Curated Optogenetic Publication Database

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 paradigm 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: Phosphatidic acid (PA) is a multifunctional lipid with important metabolic and signaling functions, and efforts to dissect its pleiotropy demand strategies for perturbing its levels with spatiotemporal precision. Previous membrane editing approaches for generating local PA pools used light-mediated induced proximity to recruit a PA-synthesizing enzyme, phospholipase D (PLD), from the cytosol to the target organelle membrane. Whereas these optogenetic PLDs exhibited high activity, their residual activity in the dark led to undesired chronic lipid production. Here, we report ultralow background membrane editors for PA wherein light directly controls PLD catalytic activity, as opposed to localization and access to substrates, exploiting a light–oxygen–voltage (LOV) domain-based conformational photoswitch inserted into the PLD sequence and enabling their stable and nonperturbative targeting to multiple organelle membranes. By coupling organelle-targeted LOVPLD activation to lipidomics analysis, we discovered different rates of metabolism for PA and its downstream products depending on the subcellular location of PA production. We also elucidated signaling roles for PA pools on different membranes in conferring local activation of AMP-activated protein kinase signaling. This work illustrates how membrane editors featuring acute, optogenetic conformational switches can provide new insights into organelle-selective lipid metabolic and signaling pathways.

Abstract: Open Reading Frame 6 (ORF6) proteins, which are unique to severe acute respiratory syndrome-related (SARS) coronavirus, inhibit the classical nuclear import pathway to antagonize host antiviral responses. Several alternative models were proposed to explain the inhibitory function of ORF6 [H. Xia et al., Cell Rep. 33, 108234 (2020); L. Miorin et al., Proc. Natl. Acad. Sci. U.S.A. 117, 28344-28354 (2020); and M. Frieman et al., J. Virol. 81, 9812-9824 (2007)]. To distinguish these models and build quantitative understanding of ORF6 function, we developed a method for scoring both ORF6 concentration and functional effect in single living cells. We combined quantification of untagged ORF6 expression level in single cells with optogenetics-based measurement of nuclear transport kinetics, using methods that could be adapted to measure concentration-dependent effects of any untagged protein. We found that SARS-CoV-2 ORF6 is ~15 times more potent than SARS-CoV-1 ORF6 in inhibiting nuclear import and export, due to differences in the C-terminal region that is required for the NUP98-RAE1 binding. The N-terminal region was required for transport inhibition. This region binds membranes but could be replaced by synthetic constructs which forced oligomerization in solution, suggesting its primary function is oligomerization. We propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the NUP98-RAE1 complexes at the nuclear pore complex, and this multivalent binding inhibits bidirectional transport.

Abstract: T cells regulate adaptive immune responses through complex signaling pathways mediated by T cell receptor (TCR). The functional domains of the TCR are combined with specific antibodies for the development of chimeric antigen receptor (CAR) T cell therapy. In this review, we first overview current understanding on the T cell signaling pathways as well as traditional methods that have been widely used for the T cell study. These methods, however, are still limited to investigating dynamic molecular events with spatiotemporal resolutions. Therefore, genetically encoded biosensors and optogenetic tools have been developed to study dynamic T cell signaling pathways in live cells. We review these cutting-edge technologies that revealed dynamic and complex molecular mechanisms at each stage of T cell signaling pathways. They have been primarily applied to the study of dynamic molecular events in TCR signaling, and they will further aid in understanding the mechanisms of CAR activation and function. Therefore, genetically encoded biosensors and optogenetic tools offer powerful tools for enhancing our understanding of signaling mechanisms in T cells and CAR-T cells.

Abstract: Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVdeg, a tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVdeg by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVdeg tag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVdeg system. Finally, we use the LOVdeg tag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVdeg tag system, and introduce a powerful new tool for bacterial optogenetics.

Abstract: Avena Sativa phototropin 1 Light-oxygen-voltage 2 domain (AsLOV2) is the model protein of Per-Arnt-Sim (PAS) superfamily, characterized by conformational changes in response to external environmental stimuli. This conformational change is initiated by the unfolding of the N-terminal helix in the dark state followed by the unfolding of the C-terminal helix. The light state is defined by the unfolded termini and the subsequent modifications in hydrogen bond patterns. In this photoreceptor, β-sheets have been identified as crucial components for mediating allosteric signal transmission between the two termini. In this study, we combined microsecond all-atm molecular dynamics simulations and Markov state modeling of conformational states to quantify molecular basis of mutation-induced allostery in the AsLOV2 protein. Through a combination of computational investigations, we determine that the Hβ and Iβ strands are the most critical structural elements involved in the allosteric mechanism. To elucidate the role of these β-sheets, we introduced 13 distinct mutations (F490L, N492A, L493A, F494L, H495L, L496F, Q497A, R500A, F509L, Q513A, L514A, D515V, and T517V) and conducted comprehensive simulation analysis. The results highlighted the role of two hydrogen bond Asn482-Leu453 and Gln479-Val520 in the observed distinct behaviors of L493A, L496F, Q497A, and D515V mutants. The comprehensive atomistic-level analysis of the conformational landscapes revealed the critical functional role of β-sheet segments in the transmission of the allosteric signal upon the photoactivation of the light state.

Abstract: In the early 2000s, the field of neuroscience experienced a groundbreaking transformation with the advent of optogenetics. This innovative technique harnesses the properties of naturally occurring and genetically engineered rhodopsins to confer light sensitivity upon target cells. The remarkable spatiotemporal precision offered by optogenetics has provided researchers with unprecedented opportunities to dissect cellular physiology, leading to an entirely new level of investigation. Initially revolutionizing neuroscience, optogenetics quickly piqued the interest of the wider scientific community, and optogenetic applications were expanded to cardiovascular research. Over the past decade, researchers have employed various optical tools to observe, regulate, and steer the membrane potential of excitable cells in the heart. Despite these advancements, achieving control over specific signaling pathways within the heart has remained an elusive goal. Here, we review the optogenetic tools suitable to control cardiac signaling pathways with a focus on GPCR signaling, and delineate potential applications for studying these pathways, both in healthy and diseased hearts. By shedding light on these exciting developments, we hope to contribute to the ongoing progress in basic cardiac research to facilitate the discovery of novel therapeutic possibilities for treating cardiovascular pathologies.

Abstract: Anti-CRISPR (Acr) proteins are encoded by mobile genetic elements to overcome the CRISPR immunity of prokaryotes, displaying promises as controllable tools for modulating CRISPR-based applications. However, characterizing novel anti-CRISPR proteins and exploiting Acr-related technologies is a rather long and tedious process. Here, we established a versatile plasmid interference with CRISPR interference (PICI) system in Escherichia coli for rapidly characterizing Acrs and developing Acr-based technologies. Utilizing the PICI system, we discovered two novel type II-A Acrs (AcrIIA33 and AcrIIA34), which can inhibit the activity of SpyCas9 by affecting DNA recognition of Cas9. We further constructed a circularly permuted AcrIIA4 (cpA4) protein and developed optogenetically engineered, robust AcrIIA4 (OPERA4) variants by combining cpA4 with the light-oxygen-voltage 2 (LOV2) blue light sensory domain. OPERA4 variants are robust light-dependent tools for controlling the activity of SpyCas9 by approximately 1000-fold change under switching dark-light conditions in prokaryotes. OPERA4 variants can achieve potent light-controllable genome editing in human cells as well. Together, our work provides a versatile screening system for characterizing Acrs and developing the Acr-based controllable tools.

Abstract: Optogenetics is a powerful approach that enables researchers to use light to dynamically manipulate cellular behavior. Since the first published use of optogenetics in synthetic biology, the field has expanded rapidly, yielding a vast array of tools and applications. Despite its immense potential for achieving high spatiotemporal precision, optogenetics has predominantly been employed as a substitute for conventional chemical inducers. In this short review, we discuss key features of microbial optogenetics and highlight applications for understanding biology, cocultures, bioproduction, biomaterials, and therapeutics, in which optogenetics is more fully utilized to realize goals not previously possible by other methods.

Abstract: Naturally occurring and engineered flavin-binding, blue-light-sensing, light, oxygen, voltage (LOV) photoreceptor domains have been used widely to design fluorescent reporters, optogenetic tools, and photosensitizers for the visualization and control of biological processes. In addition, natural LOV photoreceptors with engineered properties were recently employed for optimizing plant biomass production in the framework of a plant-based bioeconomy. Here, the understanding and fine-tuning of LOV photoreceptor (kinetic) properties is instrumental for application. In response to blue-light illumination, LOV domains undergo a cascade of photophysical and photochemical events that yield a transient covalent FMN-cysteine adduct, allowing for signaling. The rate-limiting step of the LOV photocycle is the dark-recovery process, which involves adduct scission and can take between seconds and days. Rational engineering of LOV domains with fine-tuned dark recovery has been challenging due to the lack of a mechanistic model, the long time scale of the process, which hampers atomistic simulations, and a gigantic protein sequence space covering known mutations (combinatorial challenge). To address these issues, we used machine learning (ML) trained on scarce literature data and iteratively generated and implemented experimental data to design LOV variants with faster and slower dark recovery. Over the three prediction–validation cycles, LOV domain variants were successfully predicted, whose adduct-state lifetimes spanned 7 orders of magnitude, yielding optimized tools for synthetic (opto)biology. In summary, our results demonstrate ML as a viable method to guide the design of proteins even with limited experimental data and when no mechanistic model of the underlying physical principles is available.

Abstract: The light-oxygen-voltage (LOV) domains of phototropins emerged as essential constituents of light-sensitive proteins, helping initiate blue light-triggered responses. Moreover, these domains have been identified across all kingdoms of life. LOV domains utilize flavin nucleotides as co-factors and undergo structural rearrangements upon exposure to blue light, which activates an effector domain that executes the final output of the photoreaction. LOV domains are versatile photoreceptors that play critical roles in cellular signaling and environmental adaptation; additionally, they can noninvasively sense and control intracellular processes with high spatiotemporal precision, making them ideal candidates for use in optogenetics, where a light signal is linked to a cellular process through a photoreceptor. The ongoing development of LOV-based optogenetic tools, driven by advances in structural biology, spectroscopy, computational methods, and synthetic biology, has the potential to revolutionize the study of biological systems and enable the development of novel therapeutic strategies.

Abstract: Optogenetics is a cutting-edge technology that merges light control and genetics to achieve targeted control of tissue cells. Compared to traditional methods, optogenetics offers several advantages in terms of time and space precision, accuracy, and reduced damage to the research object. Currently, optogenetics is primarily used in pathway research, drug screening, gene expression regulation, and the stimulation of molecule release to treat various diseases. The selection of light-sensitive proteins is the most crucial aspect of optogenetic technology; structural changes occur or downstream channels are activated to achieve signal transmission or factor release, allowing efficient and controllable disease treatment. In this review, we examine the extensive research conducted in the field of biomedicine concerning optogenetics, including the selection of light-sensitive proteins, the study of carriers and delivery devices, and the application of disease treatment. Additionally, we offer critical insights and future implications of optogenetics in the realm of clinical medicine.

Abstract: YAP is a transcriptional regulator that controls pluripotency, cell fate, and proliferation. How cells ensure the selective activation of YAP effector genes is unknown. This knowledge is essential to rationally control cellular decision-making. Here we leverage optogenetics, live-imaging of transcription, and cell fate analysis to understand and control gene activation and cell behavior. We reveal that cells decode the steady-state concentrations and timing of YAP activation to control proliferation, cell fate, and expression of the pluripotency regulators Oct4 and Nanog. While oscillatory YAP inputs induce Oct4 expression and proliferation optimally at frequencies that mimic native dynamics, cellular differentiation requires persistently low YAP levels. We identify the molecular logic of the Oct4 dynamic decoder, which acts through an adaptive change sensor. Our work reveals how YAP levels and dynamics enable multiplexing of information transmission for the regulation of developmental decision-making and establishes a platform for the rational control of these behaviors.

Abstract: Post-translational modifications of histones to a large extent determine the functional state of chromatin loci. Dynamic visualization of histone modifications with genetically encoded fluorescent sensors makes it possible to monitor changes in the epigenetic state of a single living cell. At the same time, the sensors can potentially compete with endogenous factors recognizing these modifications. Thus, prolonged binding of the sensors to chromatin can affect normal epigenetic regulation. Here, we report an optogenetic sensor for live-cell visualization of histone H3 methylated at lysine-9 (H3K9me3) named MPP8-LAMS (MPP8-based light-activated modification sensor). MPP8-LAMS consists of several fusion protein parts (from N- to C-terminus): i) nuclear export signal (NES), ii) far-red fluorescent protein Katushka, iii) H3K9me3-binding reader domain of the human M phase phosphoprotein 8 (MPP8), iv) the light-responsive AsLOV2 domain, which exposes a nuclear localization signal (NLS) upon blue light stimulation. In the dark, due to the NES, MPP8-LAMS is localized in the cytosol. Under blue light illumination, MPP8-LAMS underwent an efficient translocation from cytosol to nucleus, enabling visualization of H3K9me3-enriched loci. Such an on-demand visualization minimizes potential impact on cell physiology as most of the time the sensor is separated from its target. In general, the present work extends the application of optogenetics to the area of advanced use of genetically encoded sensors.

Abstract: Within a shared cytoplasm, filamentous actin (F-actin) plays numerous and critical roles across the cell body. Cells rely on actin-binding proteins (ABPs) to organize F-actin and to integrate its polymeric characteristics into diverse cellular processes. Yet, the multitude of ABPs that engage with and shape F-actin make studying a single ABP’s influence on cellular activities a significant challenge. Moreover, without a means of manipulating actin-binding subcellularly, harnessing the F-actin cytoskeleton for synthetic biology purposes remains elusive. Here, we describe a suite of designed proteins, Controllable Actin-binding Switch Tools (CASTs), whose actin-binding behavior can be controlled with external stimuli. CASTs were developed that respond to different external inputs, providing options for turn-on kinetics and enabling orthogonality. Being genetically encoded, we show that CASTs can be inserted into native protein sequences to control F-actin association locally and engineered into new structures to control cell and tissue shape and behavior.

Abstract: Cell contraction plays an important role in many physiological and pathophysiological processes. This includes functions in skeletal, heart, and smooth muscle cells, which lead to highly coordinated contractions of multicellular assemblies, and functions in non-muscle cells, which are often highly localized in subcellular regions and transient in time. While the regulatory processes that control cell contraction in muscle cells are well understood, much less is known about cell contraction in non-muscle cells. In this review, we focus on the mechanisms that control cell contraction in space and time in non-muscle cells, and how they can be investigated by light-based methods. The review particularly focusses on signal networks and cytoskeletal components that together control subcellular contraction patterns to perform functions on the level of cells and tissues, such as directional migration and multicellular rearrangements during development. Key features of light-based methods that enable highly local and fast perturbations are highlighted, and how experimental strategies can capitalize on these features to uncover causal relationships in the complex signal networks that control cell contraction.

Abstract: E-cadherin-based cell-cell adhesions are dynamically and locally regulated in many essential processes, including embryogenesis, wound healing and tissue organization, with dysregulation manifesting as tumorigenesis and metastasis. However, the lack of tools that would provide control of the high spatiotemporal precision observed with E-cadherin adhesions hampers investigation of the underlying mechanisms. Here, we present an optogenetic tool, opto-E-cadherin, that allows reversible control of E-cadherin-mediated cell-cell adhesions with blue light. With opto-E-cadherin, functionally essential calcium binding is photoregulated such that cells expressing opto-E-cadherin at their surface adhere to each other in the dark but not upon illumination. Consequently, opto-E-cadherin provides remote control over multicellular aggregation, E-cadherin-associated intracellular signalling and F-actin organization in 2D and 3D cell cultures. Opto-E-cadherin also allows switching of multicellular behaviour between single and collective cell migration, as well as of cell invasiveness in vitro and in vivo. Overall, opto-E-cadherin is a powerful optogenetic tool capable of controlling cell-cell adhesions at the molecular, cellular and behavioural level that opens up perspectives for the study of dynamics and spatiotemporal control of E-cadherin in biological processes.

Abstract: Photoproteins, luminescent proteins or optoproteins are a kind of light-response protein responsible for the conversion of light into biochemical energy that is used by some bacteria or fungi to regulate specific biological processes. Within these specific proteins, there are groups such as the photoreceptors that respond to a given light wavelength and generate reactions susceptible to being used for the development of high-novel applications, such as the optocontrol of metabolic pathways. Photoswitchable proteins play important roles during the development of new materials due to their capacity to change their conformational structure by providing/eliminating a specific light stimulus. Additionally, there are bioluminescent proteins that produce light during a heatless chemical reaction and are useful to be employed as biomarkers in several fields such as imaging, cell biology, disease tracking and pollutant detection. The classification of these optoproteins from bacteria and fungi as photoreceptors or photoresponse elements according to the excitation-emission spectrum (UV-Vis-IR), as well as their potential use in novel applications, is addressed in this article by providing a structured scheme for this broad area of knowledge.

Abstract: The dysregulation of protein kinases is associated with multiple diseases due to the kinases’ involvement in a variety of cell signaling pathways. Manipulating protein kinase function, by controlling the active site, is a promising therapeutic and investigative strategy to mitigate and study diseases. Kinase active sites share structural similarities making it difficult to specifically target one kinase, allosteric control allows specific regulation and study of kinase function without directly targeting the active site. Allosteric sites are distal to the active site but coupled via a dynamic network of inter-atomic interactions between residues in the protein. Establishing an allosteric control over a kinase requires understanding the allosteric wiring of the protein. Computational techniques offer effective and inexpensive mapping of the allosteric sites on a protein. Here, we discuss methods to map and regulate allosteric communications in proteins, and strategies to establish control over kinase functions in live cells and organisms. Protein molecules, or “sensors” are engineered to function as tools to control allosteric activity of the protein as these sensors have high spatiotemporal resolution and help in understanding cell phenotypes after immediate activation or inactivation of a kinase. Traditional methods used to study protein functions, such as knockout, knockdown, or mutation, cannot offer a sufficiently high spatiotemporal resolution. We discuss the modern repertoire of tools to regulate protein kinases as we enter a new era in deciphering cellular signaling and developing novel approaches to treat diseases associated with signal dysregulation.

Abstract: CRISPR-based base editors (BEs) are powerful tools for precise nucleotide substitution in a wide range of organisms, but spatiotemporal control of base editing remains a daunting challenge. Herein, we develop a photoactivatable base editor (Mag-ABE) for spatiotemporally controlled genome editing in vivo for the first time. The base editing activity of Mag-ABE can be activated by blue light for spatiotemporal regulation of both EGFP reporter gene and various endogenous genes editing. Meanwhile, the Mag-ABE prefers to edit A4 and A5 positions rather than to edit A6 position, showing the potential to decrease bystander editing of traditional adenine base editors. After integration with upconversion nanoparticles as a light transducer, the Mag-ABE is further applied for near-infrared (NIR) light-activated base editing of liver in transgenic reporter mice successfully. This study opens a promising way to improve the operability, safety, and precision of base editing.

Abstract: Cells communicate with each other to jointly regulate cellular processes during cellular differentiation and tissue morphogenesis. This multiscale coordination arises through spatiotemporal activity of morphogens to pattern cell signaling and transcriptional factor activity. This coded information controls cell mechanics, proliferation, and differentiation to shape the growth and morphogenesis of organs. While many of the molecular components and physical interactions have been identified in key model developmental systems, there are still many unresolved questions related to the dynamics involved due to challenges in precisely perturbing and quantitatively measuring signaling dynamics. Recently, a broad range of synthetic optogenetic tools have been developed and employed to quantitatively define relationships between signal transduction and downstream cellular responses. These optogenetic tools can control intracellular activities at the single cell or whole tissue scale to direct subsequent biological processes. In this brief review, we highlight a selected set of studies that develop and implement optogenetic tools to unravel quantitative biophysical mechanisms for tissue growth and morphogenesis across a broad range of biological systems through the manipulation of morphogens, signal transduction cascades, and cell mechanics. More generally, we discuss how optogenetic tools have emerged as a powerful platform for probing and controlling multicellular development.

Abstract: The progression through the cell cycle phases is driven by cyclin-dependent kinases and cyclins as their regulatory subunits. As nuclear protein, the cell cycle inhibitor p21/CDKN1A arrests the cell cycle at the growth phase G1 by inhibiting the activity of cyclin-dependent kinases. The G1 phase correlates with increased cell size and cellular productivity. Here, we applied an optogenetic approach to control the subcellular localization of p21 and its nuclear functions. To generate light-controllable p21, appropriate fusions with the blue light switch cryptochrome 2/CIBN and the AsLOV-based light-inducible nuclear localization signal, LINuS, were used. Both systems, p21-CRY2/CIB1 and p21-LINuS, increased the amounts of cells arrested in the G1 phase correlating with the increased cell-specific productivity of the reporter-protein-secreted alkaline phosphatase. Varying the intervals of blue LED light exposure and the light dose enable the fine-tuning of the systems. Light-controllable p21 implemented in producer cell lines could be applied to steer the uncoupling of cell proliferation and cell cycle arrest at the G1 phase optimizing the production of biotherapeutic proteins.

Abstract: Regulated cell death (RCD) controls the removal of dispensable, infected or malignant cells, and is thus essential for development, homeostasis and immunity of multicellular organisms. Over the last years different forms of RCD have been described (among them apoptosis, necroptosis, pyroptosis and ferroptosis), and the cellular signaling pathways that control their induction and execution have been characterized at the molecular level. It has also become apparent that different forms of RCD differ in their capacity to elicit inflammation or an immune response, and that RCD pathways show a remarkable plasticity. Biochemical and genetic studies revealed that inhibition of a given pathway often results in the activation of back-up cell death mechanisms, highlighting close interconnectivity based on shared signaling components and the assembly of multivalent signaling platforms that can initiate different forms of RCD. Due to this interconnectivity and the pleiotropic effects of 'classical' cell death inducers, it is challenging to study RCD pathways in isolation. This has led to the development of tools based on synthetic biology that allow the targeted induction of RCD using chemogenetic or optogenetic methods. Here we discuss recent advances in the development of such toolset, highlighting their advantages and limitations, and their application for the study of RCD in cells and animals.

Abstract: Engineered antibodies are essential tools for research and advanced pharmacy. In the development of therapeutics, antibodies are excellent candidates as they offer both target recognition and modulation. Thanks to the latest advances in biotechnology, light-activated antibody fragments can be constructed to control spontaneous antigen interaction with high spatiotemporal precision. To implement conditional antigen binding, several optogenetic and optochemical engineering concepts have recently been developed. Here, we highlight the various strategies and discuss the features of opto-conditional antibodies. Each concept offers intrinsic advantages beneficial to different applications. In summary, the novel design approaches constitute a complementary toolset to promote current and upcoming antibody technologies with ultimate precision.

Abstract: Dynamic metabolic engineering is a strategy to switch key metabolic pathways in microbial cell factories from biomass generation to accumulation of target products. Here, we demonstrate that optogenetic intervention in the cell cycle of budding yeast can be used to increase production of valuable chemicals, such as the terpenoid β-carotene or the nucleoside analog cordycepin. We achieved optogenetic cell-cycle arrest in the G2/M phase by controlling activity of the ubiquitin-proteasome system hub Cdc48. To analyze the metabolic capacities in the cell cycle arrested yeast strain, we studied their proteomes by timsTOF mass spectrometry. This revealed widespread, but highly distinct abundance changes of metabolic key enzymes. Integration of the proteomics data in protein-constrained metabolic models demonstrated modulation of fluxes directly associated with terpenoid production as well as metabolic subsystems involved in protein biosynthesis, cell wall synthesis, and cofactor biosynthesis. These results demonstrate that optogenetically triggered cell cycle intervention is an option to increase the yields of compounds synthesized in a cellular factory by reallocation of metabolic resources.