Curated Optogenetic Publication Database

Abstract: Saccharomyces cerevisiae is a widely used chassis in metabolic engineering. Due to the Crabtree effect, it preferentially produces ethanol under high-glucose conditions, limiting the synthesis of other valuable metabolites. Conventional metabolic engineering approaches typically rely on irreversible genetic modifications, making it insufficient for dynamic metabolic control. In contrast, optogenetics offers a reversible and tunable method for regulating cellular metabolism with high temporal precision. In this study, we engineered the pyruvate decarboxylase isozyme 1 (Pdc1) by inserting the photosensory modules (AsLOV2 and cpLOV2 domains) into rationally selected positions within the enzyme. Through a growth phenotype-based screening system, we identified two blue light-responsive variants, OptoPdc1D1 and OptoPdc1D2, which enable light-dependent control of enzymatic activity. Leveraging these OptoPdc1 variants, we developed opto-S. cerevisiae strains, MLy-9 and MLy-10, which demonstrated high efficiency in modulating both cell growth and ethanol production. These strains allow reliable regulation of ethanol biosynthesis in response to blue light, achieving a dynamic control range of approximately 20- to 120-fold. The opto-S. cerevisiae strains exhibited dose-dependent production in response to blue light intensity and pulse patterns, confirming their potential for precise metabolic control. This work establishes a novel protein-level strategy for regulating metabolic pathways in S. cerevisiae and introduces an effective method for controlling ethanol metabolism via optogenetic regulation.

Abstract: The efficacy of antitumor immunotherapy is closely associated with the expansion of tumor-infiltrating CD8+ T cells. However, within the tumor microenvironment, CD8+ T cells often exhibit reduced proliferation due to persistent exposure to tumor antigens. The cytokine IL-2 is a potent growth factor that can drive the expansion of tumor-infiltrating lymphocytes. While its clinical application has been severely limited by systemic toxicity and in vivo instability. To address these challenges, we have developed a dual-responsive system (EcNIL-2@UCNP/Gel-CTX) leveraging the hypoxic tropisms of E. coli Nissle 1917(EcN). This system is capable of producing IL-2 in situ upon near-infrared (NIR) irradiation and releasing low-dose cyclophosphamide (CTX) in response to matrix metalloproteinase-2 (MMP-2) in the tumor microenvironment. The EcNIL-2@UCNP/Gel-CTX system not only drives the expansion of CD8+ T cells and boost the activity of NK cells but also reduces Treg cell populations, thereby remodeling the immune microenvironment and eliciting robust tumor-specific immune responses in H22 subcutaneous tumors in mice and confers long-term protection against tumor rechallenge by promoting the generation of durable memory T cells. Our findings provide an both light and tumor microenvironment responsive platform for enhanced cancer immunotherapy.

Abstract: Coenzyme Q10 biosynthesis in Escherichia coli is constrained by kinetic mismatches between precursor synthesis and methylation, alongside bioenergetic uncoupling. We implemented an optogenetic phase-control strategy integrating dynamic light induction, ribosome binding site (RBS) engineering, and real-time membrane potential (ΔΨ) feedback. Temporal coordination of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and UbiG methyltransferase (UbiG) via a 6-h phase delay reduced methylglyoxal shunt flux by 41 ± 3% (p < 0.01) through enhanced precursor channeling. Membrane hyperpolarization to - 90 ± 2 mV (relative to - 70 mV in controls) triggered voltage-gated UbiG membrane localization (62 ± 3%) and ATP-driven S-adenosylmethionine regeneration, increasing methylation efficiency 2.3-fold. Multivariate modeling identified ΔΨ and acetate as critical control parameters, enabling optimized fermentation (dissolved oxygen (DO) 15-20%, pH 6.7-6.9). The engineered strain achieved 0.63 ± 0.07 g/L CoQ10 in 5-L bioreactors-a 4.3-fold improvement over the static control strain (0.15 ± 0.02 g/L)-with 82.5% carbon efficiency and 25.8% glycerol-to-product yield. This work establishes bioenergetically coupled temporal control as a scalable paradigm for membrane-bound isoprenoid biomanufacturing. KEY POINTS: • Phase-driven enzyme synchronization via optogenetics resolves kinetic mismatch. • Membrane hyperpolarization gates enzyme localization and ATP regeneration. • Model-integrated bioenergetic-process control enhances CoQ10 production efficiency.

Abstract: Precise control of gene expression is one of the fundamental goals of synthetic biology. Whether the objective is to modify endogenous cellular function or induce the expression of molecules for diagnostic and therapeutic purposes, gene regulation remains a key aspect of biological systems. Over time, advances in protein engineering and molecular biology have led to the creation of gene circuits capable of inducing the expression of specific proteins in response to external stimulus such as light. These optogenetic, or light-activated circuits hold significant potential for gene therapy as a tool for regulating the expression of therapeutic genes within cells. However, the applications of optogenetic systems can be limited by the lack of efficient ways for light delivery inside cells or tissue. Our approach to address this challenge is to harness the power of bioluminescence to produce light directly inside cells using a luminescent enzyme. Combined with a photosensitive transcription factor, we report the development of a fully genetically encoded optogenetic circuit for control of gene expression. Furthermore, we utilized a magneto sensitive protein to engineer a split protein version of this luminescent enzyme, where its reconstitution is driven by a 50mT magnetic stimulus. Thus, resulting in a first-of-its-kind gene circuit activated by a combination of light and magnetic stimulus. We expect this work to advance the implementation of light-controlled systems without the need of external light sources, as well as serve as a basis for the development of future magneto-sensitive tools.

Abstract: Precise control of covalent protein binding and cleavage in mammalian cells is crucial for manipulating cellular processes but remains challenging due to dark background, poor stability, low efficiency, or requirement of unnatural amino acids in current optogenetic tools. We introduce a photoswitchable intein (PS Intein) engineered by allosterically modulating a small autocatalytic gp41-1 intein with tandem Vivid photoreceptor. PS Intein exhibits superior functionality and low background in cells compared to existing tools. PS Intein-based systems enable light-induced covalent binding, cleavage, and release of proteins for regulating gene expression and cell fate. The high responsiveness and ability to integrate multiple inputs allow for intersectional cell targeting using cancer- and tumor microenvironment-specific promoters. PS Intein tolerates various fusions and insertions, facilitating its application in diverse cellular contexts. This versatile technology offers efficient light-controlled protein manipulation, providing a powerful tool for adding functionalities to proteins and precisely controlling protein networks in living cells.

Abstract: Localized translation broadly enables spatiotemporal control of gene expression. Here, we present LOV-domain-controlled ligase for translation localization (LOCL-TL), an optogenetic approach for monitoring translation with codon resolution at any defined subcellular location under physiological conditions. Application of LOCL-TL to mitochondrially localized translation revealed that ∼20% of human nuclear-encoded mitochondrial genes are translated on the outer mitochondrial membrane (OMM). Mitochondrially translated messages form two classes distinguished by encoded protein length, recruitment mechanism, and cellular function. An evolutionarily ancient mechanism allows nascent chains to drive cotranslational recruitment of long proteins via an unanticipated bipartite targeting signal. Conversely, mRNAs of short proteins, especially eukaryotic-origin electron transport chain (ETC) components, are specifically recruited by the OMM protein A-kinase anchoring protein 1 (AKAP1) in a translation-independent manner that depends on mRNA splicing. AKAP1 loss lowers ETC levels. LOCL-TL thus reveals a hierarchical strategy that enables preferential translation of a subset of proteins on the OMM.

Abstract: Targeted protein degradation is a powerful tool for biological research, cell therapy, and synthetic biology. However, conventional methods often depend on pre-fused degrons or chemical degraders, limiting their wider applications. Here we develop a guided protein labeling and degradation system (GPlad) in Escherichia coli, using de novo designed guide proteins and arginine kinase (McsB) for precise degradation of various proteins, including fluorescent proteins, metabolic enzymes, and human proteins. We expand GPlad into versatile tools such as antiGPlad, OptoGPlad, and GPTAC, enabling reversible inhibition, optogenetic regulation, and biological chimerization. The combination of GPlad and antiGPlad allows for programmable circuit construction, including ON/OFF switches, signal amplifiers, and oscillators. OptoGPlad-mediated degradation of MutH accelerates E. coli evolution under protocatechuic acid stress, reducing the required generations from 220 to 100. GPTAC-mediated degradation of AroE enhanced the titer of 3-dehydroshikimic acid to 92.6 g/L, a 23.8% improvement over the conventional CRISPR interference method. We provide a tunable, plug-and-play strategy for straightforward protein degradation without the need for pre-fusion, with substantial implications for synthetic biology and metabolic engineering.

Abstract: Performance of near-infrared probes and optogenetic tools derived from bacterial phytochromes is limited by availability of their biliverdin chromophore. To address this, we use a biliverdin reductase-A knock-out mouse model (Blvra-/-), which elevates endogenous biliverdin levels. We show that Blvra⁻/⁻ significantly enhances function of bacterial phytochrome-based systems. Light-controlled transcription using iLight optogenetic tool improves ~25-fold in Blvra-/- cells, compared to wild-type controls, and achieves ~100-fold activation in neurons. Light-induced insulin production in Blvra-/- mice reduces blood glucose by ~60% in diabetes model. To overcome depth limitations in imaging, we employ 3D photoacoustic, ultrasound, and two-photon fluorescence microscopy. This enables simultaneous photoacoustic imaging of DrBphP in neurons and super-resolution ultrasound localization microscopy of brain vasculature at depths of ~7 mm through intact scalp and skull. Two-photon microscopy achieves cellular resolution of miRFP720-expressing neurons at ~2.2 mm depth. Overall, Blvra-/- model represents powerful platform for improving efficacy of biliverdin-dependent tools for deep-tissue imaging and optogenetic manipulation.

Abstract: Induced pluripotent stem cells (iPSCs) share similar cellular features and various antigens profiles with cancer cells. Leveraging these characteristics, iPSCs hold great promise for developing wide-spectrum vaccines against cancers. In practice, iPSCs are typically combined with immune adjuvants to enhance antitumor immune responses; however, traditional adjuvants lack controllability and can induce systemic toxicity, which has limited their broad application. Here, a red/far-red light-controlled iPSC-based vaccine (RIVA) based on the chimeric photosensory protein FnBphP and its interaction partner LDB3 is developed; RIVA preserves the intrinsic tumor antigens of iPSCs and enables optogenetic control of an immune adjuvant's (IFN-β) expression under red light illumination. Experiments in multiple mouse tumor models demonstrate that RIVA inhibits tumor growth and improves animal survival in prophylactic and therapeutic settings, including against pulmonary metastatic 4T1 breast cancer. RIVA efficiently stimulates dendritic cell maturation, eliciting innate immune activation effects through NK cells and elicit adaptive immune anti-tumor responses through CD4+ and CD8+ T cells. Moreover, RIVA protects animals against tumor re-challenge by inducing strong immunological memory, with minimal systemic toxicity. This study demonstrates RIVA as an effective optogenetic approach for developing safe multi-antigen vaccines for the prevention and treatment of cancer.

Abstract: Optogenetic systems use light-responsive proteins to control gene expression, ion channels, protein localization, and signaling with the "flip of a switch". One such tool is the light activated CRISPR effector (LACE) system. Its ability to regulate gene expression in a tunable, reversible, and spatially resolved manner makes it attractive for many applications. However, LACE relies on delivery of four separate components on individual plasmids, which can limit its use. Here, we optimize LACE to reduce the number of plasmids needed to deliver all four components.

Abstract: Proper vertebrate development is dependent on tightly regulated expression of genes at the correct time and place. To identify normal but also dysregulated development leading to disease, in vivo interrogation methods with high spatiotemporal resolution are required. Recently, optogenetic tools to manipulate gene expression with spatiotemporal control have emerged, but their in vivo applications remain challenging. Here, we present a transgenic zebrafish strain termed zebrafish for heat-shock-inducible optogenetic recombinase expression (zHORSE) with inducible expression of a light-activatable Cre recombinase. We demonstrate that zHORSE endows robust spatiotemporal control over gene expression down to single-cell level at different developmental stages. We apply zHORSE for lineage tracing to identify caudal fin progenitors and for targeted expression of oncogenes. Surprisingly, one oncogene, EWS::FLI1, can cause ectopic fin formation when induced in permissive environments. zHORSE is compatible with existing loxP zebrafish effector strains and will enable many applications ranging from dissecting and precisely manipulating development to clonal cancer modeling.

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

Abstract: The in vivo continuous evolution system OrthoRep (orthogonal replication) is a powerful strategy for rapid enzyme evolution in Saccharomyces cerevisiae that diversifies genes at a rate exceeding the endogenous genome mutagenesis rate by several orders of magnitude. However, it is difficult to neofunctionalize genes using OrthoRep partly because of the way selection pressures are applied. Here we combine OrthoRep with optogenetics in a selection strategy we call OptoRep, which allows fine-tuning of selection pressure with light. With this capability, we evolved a truncated form of the endogenous monocarboxylate transporter JEN1 (JEN1t) into a de novo mevalonate importer. We demonstrate the functionality of the evolved JEN1t (JEN1tY180C/G) in the production of farnesene, a renewable aviation biofuel, from mevalonate fed to fermentation media or produced by microbial consortia. This study shows that the light-induced complementation of OptoRep may improve the ability to evolve functions not currently accessible for selection, while its fine tunability of selection pressure may allow the continuous evolution of genes whose desired function has a restrictive range between providing effective selection and cellular viability.

Abstract: The cofactor LIM-domain-binding protein 1 (Ldb1) is linked to many processes in gene regulation, including enhancer-promoter communication, interchromosomal interactions, and enhanceosome-cofactor-like activity. However, its functional requirement and molecular role during embryogenesis remain unclear. Here, we used optogenetics (iLEXY) to rapidly deplete Drosophila Ldb1 (Chip) from the nucleus at precise time windows. Remarkably, this pinpointed the essential window of Chip's function to just 1 h of embryogenesis, overlapping zygotic genome activation (ZGA). We show that Zelda, a pioneer factor essential for ZGA, recruits Chip to chromatin, and both factors regulate concordant changes in gene expression, suggesting that Chip is a cofactor of Zelda. Chip does not significantly impact chromatin architecture at these stages, but instead recruits CBP, and is essential for H3K27ac deposition at enhancers and promoters, and for the proper expression of co-regulated genes. These data identify Chip as a functional bridge between Zelda and the coactivator CBP to regulate gene expression in early embryogenesis.

Abstract: Komagataella phaffii, also known as Pichia pastoris, is a powerful host for recombinant protein production, in part due to its exceptionally strong and tightly controlled PAOX1 promoter. Most K. phaffii bioprocesses for recombinant protein production rely on PAOX1 to achieve dynamic control in two-phase processes. Cells are first grown under conditions that repress PAOX1 (growth phase), followed by methanol-induced recombinant protein expression (production phase). In this study, we propose a methanol-free approach for dynamic metabolic control in K. phaffii using optogenetics, which can help enhance input tunability and flexibility in process optimization and control. The light-responsive transcription factor EL222 from Erythrobacter litoralis is used to regulate protein production from the PC120 promoter in K. phaffii with blue light. We used two system designs to explore the advantages and disadvantages of coupling or decoupling EL222 integration with that of the gene of interest. We investigate the relationship between EL222 gene copy number and light dosage to improve production efficiency for intracellular and secreted proteins. Experiments in lab-scale bioreactors demonstrate the feasibility of the outlined optogenetic systems as potential alternatives to conventional methanol-inducible bioprocesses using K. phaffii.

Abstract: Optogenetic systems offer precise control over gene expression, but leaky activity in the dark limits their dynamic range and, consequently, their applicability. Here, we enhanced an optogenetic system based on a split T7 RNA polymerase fused to blue-light-inducible Magnets by incorporating a tet-controlled riboregulatory module. This module exploits the photosensitivity of anhydrotetracycline and the designability of synthetic small RNAs to digitize light-controlled gene expression, implementing a repressive action over the translation of a polymerase fragment gene that is relieved with blue light. Our engineered system exhibited 13-fold improvement in dynamic range upon blue light exposure, which even raised to 23-fold improvement when using cells preadapted to chemical induction. As a functional demonstration, we implemented light-controlled antibiotic resistance in bacteria. Such integration of regulatory layers represents a suitable strategy for engineering better circuits for light-based biotechnological applications.

Abstract: Chickens serve as an excellent model organism for developmental biology, offering unique opportunities for precise spatiotemporal access to embryos within eggs. Optogenes are light-activated proteins that regulate gene expression, offering a non-invasive method to activate genes at specific locations and developmental stages, advancing developmental biology research. This study employed the Magnet-Cre optogenetic system to control gene expression in developing chicken embryos. Magnet-Cre consists of two light-sensitive protein domains that dimerize upon light activation, each attached to an inactive half of the Cre recombinase enzyme, which becomes active upon dimerization. We developed an all-in-one plasmid containing a green fluorescent protein marker, the Magnet-Cre system, and a light-activated red fluorescent protein gene. This plasmid was electroporated into the neural tube of Hamburger and Hamilton (H&H) stage 14 chicken embryos. Embryo samples were cleared using the CUBIC protocol and imaged with a light sheet microscope to analyze optogenetic activity via red-fluorescent cells. We established a pipeline for Magnet-Cre activation in chicken embryos, demonstrating that a single 3-min exposure to blue light following incubation at 28 °C was sufficient to trigger gene activity within the neural tube, with increased activity upon additional light exposure. Finally, we showed a spatiotemporal control of gene activity using a localized laser light induction. This research lays the groundwork for further advancements in avian developmental biology and poultry research, enabling spatiotemporal control of genes in both embryos and transgenic chickens.

Abstract: To address the need for methods for tagging and manipulating neuronal ensembles underlying specific behaviors, we present an improved version of FLARE, termed cytoFLARE (cytosol-expressed FLARE). cytoFLARE incorporates cytosolic tethering of a transcription factor and expression of a more sensitive pair of calcium-sensing domains. We show that cytoFLARE captures more calcium- and light-dependent signals in HEK293T cells and higher signal-to-background ratios in neuronal cultures. We further establish cytoFLARE transgenic Drosophila models and apply cytoFLARE to label activated neurons upon sensory or optogenetic stimulation within a defined time window. Notably, through the cytoFLARE-driven expression of optogenetic actuators, we successfully reactivated and inhibited neurons involved in the larval nociceptive system. Our findings demonstrate the characterization and application of time-gated calcium integrators for both recording and manipulating neuronal activity in Drosophila larvae.

Abstract: Bacteria-based therapies hold great promise for cancer treatment due to their selective tumor colonization and proliferation. However, clinical application is hindered by the need for safe, precise control systems to regulate local therapeutic payload expression and release. Here we developed a near-infrared (NIR) light-mediated PadC-based photoswitch (NETMAP) system based on a chimeric phytochrome-activated diguanylyl cyclase (PadC) and a cyclic diguanylate monophosphate-dependent transcriptional activator (MrkH). The NETMAP-engineered bacteria exhibited antitumor performance in mouse tumor models with different levels of immunogenicity. Specifically, in immunogenic lymphoma tumors, NIR-induced PD-L1 and CTLA-4 nanobodies enhanced the activation of adaptive immunity. In low-immunogenic tumors-including mouse-derived colon cancer models, an orthotopic human breast cancer cell line-derived xenograft model and a colorectal cancer patient-derived xenograft model-NIR-induced azurin and cytolysin A predominantly led to tumor inhibition. Our study identifies an NIR light-mediated therapeutic platform for engineered bacteria-based therapies with customizable outputs and precise dosage control.

Abstract: Proteins phase-separate to form condensates that partition and concentrate biomolecules into membraneless compartments. These condensates can exhibit dichotomous behaviors in biology by supporting cellular physiology or instigating pathological protein aggregation1–3. Tau and α- synuclein (αSyn) are neuronal proteins that form heterotypic (Tau:αSyn) condensates associated with both physiological and pathological processes. Tau and αSyn functionally regulate microtubules8–12, but are also known to misfold and co-deposit in aggregates linked to various neurodegenerative diseases4,5,6,7, which highlights the paradoxically ambivalent effect of Tau:αSyn condensation in health and disease. Here, we show that tubulin modulates Tau:αSyn condensates by promoting microtubule interactions, competitively inhibiting the formation of homotypic and heterotypic pathological oligomers. In the absence of tubulin, Tau-driven protein condensation accelerates the formation of toxic Tau:αSyn heterodimers and amyloid fibrils. However, tubulin partitioning into Tau:αSyn condensates modulates protein interactions, promotes microtubule polymerization, and prevents Tau and αSyn oligomerization and aggregation. We distinguished distinct Tau and αSyn structural states adopted in tubulin-absent (pathological) and tubulin-rich (physiological) condensates, correlating compact conformations with aggregation and extended conformations with function. Furthermore, using various neuronal cell models, we showed that loss of stable microtubules, which occurs in Alzheimer’s disease and Parkinsons disease patients13,14, results in pathological oligomer formation and loss of neurites, and that functional condensation using an inducible optogenetic Tau construct resulted in microtubule stablization. Our results identify that tubulin is a critical modulator in switching Tau:αSyn pathological condensates to physiological, mechanistically relating the loss of stable microtubules with disease progression. Tubulin restoration strategies and Tau-mediated microtubule stabilization can be potential therapies targeting both Tau-specific and Tau/αSyn mixed pathologies.

Abstract: With the development of metabolic engineering, increasing requirements for efficient microbial biosynthesis call for establishment of multi-strain co-culture system. Dynamic regulation of population ratios is crucial for optimizing bioproduction performance. Optogenetic systems with high universality and flexibility have the potential to realize dynamic control of population proportion. In this study, we utilized an optimized chromatic acclimation sensor/regulator (CcaS/R) system and a blue light-activated YF1-FixJ-PhlF system as induction modules. A pair of orthogonal quorum sensing systems and a toxin-antitoxin system were employed as communication module and effector module, respectively. By integrating these modules, we developed a dual light-controlled co-culture system that enables dynamic regulation of population ratios. This co-culture system provides a universal toolkit for applications in metabolic engineering and synthetic biology.

Abstract: Flocculation in Saccharomyces cerevisiae is a critical phenotype with ecological and industrial significance. This study aimed to functionally dissect the contributions of individual FLO genes (FLO1, FLO5, FLO9, FLO10, FLO11) to flocculation by employing an optogenetic circuit (OptoQ-AMP5) for precise, light-inducible control of gene expression. A FLO-null platform yeast strain was engineered allowing the expression of individual FLO genes without native background interference. Each FLO gene was reintroduced into the FLO-null background under the control of OptoQ-AMP5. Upon light induction, strains expressing FLO1, FLO5, or FLO10 demonstrated strong flocculation, with FLO1 and FLO5 forming large and structurally distinct aggregates. FLO9 induced a weaker phenotype. Sugar inhibition assays revealed distinct sensitivities among flocculins, notably FLO9's novel sensitivity to fructose and maltotriose. Additionally, FLO-induced changes in cell surface hydrophobicity were quantified, revealing that FLO10 and FLO1 conferred the greatest hydrophobicity, correlating with their aggregation strength. This work establishes a robust platform for investigating flocculation mechanisms in yeast with temporal precision. It highlights the phenotypic diversity encoded within the FLO gene family and their differential responses to environmental cues. The optogenetic system provides a valuable tool for both fundamental studies and the rational engineering of yeast strains for industrial fermentation processes requiring controlled flocculation.

Abstract: The model liverwort Marchantia polymorpha is an emerging testbed species for plant metabolic engineering but lacks well-characterized inducible promoters, which are necessary to minimize biochemical and physiological disruption when over-accumulating target products. Here, we demonstrate the functionality of the light-inducible plant-usable light-switch elements (PULSE) optogenetic system in Marchantia and exemplify its use through the light-inducible overproduction of the bioplastic poly-3-hydroxybutyrate (PHB).

Abstract: Photosynthetic cyanobacteria can be utilised in biotechnology as environmentally sustainable cell factories to convert CO2 into a diverse range of biochemicals. However, a lack of molecular tools available for precise and dynamic control of gene expression hinders metabolic engineering and contributes to low product titres. Optogenetic tools enable light-regulated control of gene expression with high tunability and reversibility. To date, their application in cyanobacteria is limited and transferability between species remains unclear. In this study, we expressed the blue light-repressible YF1/FixJ and the green/red light-responsive CcaS/CcaR systems in Synechococcus sp. PCC 7002 and characterised their performance using GFP fluorescence assays and qRT-PCR. The YF1/FixJ system of non-cyanobacterial origin showed poor performance with a maximum dynamic range of 1.5-fold despite several steps to improve this. By contrast, the CcaS/CcaR system originating from the cyanobacterium Synechocystis sp. PCC 6803 responded well to light wavelengths and intensities, with a 6-fold increased protein fluorescence output observed after 30 min of green light. Monitoring GFP transcript levels allowed us to quantify the kinetics of transcriptional activation and deactivation and to test the effect of both multiple green/red and light/dark cycles on system performance. Finally, we increased CcaS/CcaR system activity under green light through targeted genetic modifications to the pCpcG2 output promoter. This study provides a detailed characterisation of the behaviour of the CcaS/CcaR system in Synechococcus sp. PCC 7002, as well as underlining the complexity of transferring optogenetic tools across species.

Abstract: In the immunosuppressive tumor microenvironment (TME), tumor-associated macrophages (TAMs) predominantly exhibit an immunosuppressive M2 phenotype, which facilitates tumor proliferation and metastasis. Although current strategies aimed at reprogramming TAMs hold promise, their sustainability and effectiveness are limited due to repeated injections. Herein, a bacterial therapy platform containing two engineered strains was developed. One strain was engineered to produce and secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) to promote M2-like TAMs repolarization to M1-like TAMs, while the other strain was designed to secrete small interfering RNA (siRNA) targeting signal regulatory protein α (SIRPα). The two strains can continuously and efficiently produce bioactive therapeutic agents in situ, exerting a sustained and synergistic therapeutic effect in TAMs to inhibit tumor growth. To enhance treatment efficacy, optogenetic strategy was implemented to effectively control the production of GM-CSF, and outer membrane vesicles (OMVs) produced by engineered bacteria were utilized to protect the siRNA from degradation in the external environment. The experimental results indicated that the bacterial therapy platform could continuously produce and release bioactive GM-CSF and SIRPα siRNA, exhibiting significant therapeutic activity. In vivo experiments further demonstrated that this platform showed more sustained and stable therapeutic effects compared to conventional drug therapies. Additionally, the combination of these two engineered strains yielded the highest ratio of M1/M2 TAMs (0.80) and the lowest ratio of F4/80+SIRPα+TAMs (3.46 %) than single strain therapy. Our study expanded the potential of engineered bacteria as pharmaceutical factories for in vivo therapeutic applications.