, providing a perfect strategy for spatiotemporally controlling corresponding protein activity in subcellular, cellular or tissue levels. In this review, we introduce the basic mechanisms underlying different optogenetic tools. Then, we demonstrate how optogenetic methods have been applied in vivo to dissect collective cell migration and tissue morphogenesis during development. Additionally, we describe some promising optogenetic approaches for advancing this field. Together, this review will guide and facilitate future studies of collective cell migration in vivo and tissue morphogenesis by optogenetics.

Abstract: This volume explores the latest advancements in the field of optogenetics and how it uses cellular light-sensing components and genetic engineering to control proteins and biological processes. The book chapters are organized into four parts. Part One focuses on intracellular optogenetic components for control of specific cell functions; Part Two looks at externally supplied light regulators that do not require genetic manipulation of target cells; Part Three highlights optogenetic control of organelles, and Part Four introduces technical tools required for light induction in optogenetic experiments, as well as a method for performing and analyzing optogenetic cell-cell adhesion. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and practical, Optogenetics: Methods and Protocols is a valuable resource to help researchers understand and apply the concepts of optogenetics and the underlying bioengineering principles, and establish the required technical light-illumination setups for administering light inputs and analysis of experimental outcomes.

Abstract: The CRISPR/Cas9 gene-editing technology, derived from the adaptive immune mechanisms of bacteria, has demonstrated remarkable advantages in fields such as gene function research and the treatment of genetic diseases due to its simplicity in design, precise targeting, and ease of use. Despite challenges such as off-target effects and cytotoxicity, effective spatiotemporal control strategies have been achieved for the CRISPR/Cas9 system through precise regulation of Cas9 protein activity as well as engineering of guide RNAs (gRNAs). This review provides a comprehensive analysis of the core components and functional mechanisms underlying the CRISPR/Cas9 system, highlights recent advancements in spatiotemporal control strategies, and discusses future directions for development.

Abstract: As synthetic biology advances, the necessity for robust biocontainment strategies for genetically engineered organisms (GEOs) grows increasingly critical to mitigate biosafety risks related to their potential environmental release. This paper aims to evaluate environment signal-dependent biocontainment systems for engineered organisms, focusing specifically on leveraging triggered responses and combinatorial systems. There are different types of triggers—chemical, light, temperature, and pH—this review illustrates how these systems can be designed to respond to environmental signals, ensuring a higher safety profile. It also focuses on combinatorial biocontainment to avoid consequences of unintended GEO release into an external environment. Case studies are discussed to demonstrate the practical applications of these systems in real-world scenarios.

Abstract: Light as an environmental signal can effectively regulate various biological processes in microbial systems. Optical and optogenetic tools are able to utilize light for precise control methods with minimal interference. Recently, research on these tools has extended to the field of microbiology. Distinguishing from existing reviews, this review narrows the scope of application into food sector, focusing on advances in optical and optogenetic tools for microbial control, including optical tools targeting pathogenic or probiotic bacteria for non-thermal sterilization, antimicrobial photodynamic therapy, or photobiomodulation, combined with nanomaterials as photosensors for food analysis. As well as using optogenetic tools for more convenient and precise control in food production processes, covering reversible induction, metabolic flux regulation, biofilm formation, and inhibition. These tools offer new solutions to goals that cannot be achieved by traditional methods, and they are still maturing to explore other uses in the food field.

Abstract: Corynebacterium glutamicum is a key industrial workhorse for producing amino acids and high-value chemicals. Balancing metabolic flow between cell growth and product synthesis is crucial for enhancing production efficiency. Developing dynamic, broadly applicable, and minimally toxic gene regulation tools for C. glutamicum remains challenging, as optogenetic tools ideal for dynamic regulatory strategies have not yet been developed. This study introduces an advanced light-controlled gene expression system using light-controlled RNA-binding proteins (RBP), a first for Corynebacterium glutamicum. We established a gene expression regulation system, 'LightOnC.glu', utilizing the light-controlled RBP to construct light-controlled transcription factors in C. glutamicum. Simultaneously, we developed a high-performance light-controlled gene interference system using CRISPR/Cpf1 tools. The metabolic flow in the synthesis network was designed to enable the production of chitin oligosaccharides (CHOSs) and chondroitin sulphate oligosaccharides A (CSA) for the first time in C. glutamicum. Additionally, a light-controlled bioreactor was constructed, achieving a CHOSs production concentration of 6.2 g/L, the highest titer recorded for CHOSs biosynthesis to date. Herein, we have established a programmable light-responsive genetic circuit in C. glutamicum, advancing the theory of dynamic regulation based on light signaling. This breakthrough has potential applications in optimizing metabolic modules in other chassis cells and synthesizing other compounds.

Abstract: Research for biopharmaceutical production processes with mammalian cells steadily aims to enhance the cell-specific productivity as a means for optimizing total productivities of bioreactors. Whereas current technologies such as pH, temperature, and osmolality shift require modifications of the cultivation medium, the use of optogenetic switches in recombinant producer cells might be a promising contact-free alternative. However, the proper application of optogenetically engineered cells requires a detailed understanding of basic cellular responses of cells that do not yet contain the optogenetic switches. The knowhow of ideal light exposure to enable the optimum use of related approaches is missing so far. Consequently, the current study set out to find optimum conditions for IgG1 producing Chinese hamster ovary (CHO) cells which were exposed to blue LED light. Growth characteristics, cell-specific productivity using enzyme-linked immunosorbent assay, as well as cell cycle distribution using flow cytometry were analyzed. Whereas too harsh light exposure causes detrimental growth effects that could be compensated with antioxidants, a surprising boost of cell-specific productivity by 57% occurred at optimum high light doses. The increase coincided with an increased number of cells in the G1 phase of the cell cycle after 72 h of illumination. The results present a promising new approach to boost biopharmaceutical productivity of mammalian cells simply by proper light exposure without any further optogenetic engineering. KEY POINTS: • Blue LED light hinders growth in CHO DP-12 cells • Antioxidants protect to a certain degree from blue light effects • Illumination with blue LED light raises cell-specific productivity.

Abstract: Light-Oxygen-Voltage (LOV) domains are the protein-based light switches used in nature to trigger and regulate various processes. They allow light signals to be converted into metabolic signaling cascades. Various LOV-domain proteins have been characterized in the last few decades and have been used to develop light-sensitive tools in cell biology research. LOV-based applications exploit the light-driven regulation of effector elements to activate signaling pathways, activate genes, or locate proteins within cells. A relatively new application of an engineered small LOV-domain protein called miniSOG (mini singlet oxygen generator) is based on the light-induced formation of reactive oxygen species (ROS). The first miniSOG was engineered from a LOV domain from Arabidopsis thaliana. This engineered 14 kDa light-responsive flavin-containing protein can be exploited as protein tag for the light-triggered localized production of ROS. Such tunable ROS production by miniSOG or similarly redesigned LOV-domains can be of use in studies focused on subcellular phenomena but may also allow new light-fueled catalytic processes. This review provides an overview of the discovery of LOV domains and their development into tools for cell biology. It also highlights recent advancements in engineering LOV domains for various biotechnological applications and cell biology studies.

Abstract: Optogenetic tools have been used in a wide range of microbial engineering applications that benefit from the tunable, spatiotemporal control that light affords. However, the majority of current optogenetic constructs for bacteria respond to blue light, limiting the potential for multichromatic control. In addition, other wavelengths offer potential benefits over blue light, including improved penetration of dense cultures and reduced potential for toxicity. In this study, we introduce OptoCre-REDMAP, a red light inducible Cre recombinase system in Escherichia coli. This system harnesses the plant photoreceptors PhyA and FHY1 and a split version of Cre recombinase to achieve precise control over gene expression and DNA excision. We optimized the design by modifying the start codon of Cre and characterized the impact of different levels of induction to find conditions that produced minimal basal expression in the dark and induced full activation within 4 h of red light exposure. We characterized the system's sensitivity to ambient light, red light intensity, and exposure time, finding OptoCre-REDMAP to be reliable and flexible across a range of conditions. In coculture experiments with OptoCre-REDMAP and the blue light responsive OptoCre-VVD, we found that the systems responded orthogonally to red and blue light inputs. Direct comparisons between red and blue light induction with OptoCre-REDMAP and OptoCre-VVD demonstrated the superior penetration properties of red light. OptoCre-REDMAP's robust and selective response to red light makes it suitable for advanced synthetic biology applications, particularly those requiring precise multichromatic control.

Abstract: Overexpression of a single enzyme in a multigene heterologous pathway may be out of balance with the other enzymes in the pathway, leading to accumulated toxic intermediates, imbalanced carbon flux, reduced productivity of the pathway, or an inhibited growth phenotype. Therefore, optimal, balanced, and synchronized expression levels of enzymes in a particular metabolic pathway is critical to maximize production of desired compounds while maintaining cell fitness in a growing culture. Furthermore, the optimal intracellular concentration of an enzyme is determined by the expression strength, specific timing/duration, and degradation rate of the enzyme. Here, we modulated the intracellular concentration of a key enzyme, namely HMG-CoA synthase (HMGS), in the heterologous mevalonate pathway by tuning its expression level and period of transcription to enhance limonene production in Escherichia coli. Facilitated by the tuned blue-light inducible BLADE/pBad system, we observed that limonene production was highest (160 mg/L) with an intermediate transcription level of HMGS from moderate light illumination (41 au, 150 s ON/150 s OFF) throughout the growth. Owing to the easy penetration and removal of blue-light illumination from the growing culture which is hard to obtain using conventional chemical-based induction, we further explored different induction patterns of HMGS under strong light illumination (2047 au, 300 s ON) for different durations along the growth phases. We identified a specific timing of HMGS expression in the log phase (3-9 h) that led to optimal limonene production (200 mg/L). This is further supported by a mathematical model that predicts several periods of blue-light illumination (3-9 h, 0-9 h, 3-12 h, 0-12 h) to achieve an optimal expression level of HMGS that maximizes limonene production and maintains cell fitness. Compared to moderate and prolonged transcription (41 au, 150 s ON/150 s OFF, 0-73 h), strong but time-limited transcription (2047 au, 300 s ON, 3-9 h) of HMGS could maintain its optimal intracellular concentration and further increased limonene production up to 92% (250 mg/L) in the longer incubation (up to 73 h) without impacting cell fitness. This work has provided new insight into the "right amount" and "just-in-time" expression of a critical metabolite enzyme in the upper module of the mevalonate pathway using optogenetics. This study would complement previous findings in modulating HMGS expression and potentially be applicable to heterologous production of other terpenoids in E. coli.

Abstract: Tight coordination of cell-cell signaling in space and time is vital for self-organization in tissue patterning. During vertebrate development, the segmentation clock drives oscillatory gene expression in the presomitic mesoderm (PSM), leading to the periodic formation of somites. Oscillatory gene expression is synchronized at the cell population level; inhibition of Delta-Notch signaling results in the loss of synchrony and the fusion of somites. However, it remains unclear how cell-cell signaling couples oscillatory gene expression and controls synchronization. Here, we report the reconstitution of synchronized oscillation in PSM organoids by synthetic cell-cell signaling with designed ligand-receptor pairs. Optogenetic assays uncovered that the intracellular domains of synthetic ligands play key roles in dynamic cell-cell communication. Oscillatory coupling using synthetic cell-cell signaling recovered the synchronized oscillation in PSM cells deficient for Delta-Notch signaling; non-oscillatory coupling did not induce recovery. This study reveals the mechanism by which ligand-receptor molecules coordinate the synchronization of the segmentation clock, and provides direct evidence of oscillatory cell-cell communication in the segmentation clock.

Abstract: RNA molecules play a vital role in linking genetic information with various cellular processes. In recent years, a variety of optogenetic tools have been engineered for regulating cellular RNA metabolism and functions. These highly desirable tools can offer non-intrusive control with spatial precision, remote operation, and biocompatibility. Here, we would like to review these currently available approaches that can regulate RNAs with light: from non-genetically encodable chemically modified oligonucleotides to genetically encoded RNA aptamers that recognize photosensitive small-molecule or protein ligands. Some key applications of these optogenetic tools will also be highlighted to illustrate how they have been used for regulating all aspects of the RNA life cycle: from RNA synthesis, maturation, modification, and translation to their degradation, localization, and phase separation control. Some current challenges and potential practical utilizations of these RNA optogenetic tools will also be discussed.

Abstract: Genetic, colocalization, and biochemical studies suggest that the ankyrin repeat-containing proteins Inversin (INVS) and ANKS6 function with the NEK8 kinase to control tissue patterning and maintain organ physiology. It is unknown whether these three proteins assemble into a static “Inversin complex” or one that adopts multiple bioactive forms. Through characterization of hyperactive alleles in C. elegans, we discovered that the Inversin complex is activated by dimerization. Genome engineering of an RFP tag onto the nematode homologues of INVS (MLT-4) and NEK8 (NEKL-2) induced a gain-of-function, cyst-like phenotype that was suppressed by monomerization of the fluorescent tag. Stimulated dimerization of MLT-4 or NEKL-2 using optogenetics was sufficient to recapitulate the phenotype of a constitutively active Inversin complex. Further, dimerization of NEKL-2 bypassed a lethal MLT-4 mutant, demonstrating that the dimeric form is required for function. We propose that dynamic switching between at least two functionally distinct states–-an active dimer and an inactive monomer–-gates the output of the Inversin complex.

Abstract: Cytokines have attracted sustained attention due to their multi-functional cellular response in immunotherapy. However, their application was limited to their short half-time, narrow therapeutic window, and undesired side effects. To address this issue, we developed a portable smart blue-light controlled (PSLC) device based on optogenetic technology. By combining this PSLC device with blue-light controlled gene modules, we successfully achieved the targeted regulation of cytokine expression within the tumor microenvironment. To alter the tumor microenvironment of solid tumors, pro-inflammatory cytokines were selected as blue-light controlled molecules. The results show that blue-light effectively regulates the expression of pro-inflammatory cytokines both in vitro and in vivo. This strategy leads to enhanced and activated tumor-infiltrating immune cells, which facilitated to overcome the immunosuppressive microenvironment, resulting in significant tumor shrinkage in tumor-bearing mice. Hence, our study offers a unique strategy for cytokine therapy and a convenient device for animal studies in optogenetic immunotherapy.

Abstract: Nitrogen limitations in the grape must is the main cause of stuck fermentations during the winemaking process. In Saccharomyces cerevisiae, a genetic segment known as region A, which harbors 12 protein-coding genes, was acquired horizontally from a phylogenetically distant yeast species. This region is mainly present in the genome of wine yeast strains, carrying genes that have been associated with nitrogen utilization. Despite the putative importance of region A in yeast fermentation, its contribution to the fermentative process is largely unknown. In this work, we used a wine yeast strain to evaluate the contribution of region A to the fermentation process. To do this, we first sequenced the genome of the wine yeast strain known as ‘ALL’ using long-read sequencing and determined that region A is present in a single copy with two possible subtelomeric locations. We then implemented an optogenetic system in this wine yeast strain to precisely regulate the expression of each gene inside this region, generating a collection of 12 strains that allow for light- activated gene expression. To evaluate the role of these genes during fermentation, we assayed this collection using microculture and fermentation experiments in synthetic must with varying amounts of nitrogen concentration. Our results show that changes in gene expression for genes within this region can impact growth parameters and fermentation rate. We additionally found that the expression of various genes in region A is necessary to complete the fermentation process and prevent stuck fermentations under low nitrogen conditions. Altogether, our optogenetics-based approach demonstrates the importance of region A in completing fermentation under nitrogen-limited conditions.

Abstract: The Islets of Langerhans reside within the endocrine pancreas as highly vascularised micro-organs that are responsible for the secretion of key hormones, such as insulin and glucagon. Islet function relies on a range of dynamic molecular processes that include calcium (Ca2+) waves, hormone pulses, and complex interactions between islet cell types. Dysfunction of these processes results in poor maintenance of blood glucose homeostasis and is a hallmark of diabetes. Very recently, the development of optogenetic methods that rely on light-sensitive molecular actuators has allowed perturbing islet function with near physiological spatio-temporal acuity. These actuators harness natural photoreceptor proteins and their engineered variants to manipulate mouse and human cells that are not normally light-responsive. Until recently, optogenetics in islet biology has primarily focused on hormone production and secretion; however, studies on further aspects of islet function, including paracrine regulation between islet cell types and dynamics within intracellular signaling pathways are emerging. Here, we discuss the applicability of optogenetics to islets cells and comprehensively review seminal as well as recent work on optogenetic actuators and their effects in islet function and diabetes mellitus (DM).

Abstract: Cells use dynamic spatial and temporal cues to instruct cell fate decisions during development. Morphogens are key examples, where the concentration and duration of morphogen exposure produce distinct cell fates that drive tissue patterning. Studying the dynamics of these processes has been challenging. Here, we establish an optogenetic system for morphogen production that enables the investigation of developmental patterning in vitro. Using a tunable light-inducible gene expression system, we generate long-range Shh gradients that pattern neural progenitors into spatially distinct progenitor domains mimicking the spatial arrangement of neural progenitors found in vivo during vertebrate neural tube development. With this system, we investigate how biochemical features of Shh and the presence of morphogen-interacting proteins affect the patterning length scale. We measure tissue clearance rates, revealing that Shh has an extracellular half-life of about 1h, and we probe how the level and duration of morphogen exposure govern the acquisition and maintenance of cell fates. The rate of Shh turnover is substantially faster than the downstream gene expression dynamics, indicating that the gradient is continually renewed during patterning. Together the optogenetic approach establishes a simple experimental system for the quantitative interrogation of morphogen patterning. Controlling morphogen dynamics in a reproducible manner provides a framework to dissect the interplay between biochemical cues, the biophysics of gradient formation, and the transcriptional programmes underlying developmental patterning.

Abstract: Diabetes mellitus (DM) is a common chronic disease affecting humans globally. It is characterized by abnormally elevated blood glucose levels due to the failure of insulin production or reduction of insulin sensitivity and functionality. Insulin and glucagon-like peptide (GLP)-1 replenishment or improvement of insulin resistance are the two major strategies to treat diabetes. Recently, optogenetics that uses genetically encoded light-sensitive proteins to precisely control cell functions has been regarded as a novel therapeutic strategy for diabetes. Here, we summarize the latest development of optogenetics and its integration with synthetic biology approaches to produce light-responsive cells for insulin/GLP-1 production, amelioration of insulin resistance and neuromodulation of insulin secretion. In addition, we introduce the development of cell encapsulation and delivery methods and smart bioelectronic devices for the in vivo application of optogenetics-based cell therapy in diabetes. The remaining challenges for optogenetics-based cell therapy in the clinical translational study are also discussed.

Abstract: Optogenetic, known as the method of 21 centuries, combines optic and genetic engineering to precisely control photosensitive proteins for manipulation of a broad range of cellular functions, such as flux of ions, protein oligomerization and dissociation, cellular intercommunication, and so on. In this technique, light is conventionally delivered to targeted cells through optical fibers or micro light-emitting diodes, always suffering from high invasiveness, wide-field illumination facula, strong absorption, and scattering by nontargeted endogenous substance. Light-transducing nanomaterials with advantages of high spatiotemporal resolution, abundant wireless-excitation manners, and easy functionalization for recognition of specific cells, recently have been widely explored in the field of optogenetics; however, there remain a few challenges to restrain its clinical applications. This review summarized recent progress on light-responsive genetically encoded proteins and the myriad of activation strategies by use of light-transducing nanomaterials and their disease-treatment applications, which is expected for sparking helpful thought to push forward its preclinical and translational uses.

Abstract: Light-dependent adaptations of organismal physiology, development, and behavior abound in nature and depend on sensory photoreceptors. As one class, light–oxygen–voltage (LOV) photoreceptors harness flavin-nucleotide chromophores to sense blue light. Photon absorption drives the LOV receptor to its signaling state, characterized by a metastable thioadduct between the flavin and a conserved cysteine residue. With this cysteine absent, LOV receptors instead undergo photoreduction to the flavin semiquinone which however can still elicit downstream physiological responses. Irrespective of the cysteine presence, the LOV photochemical response thus entails a formal reduction of the flavin. Against this backdrop, we here investigate the reduction midpoint potential E0 in the paradigmatic LOV2 domain from Avena sativa phototropin 1 (AsLOV2), and how it can be deliberately varied. Replacements of residues at different sites near the flavin by methionine consistently increase E0 from its value of around −280 mV by up to 40 mV. Moreover, methionine introduction invariably impairs photoactivation efficiency and thus renders the resultant AsLOV2 variants less light-sensitive. Although individual methionine substitutions also affect the stability of the signaling state and downstream allosteric responses, no clear-cut correlation with the redox properties emerges. With a reduction midpoint potential near −280 mV, AsLOV2 and, by inference, other LOV receptors may be partially reduced inside cells which directly affects their light responsiveness. The targeted modification of the chromophore environment, as presently demonstrated, may mitigate this effect and enables the design of LOV receptors with stratified redox sensitivities.

Abstract: Molecular optogenetics utilizes genetically encoded, light-responsive protein switches to control the function of molecular processes. Over the last two years, there have been notable advances in the development of novel optogenetic switches, their utilization in elucidating intricate signaling pathways, and their progress toward practical applications in biotechnological processes, material sciences, and therapeutic applications. In this review, we discuss these areas, offer insights into recent developments, and contemplate future directions.

Abstract: [This corrects the article DOI: 10.1002/mco2.226.].

Abstract: The tumor microenvironment is a barrier to breast cancer therapy. Cancer-associated fibroblast cells (CAFs) can support tumor proliferation, metastasis, and drug resistance by secreting various cytokines and growth factors. Abnormal angiogenesis provides sufficient nutrients for tumor proliferation. Considering that CAFs express the sigma receptor (which recognizes anisamide, AA), we developed a CAFs and breast cancer cells dual-targeting nano drug delivery system to transport the LightOn gene express system, a spatiotemporal controlled gene expression consisting of a light-sensitive transcription factor and a specific minimal promoter. We adopted RGD (Arg-Gly-Asp) to selectively bind to the αvβ3 integrin on activated vascular endothelial cells and tumor cells. After the LightOn system has reached the tumor site, LightOn gene express system can spatiotemporal controllably express toxic Pseudomonas exotoxin An under blue light irradiation. The LightOn gene express system, combined with multifunctional nanoparticles, achieved high targeting delivery efficiency both in vitro and in vivo. It also displayed strong tumor and CAFs inhibition, anti-angiogenesis ability and anti-metastasis ability, with good safety. Moreover, it improved survival rate, survival time, and lung metastasis rate in a mouse breast cancer model. This study proves the efficacy of combining the LightOn system with targeted multifunctional nanoparticles in tumor and anti-metastatic therapy and provides new insights into tumor microenvironment regulation.

Abstract: Optogenetics has opened new possibilities in the remote control of diverse cellular functions with high spatiotemporal precision using light. However, delivering light to optically non-transparent systems remains a challenge. Here, we describe the photoactivation of light-oxygen-voltage-sensing domains (LOV domains) with in situ generated light from a chemiluminescence reaction between luminol and H2O2. This activation is possible due to the spectral overlap between the blue chemiluminescence emission and the absorption bands of the flavin chromophore in LOV domains. All four LOV domain proteins with diverse backgrounds and structures (iLID, BcLOV4, nMagHigh/pMagHigh, and VVDHigh) were photoactivated by chemiluminescence as demonstrated using a bead aggregation assay. The photoactivation with chemiluminescence required a critical light-output below which the LOV domains reversed back to their dark state with protein characteristic kinetics. Furthermore, spatially confined chemiluminescence produced inside giant unilamellar vesicles (GUVs) was able to photoactivate proteins both on the membrane and in solution, leading to the recruitment of the corresponding proteins to the GUV membrane. Finally, we showed that reactive oxygen species produced by neutrophil like cells can be converted into sufficient chemiluminescence to recruit the photoswitchable protein BcLOV4-mCherry from solution to the cell membrane. The findings highlight the utility of chemiluminescence as an endogenous light source for optogenetic applications, offering new possibilities for studying cellular processes in optically non-transparent systems.