Curated Optogenetic Publication Database

Abstract: Several strategies, including inducer addition and biosensor use, have been developed for dynamical regulation. However, the toxicity, cost, and inflexibility of existing strategies have created a demand for superior technology. In this study, we designed an optogenetic dual-switch system and applied it to increase polyhydroxybutyrate (PHB) production. First, an optimized chromatic acclimation sensor/regulator (RBS10-CcaS#10-CcaR) system (comprising an optimized ribosomal binding site (RBS), light sensory protein CcaS, and response regulator CcaR) was selected for a wide sensing range of approximately 10-fold between green-light activation and red-light repression. The RBS10-CcaS#10-CcaR system was combined with a blue light-activated YF1-FixJ-PhlF system (containing histidine kinase YF1, response regulator FixJ, and repressor PhlF) engineered with reduced crosstalk. Finally, the optogenetic dual-switch system was used to rewire the metabolic flux for PHB production by regulating the sequences and intervals of the citrate synthase gene (gltA) and PHB synthesis gene (phbCAB) expression. Consequently, the strain RBS34, which has high gltA expression and a time lag of 3 h, achieved the highest PHB content of 16.6 wt%, which was approximately 3-fold that of F34 (expressed at 0 h). The results indicate that the optogenetic dual-switch system was verified as a practical and convenient tool for increasing PHB production.

Abstract: Advances in genetic engineering, combined with the development of optical technologies, have allowed optogenetics to broaden its area of possible applications in recent years. However, the application of optogenetic tools in industry, including biotechnology and the production of biomaterials, is still limited, because each practical task requires the engineering of a specific optogenetic system. In this review, we discuss recent advances in the use of optogenetic tools in the production of biofuels and valuable chemicals, the synthesis of biomedical and polymer materials, and plant agrobiology. We also offer a comprehensive analysis of the properties and industrial applicability of light-controlled and other smart biomaterials. These data allow us to outline the prospects for the future use of optogenetics in bioindustry.

Abstract: Owing to its ubiquity and easy availability in nature, light has been widely employed to control complex cellular behaviors. Light-sensitive proteins are the foundation to such diverse and multilevel adaptive regulations in a large range of organisms. Due to their remarkable properties and potential applications in engineered systems, exploration and engineering of natural light-sensitive proteins have significantly contributed to expand optogenetic toolboxes with tailor-made performances in synthetic genetic circuits. Progressively, more complex systems have been designed in which multiple photoreceptors, each sensing its dedicated wavelength, are combined to simultaneously coordinate cellular responses in a single cell. In this review, we highlight recent works and challenges on multiplexed optogenetic circuits in natural and engineered systems for a dynamic regulation breakthrough in biotechnological applications.

Abstract: Optogenetics holds the promise of controlling biological processes with superb temporal and spatial resolution at minimal perturbation. Although many of the light-reactive proteins used in optogenetic systems are derived from prokaryotes, applications were largely limited to eukaryotes for a long time. In recent years, however, an increasing number of microbiologists use optogenetics as a powerful new tool to study and control key aspects of bacterial biology in a fast and often reversible manner. After a brief discussion of optogenetic principles, this review provides an overview of the rapidly growing number of optogenetic applications in bacteria, with a particular focus on studies venturing beyond transcriptional control. To guide future experiments, we highlight helpful tools, provide considerations for successful application of optogenetics in bacterial systems, and identify particular opportunities and challenges that arise when applying these approaches in bacteria.

Abstract: This review adds the bilin-binding phytochromes to the Chemical Reviews thematic issue "Optogenetics and Photopharmacology". The work is structured into two parts. We first outline the photochemistry of the covalently bound tetrapyrrole chromophore and summarize relevant spectroscopic, kinetic, biochemical, and physiological properties of the different families of phytochromes. Based on this knowledge, we then describe the engineering of phytochromes to further improve these chromoproteins as photoswitches and review their employment in an ever-growing number of different optogenetic applications. Most applications rely on the light-controlled complex formation between the plant photoreceptor PhyB and phytochrome-interacting factors (PIFs) or C-terminal light-regulated domains with enzymatic functions present in many bacterial and algal phytochromes. Phytochrome-based optogenetic tools are currently implemented in bacteria, yeast, plants, and animals to achieve light control of a wide range of biological activities. These cover the regulation of gene expression, protein transport into cell organelles, and the recruitment of phytochrome- or PIF-tagged proteins to membranes and other cellular compartments. This compilation illustrates the intrinsic advantages of phytochromes compared to other photoreceptor classes, e.g., their bidirectional dual-wavelength control enabling instant ON and OFF regulation. In particular, the long wavelength range of absorption and fluorescence within the "transparent window" makes phytochromes attractive for complex applications requiring deep tissue penetration or dual-wavelength control in combination with blue and UV light-sensing photoreceptors. In addition to the wide variability of applications employing natural and engineered phytochromes, we also discuss recent progress in the development of bilin-based fluorescent proteins.

Abstract: Optogenetics involves the use of light to control cellular functions and has become increasingly popular in various areas of research, especially in the precise control of gene expression. While this technology is already well established in neurobiology and basic research, its use in bioprocess development is still emerging. Some optogenetic switches have been implemented in yeasts for different purposes, taking advantage of a wide repertoire of biological parts and relatively easy genetic manipulation. In this review, we cover the current strategies used for the construction of yeast strains to be used in optogenetically controlled protein or metabolite production, as well as the operational aspects to be considered for the scale-up of this type of process. Finally, we discuss the main applications of optogenetic switches in yeast systems and highlight the main advantages and challenges of bioprocess development considering future directions for this field.

Abstract: Optogenetics initially used plant photoreceptors to monitor neural circuits, later it has expanded to include engineered plant photoreceptors. Recently photoreceptors from bacteria, algae and cyanobacteria have been used as an optogenetic tool. Bilin-based photoreceptors are common light-sensitive photoswitches in plants, algae, bacteria and cyanobacteria. Here we discuss the photoreceptors from cyanobacteria. Several new photoreceptors have been explored in cyanobacteria which are now proposed as cyanobacteriochrome. The domains in the cyanobacteriochrome, light-induced signaling transduction, photoconversion, are the most attractive features for the optogenetic system. The wider spectral feature of cyanobacteriochrome from UV to visible radiation makes it a light potential sensitive optogenetic tool. Besides, cyanobacterial phytochrome responses to yellow, orange and blue light have more application in optogenetics. This chapter summarizes the photoconversion, phototaxis, cell aggregation, cell signaling mediated by cyanobacteriochrome and cyanophytochrome. As there is a wide range of cyanobacteriochrome and its combination delivers a varied light-sensitive response. Besides coordination among cyanobacteriochromes in cell signaling reduces the engineering of photoreceptors for the optogenetic system.

Abstract: Cyanobacteriochromes (CBCRs) are small, linear tetrapyrrole (bilin)-binding photoreceptors in the phytochrome superfamily that regulate diverse light-mediated adaptive processes in cyanobacteria. More spectrally diverse than canonical red/far-red-sensing phytochromes, CBCRs were thought to be restricted to sensing visible and near UV light until recently when several subfamilies with far-red-sensing representatives (frCBCRs) were discovered. Two of these frCBCRs subfamilies have been shown to incorporate bilin precursors with larger pi-conjugated chromophores, while the third frCBCR subfamily uses the same phycocyanobilin precursor found in the bulk of the known CBCRs. To elucidate the molecular basis of far-red light perception by this third frCBCR subfamily, we determined the crystal structure of the far-red-absorbing dark state of one such frCBCR Anacy_2551g3 from Anabaena cylindrica PCC 7122 which exhibits a reversible far-red/orange photocycle. Determined by room temperature serial crystallography and cryocrystallography, the refined 2.7-Å structure reveals an unusual all-Z,syn configuration of the phycocyanobilin (PCB) chromophore that is considerably less extended than those of previously characterized red-light sensors in the phytochrome superfamily. Based on structural and spectroscopic comparisons with other bilin-binding proteins together with site-directed mutagenesis data, our studies reveal protein-chromophore interactions that are critical for the atypical bathochromic shift. Based on these analyses, we propose that far-red absorption in Anacy_2551g3 is the result of the additive effect of two distinct red-shift mechanisms involving cationic bilin lactim tautomers stabilized by a constrained all-Z,syn conformation and specific interactions with a highly conserved anionic residue.

Abstract: Light has become established as a tool not only to visualize and investigate but also to steer biological systems. This review starts by discussing the unique features that make light such an effective control input in biology. It then gives an overview of how light‐control came to progress, starting with photoactivatable compounds and leading up to current genetic implementations using optogenetic approaches. The review then zooms in on optogenetics, focusing on photosensitive proteins, which form the basis for optogenetic engineering using synthetic biological approaches. As the regulation of transcription provides a highly versatile means for steering diverse biological functions, the focus of this review then shifts to transcriptional light regulators, which are presented in the biotechnologically highly relevant model organism Escherichia coli.

Abstract: Optimization of engineered biological systems requires precise control over the rates and timing of gene expression. Optogenetics is used to dynamically control gene expression as an alternative to conventional chemical-based methods since it provides a more convenient interface between digital control software and microbial culture. Here, we describe the construction of a real-time optogenetics platform, which performs closed-loop control over the CcaR-CcaS two-plasmid system in Escherichia coli. We showed the first model-based design approach by constructing a nonlinear representation of the CcaR-CcaS system, tuned the model through open-loop experimentation to capture the experimental behavior, and applied the model in silico to inform the necessary changes to build a closed-loop optogenetic control system. Our system periodically induces and represses the CcaR-CcaS system while recording optical density and fluorescence using image processing techniques. We highlight the facile nature of constructing our system and how our model-based design approach will potentially be used to model other systems requiring closed-loop optogenetic control.

Abstract: Optogenetics utilizes photosensitive proteins to manipulate the localization and interaction of molecules in living cells. Because light can be rapidly switched and conveniently confined to the sub‐micrometer scale, optogenetics allows for controlling cellular events with an unprecedented resolution in time and space. The past decade has witnessed an enormous progress in the field of optogenetics within the biological sciences. The ever‐increasing amount of optogenetic tools, however, can overwhelm the selection of appropriate optogenetic strategies. Considering that each optogenetic tool may have a distinct mode of action, a comparative analysis of the current optogenetic toolbox can promote the further use of optogenetics, especially by researchers new to this field. This review provides such a compilation that highlights the spatiotemporal accuracy of current optogenetic systems. Recent advances of optogenetics in live cells and animal models are summarized, the emerging work that interlinks optogenetics with other research fields is presented, and exciting clinical and industrial efforts to employ optogenetic strategy toward disease intervention are reported.

Abstract: Gut microbial metabolism is associated with host longevity. However, because it requires direct manipulation of microbial metabolism in situ, establishing a causal link between these two processes remains challenging. We demonstrate an optogenetic method to control gene expression and metabolite production from bacteria residing in the host gut. We genetically engineer an Escherichia coli strain that secretes colanic acid (CA) under the quantitative control of light. Using this optogenetically-controlled strain to induce CA production directly in the Caenorhabditis elegans gut, we reveal the local effect of CA in protecting intestinal mitochondria from stress-induced hyper-fragmentation. We also demonstrate that the lifespan-extending effect of this strain is positively correlated with the intensity of green light, indicating a dose-dependent CA benefit on the host. Thus, optogenetics can be used to achieve quantitative and temporal control of gut bacterial metabolism in order to reveal its local and systemic effects on host health and aging.

Abstract: Progress in metabolic engineering and synthetic and systems biology has made bioproduction an increasingly attractive and competitive strategy for synthesizing biomolecules, recombinant proteins and biofuels from renewable feedstocks. Yet, due to poor productivity, it remains difficult to make a bioproduction process economically viable at large scale. Achieving dynamic control of cellular processes could lead to even better yields by balancing the two characteristic phases of bioproduction, namely, growth versus production, which lie at the heart of a trade-off that substantially impacts productivity. The versatility and controllability offered by light will be a key element in attaining the level of control desired. The popularity of light-mediated control is increasing, with an expanding repertoire of optogenetic systems for novel applications, and many optogenetic devices have been designed to test optogenetic strains at various culture scales for bioproduction objectives. In this review, we aim to highlight the most important advances in this direction. We discuss how optogenetics is currently applied to control metabolism in the context of bioproduction, describe the optogenetic instruments and devices used at the laboratory scale for strain development, and explore how current industrial-scale bioproduction processes could be adapted for optogenetics or could benefit from existing photobioreactor designs. We then draw attention to the steps that must be undertaken to further optimize the control of biological systems in order to take full advantage of the potential offered by microbial factories.

Abstract: Signaling networks control the flow of information through biological systems and coordinate the chemical processes that constitute cellular life. Optogenetic actuators - genetically encoded proteins that undergo light-induced changes in activity or conformation - are useful tools for probing signaling networks over time and space. They have permitted detailed dissections of cellular proliferation, differentiation, motility, and death, and enabled the assembly of synthetic systems with applications in areas as diverse as photography, chemical synthesis, and medicine. In this review, we provide a brief introduction to optogenetic systems and describe their application to molecular-level analyses of cell signaling. Our discussion highlights important research achievements and speculates on future opportunities to exploit optogenetic systems in the study and assembly of complex biochemical networks.

Abstract: Cybergenetic systems use computer interfaces to enable feed-back controls over biological processes in real time. The complex and dynamic nature of cellular metabolism makes cybergenetics attractive for controlling engineered metabolic pathways in microbial fermentations. Cybergenetics would not only create new avenues of research into cellular metabolism, it would also enable unprecedented strategies for pathway optimization and bioreactor operation and automation. Implementation of metabolic cybergenetics, however, will require new capabilities from actuators, biosensors, and control algorithms. The recent application of optogenetics in metabolic engineering, the expanding role of genetically encoded biosensors in strain development, and continued progress in control algorithms for biological processes suggest that this technology will become available in the not so distant future.

Abstract: The expression of a gene to a protein is one of the most vital biological processes. The use of light to control biology offers unparalleled spatiotemporal resolution from an external, orthogonal signal. A variety of methods have been developed that use light to control the steps of transcription and translation of specific genes into proteins, for cell-free to in vivo biotechnology applications. These methods employ techniques ranging from the modification of small molecules, nucleic acids and proteins with photocages, to the engineering of proteins involved in gene expression using naturally light-sensitive proteins. Although the majority of currently available technologies employ ultraviolet light, there has been a recent increase in the use of functionalities that work at longer wavelengths of light, to minimise cellular damage and increase tissue penetration. Here, we discuss the different chemical and biological methods employed to control gene expression, while also highlighting the central themes and the most exciting applications within this diverse field.

Abstract: The precision and repeatability of in vivo biological studies is predicated upon methods for isolating a targeted subsystem from external sources of noise and variability. However, in many experimental frameworks, this is made challenging by nonstatic environments during host cell growth, as well as variability introduced by manual sampling and measurement protocols. To address these challenges, we developed Chi.Bio, a parallelised open-source platform that represents a new experimental paradigm in which all measurement and control actions can be applied to a bulk culture in situ. In addition to continuous-culturing capabilities, it incorporates tunable light outputs, spectrometry, and advanced automation features. We demonstrate its application to studies of cell growth and biofilm formation, automated in silico control of optogenetic systems, and readout of multiple orthogonal fluorescent proteins in situ. By integrating precise measurement and actuation hardware into a single low-cost platform, Chi.Bio facilitates novel experimental methods for synthetic, systems, and evolutionary biology and broadens access to cutting-edge research capabilities.

Abstract: Syntheses of many commodities that are produced using microorganisms require cofactors such as ATP and NAD(P)H. Thus, optimization of the flux distribution in central carbon metabolism, which plays a key role in cofactor regeneration, is critical for enhancing the production of the target compounds. Since the intracellular and extracellular conditions change over time in the fermentation process, dynamic control of the metabolic system for maintaining the cellular state appropriately is necessary. Here, we review techniques for detecting the intracellular metabolic state with fluorescent sensors and controlling the flux of central carbon metabolism with optogenetic tools, as well as present a prospect of bio-production processes for fine-tuning the flux distribution.

Abstract: To perceive fluctuations in light quality, quantity, and timing, higher plants have evolved diverse photoreceptors including UVR8 (a UV-B photoreceptor), cryptochromes, phototropins, and phytochromes (Phys). In contrast to plants, prokaryotic oxygen-evolving photosynthetic organisms, cyanobacteria, rely mostly on bilin-based photoreceptors, namely, cyanobacterial phytochromes (Cphs) and cyanobacteriochromes (CBCRs), which exhibit structural and functional differences compared with plant Phys. CBCRs comprise varying numbers of light sensing domains with diverse color-tuning mechanisms and signal transmission pathways, allowing cyanobacteria to respond to UV-A, visible, and far-red lights. Recent genomic surveys of filamentous cyanobacteria revealed novel CBCRs with broader chromophore-binding specificity and photocycle protochromicity. Furthermore, a novel Cph lineage has been identified that absorbs blue-violet/yellow-orange light. In this minireview, we briefly discuss the diversity in color sensing and signal transmission mechanisms of Cphs and CBCRs, along with their potential utility in the field of optogenetics.

Abstract: In this chapter, we summarize the molecular mechanisms of the linear tetrapyrrole-binding photoreceptors, phytochromes, and cyanobacteriochromes. We especially focus on the color-tuning mechanisms and conformational changes during the photoconversion process. Furthermore, we introduce current status of development of the optogenetic tools based on these molecules. Huge repertoire of these photoreceptors with diverse spectral properties would contribute to development of multiplex optogenetic regulation. Among them, the photoreceptors incorporating the biliverdin IXα chromophore is advantageous for in vivo optogenetics because this is intrinsic in the mammalian cells, and absorbs far-red light penetrating into deep mammalian tissues.

Abstract: The features of the light-responsive cyanobacterial CcaSR regulatory module that determine interoperability of this optogenetic device between Escherichia coli and Pseudomonas putida have been examined. For this, all structural parts (i.e. ho1 and pcyA genes for synthesis of phycocyanobilin, the ccaS/ccaR system from Synechocystis and its cognate downstream promoter) were maintained but their expression levels and stoichiometry diversified by [i] reassembling them together in a single broad host range, standardized vector and [ii] subjecting the non-coding regulatory sequences to multiple cycles of directed evolution with random genomic mutations (DIvERGE), a recombineering method that intensifies mutation rates within discrete DNA segments. Once passed to P. putida, various clones displayed a wide dynamic range, insignificant leakiness and excellent capacity in response to green light. Inspection of the evolutionary intermediates pinpointed translational control as the main bottleneck for interoperability and suggested a general approach for easing the exchange of genetic cargoes between different species i.e. optimization of relative expression levels and upturning of subcomplex stoichiometry.

Abstract: Recent advances in synthetic biology and an emerging algal biotechnology market have spurred a prolific increase in the availability of molecular tools for cyanobacterial research. Nevertheless, work to date has focused primarily on only a small subset of model species, which arguably limits fundamental discovery and applied research towards wider commercialisation. Here, we review the requirements for uptake of new strains, including several recently characterised fast-growing species and promising non-model species. Furthermore, we discuss the potential applications of new techniques available for transformation, genetic engineering and regulation, including an up-to-date appraisal of current Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas) and CRISPR interference (CRISPRi) research in cyanobacteria. We also provide an overview of several exciting molecular tools that could be ported to cyanobacteria for more advanced metabolic engineering approaches (e.g., genetic circuit design). Lastly, we introduce a forthcoming mutant library for the model species Synechocystis sp. PCC 6803 that promises to provide a further powerful resource for the cyanobacterial research community.

Abstract: An engineering tool for controlling flux distribution on metabolic pathways to an appropriate state is highly desirable in bio-production. An optogenetic switch, which regulates gene expression by light illumination is an attractive on/off switchable system, and is a promising way for flux control with an external stimulus. We demonstrated a light-inducible flux control between glycolysis and the methylglyoxal (MGO) pathway in Escherichia coli using a CcaS/CcaR system. CcaR is phosphorylated by green light and is dephosphorylated by red light. Phosphorylated CcaR induces gene expression under the cpcG2 promoter. The tpiA gene was expressed under the cpcG2 promoter in a genomic tpiA deletion strain. The strain was then cultured with glucose minimum medium under green or red light. We found that tpiA mRNA level under green light was four times higher than that under red light. The repression of tpiA expression led to a decrease in glycolytic flux, resulting in slower growth under red light (0.25 h-1 ) when compared to green light (0.37 h-1 ). The maximum extracellular MGO concentration under red light (0.2 mM) was higher than that under green light (0.05 mM). These phenotypes confirm that the MGO pathway flux was enhanced under red light. This article is protected by copyright. All rights reserved.

Abstract: The Gram-positive bacterium Bacillus subtilis exhibits complex spatial and temporal gene expression signals. Although optogenetic tools are ideal for studying such processes, none has been engineered for this organism. Here, we port a cyanobacterial light sensor pathway comprising the green/red photoreversible two-component system CcaSR, two metabolic enzymes for production of the chromophore phycocyanobilin (PCB), and an output promoter to control transcription of a gene of interest into B. subtilis. Following an initial non-functional design, we optimize expression of pathway genes, enhance PCB production via a translational fusion of the biosynthetic enzymes, engineer a strong chimeric output promoter, and increase dynamic range with a miniaturized photosensor kinase. Our final design exhibits over 70-fold activation and rapid response dynamics, making it well-suited to studying a wide range of gene regulatory processes. In addition, the synthetic biology methods we develop to port this pathway should make B. subtilis easier to engineer in the future.

Abstract: Dynamically controlling cellular metabolism can improve a cell's yield and productivity towards a target compound. However, the application of this strategy is currently limited by the availability of reversible metabolic switches. Unlike chemical inducers, light can readily be applied and removed from the medium multiple times without causing chemical changes. This makes light-inducible systems a potent tool to dynamically control cellular metabolism. Here we describe the construction of a light-inducible metabolic switch to regulate flux distribution between two glycolytic pathways, the Embden-Meyerhof-Parnas (EMP) and oxidative pentose phosphate (oxPP) pathways. This was achieved by using chromatic acclimation sensor/regulator (CcaSR) optogenetic system to control the expression of pgi, a metabolic gene which expression determines flux distribution between EMP and oxPP pathways. Control over these pathways may allow us to maximize Escherichia coli's yield on highly-reduced compounds such as mevalonate. Background pgi expression of the initial CcaSR construct was too high to significantly reduce pgi expression during the OFF-state. Therefore, we attenuated the system's output leakage by adjusting plasmid copy number and by tagging Pgi with ssRA protein degradation signal. Using our CcaSR-pgi ver.3, we could control EMP:oxPP flux ratio to 50:49 and 0.5:99 (of total glycolytic flux) by exposure to green and red light, respectively.