Disease modeling and pharmacological rescue of autosomal dominant retinitis pigmentosa associated with RHO copy number variation.

Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder, causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene (RHO) account for ~25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first-ever reported mono-allelic copy number variation (CNV) in RHO as a novel cause of adRP. We (a) show advanced retinal degeneration in a male patient (68 years of age) harboring four transcriptionally active intact copies of rhodopsin, (b) recapitulated the clinical phenotypes using retinal organoids, and (c) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with RHO-CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased RHO mRNA expression (quantitative real-time PCR [qRT-PCR] and bulk RNA sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300 days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target NR2E3, an upstream regulator of RHO, to alter RHO expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that RHO expression requires precise control. Taken together, this study supports the clinical data indicating that RHO-CNV associated adRPdevelops as a result of protein overexpression, thereby overloading the photoreceptor post-translational modification machinery.

Deciphering the role of glycosaminoglycans in GPCR signaling.

G protein-coupled receptors (GPCR) and glycosaminoglycans (GAGs) are two essential components of the cell surface that regulate physiological processes in the body. GPCRs are the most extensive family of transmembrane receptors that control cellular responses to extracellular stimuli, while GAGs are polysaccharides that contribute to the function of the extracellular matrix (ECM). Due to their proximity to the plasma membrane, GAGs participate in signal transduction by interacting with various extracellular molecules and cell surface receptors. GAGs can directly interact with certain GPCRs or their ligands (chemokines, peptide hormones and neuropeptides, structural proteins, and enzymes) from the glutamate receptor family, the rhodopsin receptor family, the adhesion receptor family, and the secretin receptor family. These interactions have recently become an emerging topic, providing a new avenue for understanding how GPCR signaling is regulated. This review discusses our current state of knowledge about the role of GAGs in GPCR signaling and function.

Multiple retinal isomerizations during the early phase of the bestrhodopsin photoreaction.

Bestrhodopsins constitute a class of light-regulated pentameric ion channels that consist of one or two rhodopsins in tandem fused with bestrophin ion channel domains. Here, we report on the isomerization dynamics in the rhodopsin tandem domains of Phaeocystis antarctica bestrhodopsin, which binds all-trans retinal Schiff-base (RSB) absorbing at 661 nm and, upon illumination, converts to the meta-stable P540 state with an unusual 11-cis RSB. The primary photoproduct P682 corresponds to a mixture of highly distorted 11-cis and 13-cis RSB directly formed from the excited state in 1.4 ps. P673 evolves from P682 in 500 ps and contains highly distorted 13-cis RSB, indicating that the 11-cis fraction in P682 converts to 13-cis. Next, P673 establishes an equilibrium with P595 in 1.2 µs, during which RSB converts to 11-cis and then further proceeds to P560 in 48 µs and P540 in 1.0 ms while remaining 11-cis. Hence, extensive isomeric switching occurs on the early ground state potential energy surface (PES) on the hundreds of ps to µs timescale before finally settling on a metastable 11-cis photoproduct. We propose that P682 and P673 are trapped high up on the ground-state PES after passing through either of two closely located conical intersections that result in 11-cis and 13-cis RSB. Co-rotation of C11=C12 and C13=C14 bonds results in a constricted conformational landscape that allows thermal switching between 11-cis and 13-cis species of highly strained RSB chromophores. Protein relaxation may release RSB strain, allowing it to evolve to a stable 11-cis isomeric configuration in microseconds.

Voluntary exercise preserves visual function and reduces inflammatory response in an adult mouse model of autosomal dominant retinitis pigmentosa.

Whole-body physical exercise has been shown to promote retinal structure and function preservation in animal models of retinal degeneration. It is currently unknown how exercise modulates retinal inflammatory responses. In this study, we investigated cytokine alterations associated with retinal neuroprotection induced by voluntary running wheel exercise in a retinal degeneration mouse model of class B1 autosomal dominant retinitis pigmentosa, I307N Rho. I307N Rho mice undergo rod photoreceptor degeneration when exposed to bright light (induced). Our data show, active induced mice exhibited significant preservation of retinal and visual function compared to inactive induced mice after 4 weeks of exercise. Retinal cytokine expression revealed significant reductions of proinflammatory chemokines, keratinocyte-derived chemokine (KC) and interferon gamma inducible protein-10 (IP-10) expression in active groups compared to inactive groups. Through immunofluorescence, we found KC and IP-10 labeling localized to retinal vasculature marker, collagen IV. These data show that whole-body exercise lowers specific retinal cytokine expression associated with retinal vasculature. Future studies should determine whether suppression of inflammatory responses is requisite for exercise-induced retinal protection.

Light regulation of rhodopsin distribution during outer segment renewal in murine rod photoreceptors.

Vision under dim light relies on primary cilia elaborated by rod photoreceptors in the retina. This specialized sensory structure, called the rod outer segment (ROS), comprises hundreds of stacked, membranous discs containing the light-sensitive protein rhodopsin, and the incorporation of new discs into the ROS is essential for maintaining the rod's health and function. ROS renewal appears to be primarily regulated by extrinsic factors (light); however, results vary depending on different model organisms. We generated two independent transgenic mouse lines where rhodopsin's fate is tracked by a fluorescently labeled rhodopsin fusion protein (Rho-Timer) and show that rhodopsin incorporation into nascent ROS discs appears to be regulated by both external lighting cues and autonomous retinal clocks. Live-cell imaging of the ROS isolated from mice exposed to six unique lighting conditions demonstrates that ROS formation occurs in a periodic manner in cyclic light, constant darkness, and artificial light/dark cycles. This alternating bright/weak banding of Rho-Timer along the length of the ROS relates to inhomogeneities in rhodopsin density and potential points of structural weakness. In addition, we reveal that prolonged dim ambient light exposure impacts not only the rhodopsin content of new discs but also that of older discs, suggesting a dynamic interchange of material between new and old discs. Furthermore, we show that rhodopsin incorporation into the ROS is greatly altered in two autosomal recessive retinitis pigmentosa mouse models, potentially contributing to the pathogenesis. Our findings provide insights into how extrinsic (light) and intrinsic (retinal clocks and genetic mutation) factors dynamically regulate mammalian ROS renewal.

Adaptive Evolution of Nearctic Deepwater Fish Vision: Implications for Assessing Functional Variation for Conservation.

Intraspecific functional variation is critical for adaptation to rapidly changing environments. For visual opsins, functional variation can be characterized in vitro and often reflects a species' ecological niche but is rarely considered in the context of intraspecific variation or the impact of recent environmental changes on species of cultural or commercial significance. Investigation of adaptation in postglacial lakes can provide key insight into how rapid environmental changes impact functional evolution. Here, we report evidence for molecular adaptation in vision in 2 lineages of Nearctic fishes that are deep lake specialists: ciscoes and deepwater sculpin. We found depth-related variation in the dim-light visual pigment rhodopsin that evolved convergently in these 2 lineages. In vitro characterization of spectral sensitivity of the convergent deepwater rhodopsin alleles revealed blue-shifts compared with other more widely distributed alleles. These blue-shifted rhodopsin alleles were only observed in deep clear postglacial lakes with underwater visual environments enriched in blue light. This provides evidence of remarkably rapid and convergent visual adaptation and intraspecific functional variation in rhodopsin. Intraspecific functional variation has important implications for conservation, and these fishes are of conservation concern and great cultural, commercial, and nutritional importance to Indigenous communities. We collaborated with the Saugeen Ojibway Nation to develop and test a metabarcoding approach that we show is efficient and accurate in recovering the ecological distribution of functionally relevant variation in rhodopsin. Our approach bridges experimental analyses of protein function and genetics-based tools used in large-scale surveys to better understand the ecological extent of adaptive functional variation.

Aggregation of rhodopsin mutants in mouse models of autosomal dominant retinitis pigmentosa.

Mutations in rhodopsin can cause it to misfold and lead to retinal degeneration. A distinguishing feature of these mutants in vitro is that they mislocalize and aggregate. It is unclear whether or not these features contribute to retinal degeneration observed in vivo. The effect of P23H and G188R misfolding mutations were examined in a heterologous expression system and knockin mouse models, including a mouse model generated here expressing the G188R rhodopsin mutant. In vitro characterizations demonstrate that both mutants aggregate, with the G188R mutant exhibiting a more severe aggregation profile compared to the P23H mutant. The potential for rhodopsin mutants to aggregate in vivo was assessed by PROTEOSTAT, a dye that labels aggregated proteins. Both mutants mislocalize in photoreceptor cells and PROTEOSTAT staining was detected surrounding the nuclei of photoreceptor cells. The G188R mutant promotes a more severe retinal degeneration phenotype and greater PROTEOSTAT staining compared to that promoted by the P23H mutant. Here, we show that the level of PROTEOSTAT positive cells mirrors the progression and level of photoreceptor cell death, which suggests a potential role for rhodopsin aggregation in retinal degeneration.

Scaling up Functional Analyses of the G Protein-Coupled Receptor Rhodopsin.

Eukaryotic cells use G protein-coupled receptors (GPCRs) to convert external stimuli into internal signals to elicit cellular responses. However, how mutations in GPCR-coding genes affect GPCR activation and downstream signaling pathways remain poorly understood. Approaches such as deep mutational scanning show promise in investigations of GPCRs, but a high-throughput method to measure rhodopsin activation has yet to be achieved. Here, we scale up a fluorescent reporter assay in budding yeast that we engineered to study rhodopsin's light-activated signal transduction. Using this approach, we measured the mutational effects of over 1200 individual human rhodopsin mutants, generated by low-frequency random mutagenesis of the GPCR rhodopsin (RHO) gene. Analysis of the data in the context of rhodopsin's three-dimensional structure reveals that transmembrane helices are generally less tolerant to mutations compared to flanking helices that face the lipid bilayer, which suggest that mutational tolerance is contingent on both the local environment surrounding specific residues and the specific position of these residues in the protein structure. Comparison of functional scores from our screen to clinically identified rhodopsin disease variants found many pathogenic mutants to be loss of function. Lastly, functional scores from our assay were consistent with a complex counterion mechanism involved in ligand-binding and rhodopsin activation. Our results demonstrate that deep mutational scanning is possible for rhodopsin activation and can be an effective method for revealing properties of mutational tolerance that may be generalizable to other transmembrane proteins.

Transforming yeast into a facultative photoheterotroph via expression of vacuolar rhodopsin.

Phototrophic metabolism, the capture of light for energy, was a pivotal biological innovation that greatly increased the total energy available to the biosphere. Chlorophyll-based photosynthesis is the most familiar phototrophic metabolism, but retinal-based microbial rhodopsins transduce nearly as much light energy as chlorophyll does,1 via a simpler mechanism, and are found in far more taxonomic groups. Although this system has apparently spread widely via horizontal gene transfer,2,3,4 little is known about how rhodopsin genes (with phylogenetic origins within prokaryotes5,6) are horizontally acquired by eukaryotic cells with complex internal membrane architectures or the conditions under which they provide a fitness advantage. To address this knowledge gap, we sought to determine whether Saccharomyces cerevisiae, a heterotrophic yeast with no known evolutionary history of phototrophy, can function as a facultative photoheterotroph after acquiring a single rhodopsin gene. We inserted a rhodopsin gene from Ustilago maydis,7 which encodes a proton pump localized to the vacuole, an organelle normally acidified via a V-type rotary ATPase, allowing the rhodopsin to supplement heterotrophic metabolism. Probes of the physiology of modified cells show that they can deacidify the cytoplasm using light energy, demonstrating the ability of rhodopsins to ameliorate the effects of starvation and quiescence. Further, we show that yeast-bearing rhodopsins gain a selective advantage when illuminated, proliferating more rapidly than their non-phototrophic ancestor or rhodopsin-bearing yeast cultured in the dark. These results underscore the ease with which rhodopsins may be horizontally transferred even in eukaryotes, providing novel biological function without first requiring evolutionary optimization.

Cation-π Interactions Greatly Influence Ion Transportability of the Light-Driven Sodium Pump KR2: A Molecular Dynamics Study.

Krokinobacter eikastus rhodopsin 2 (KR2) is a typical light-driven sodium pump. Although wild-type KR2 exhibits high Na+ selectivity, mutagenesis performed on the residues constituting the entrance enables permeation of K+ and Cs+, while the underlying mechanism remains elusive. This study presents a comprehensive molecular dynamics investigation, including force field optimization, metadynamics, and alchemical free energy methods, to explore the N61L/G263F mutant of KR2, which exhibits transportability for K+ and Cs+. The introduced Phe263 residue can directly promote ion binding at the entrance through cation-π interactions, while the N61L mutation can enhance ion binding at Phe46 by relieving steric hindrance. These results suggest that cation-π interactions may significantly influence the ion transportability and selectivity of KR2, which can provide important insights for protein engineering and the design of artificial ion transporters.

Hijacking of internal calcium dynamics by intracellularly residing viral rhodopsins.

Rhodopsins are ubiquitous light-driven membrane proteins with diverse functions, including ion transport. Widely distributed, they are also coded in the genomes of giant viruses infecting phytoplankton where their function is not settled. Here, we examine the properties of OLPVR1 (Organic Lake Phycodnavirus Rhodopsin) and two other type 1 viral channelrhodopsins (VCR1s), and demonstrate that VCR1s accumulate exclusively intracellularly, and, upon illumination, induce calcium release from intracellular IP3-dependent stores. In vivo, this light-induced calcium release is sufficient to remote control muscle contraction in VCR1-expressing tadpoles. VCR1s natively confer light-induced Ca2+ release, suggesting a distinct mechanism for reshaping the response to light of virus-infected algae. The ability of VCR1s to photorelease calcium without altering plasma membrane electrical properties marks them as potential precursors for optogenetics tools, with potential applications in basic research and medicine.

Engineering yeast with a light-driven proton pump system in the vacuolar membrane.

BACKGROUND: The supply of ATP is a limiting factor for cellular metabolism. Therefore, cell factories require a sufficient ATP supply to drive metabolism for efficient bioproduction. In the current study, a light-driven proton pump in the vacuolar membrane was constructed in yeast to reduce the ATP consumption required by V-ATPase to maintain the acidification of the vacuoles and increase the intracellular ATP supply for bioproduction. RESULTS: Delta rhodopsin (dR), a microbial light-driven proton-pumping rhodopsin from Haloterrigena turkmenica, was expressed and localized in the vacuolar membrane of Saccharomyces cerevisiae by conjugation with a vacuolar membrane-localized protein. Vacuoles with dR were isolated from S. cerevisiae, and the light-driven proton pumping activity was evaluated based on the pH change outside the vacuoles. A light-induced increase in the intracellular ATP content was observed in yeast harboring vacuoles with dR. CONCLUSIONS: Yeast harboring the light-driven proton pump in the vacuolar membrane developed in this study are a potential optoenergetic cell factory suitable for various bioproduction applications.

Optimizing cryo-EM structural analysis of Gi-coupling receptors via engineered Gt and Nb35 application.

Cryo-EM single particle analysis has recently facilitated the high-resolution structural determination of numerous GPCR-G complexes. Diverse methodologies have been devised with this trend, and in the case of GPCR-Gi complexes, scFv16, an antibody that recognizes the intricate interface of the complex, has been mainly implemented to stabilize the complex. However, owing to their flexibility and heterogeneity, structural determinations of GPCR-Gi complexes remain both challenging and resource-intensive. By employing eGαt, which exhibits binding affinity to modified nanobody Nb35, the cryo-EM structure of Rhodopsin-eGαt complex was previously reported. Using this modified G protein, we determined the structure of the ETB-eGt complex bound to the modified Nb35. The determined structure of ETB receptor was the same as the previously reported ETB-Gi complex, and the resulting dataset demonstrated significantly improved anisotropy. This modified G protein will be utilized for the structural determination of other GPCR-Gi complexes.

A rapid, tag-free way to purify functional GPCRs.

G protein-coupled receptors (GPCRs) play diverse signaling roles and represent major pharmaceutical targets. Consequently, they are the focus of intense study, and numerous advances have been made in their handling and analysis. However, a universal way to purify GPCRs has remained elusive, in part because of their inherent instability when isolated from cells. To address this, we have developed a general, rapid, and tag-free way to purify GPCRs. The method uses short peptide analogs of the Gα subunit C terminus (Gα-CT) that are attached to chromatography beads (Gα-CT resin). Because the Gα-CT peptides bind active GPCRs with high affinity, the Gα-CT resin selectively purifies only active functional receptors. We use this method to purify both rhodopsin and the ß2-adrenergic receptor and show they can be purified in either active conformations or inactive conformations, simply by varying elution conditions. While simple in concept-leveraging the conserved GPCR-Gα-CT binding interaction for the purpose of GPCR purification-we think this approach holds excellent potential to isolate functional receptors for a myriad of uses, from structural biology to proteomics.

Engineering artificial photosynthesis based on rhodopsin for CO2 fixation.

Microbial rhodopsin, a significant contributor to sustaining life through light harvesting, holds untapped potential for carbon fixation. Here, we construct an artificial photosynthesis system which combines the proton-pumping ability of rhodopsin with an extracellular electron uptake mechanism, establishing a pathway to drive photoelectrosynthetic CO2 fixation by Ralstonia eutropha (also known as Cupriavidus necator) H16, a facultatively chemolithoautotrophic soil bacterium. R. eutropha is engineered to heterologously express an extracellular electron transfer pathway of Shewanella oneidensis MR-1 and Gloeobacter rhodopsin (GR). Employing GR and the outer-membrane conduit MtrCAB from S. oneidensis, extracellular electrons and GR-driven proton motive force are integrated into R. eutropha's native electron transport chain (ETC). Inspired by natural photosynthesis, the photoelectrochemical system splits water to supply electrons to R. eutropha via the Mtr outer-membrane route. The light-activated proton pump - GR, supported by canthaxanthin as an antenna, powers ATP synthesis and reverses the ETC to regenerate NADH/NADPH, facilitating R. eutropha's biomass synthesis from CO2. Overexpression of a carbonic anhydrase further enhances CO2 fixation. This artificial photosynthesis system has the potential to advance the development of efficient photosynthesis, redefining our understanding of the ecological role of microbial rhodopsins in nature.

Fluorescence of the Retinal Chromophore in Microbial and Animal Rhodopsins.

Fluorescence of the vast majority of natural opsin-based photoactive proteins is extremely low, in accordance with their functions that depend on efficient transduction of absorbed light energy. However, several recently proposed classes of engineered rhodopsins with enhanced fluorescence, along with the discovery of a new natural highly fluorescent rhodopsin, NeoR, opened a way to exploit these transmembrane proteins as fluorescent sensors and draw more attention to studies on this untypical rhodopsin property. Here, we review the available data on the fluorescence of the retinal chromophore in microbial and animal rhodopsins and their photocycle intermediates, as well as different isomers of the protonated retinal Schiff base in various solvents and the gas phase.

K+-Dependent Photocycle and Photocurrent Reveal the Uptake of K+ in Light-Driven Sodium Pump.

Engineering light-controlled K+ pumps from Na+-pumping rhodopsins (NaR) greatly expands the scope of optogenetic applications. However, the limited knowledge regarding the kinetic and selective mechanism of K+ uptake has significantly impeded the modification and design of light-controlled K+ pumps, as well as their practical applications in various fields, including neuroscience. In this study, we presented K+-dependent photocycle kinetics and photocurrent of a light-driven Na+ pump called Nonlabens dokdonensis rhodopsin 2 (NdR2). As the concentration of K+ increased, we observed the accelerated decay of M intermediate in the wild type (WT) through flash photolysis. In 100 mM KCl, the lifetime of the M decay was approximately 1.0 s, which shortened to around 0.6 s in 1 M KCl. Additionally, the K+-dependent M decay kinetics were also observed in the G263W/N61P mutant, which transports K+. In 100 mM KCl, the lifetime of the M decay was approximately 2.5 s, which shortened to around 0.2 s in 1 M KCl. According to the competitive model, in high KCl, K+ may be taken up from the cytoplasmic surface, competing with Na+ or H+ during M decay. This was further confirmed by the K+-dependent photocurrent of WT liposome. As the concentration of K+ increased to 500 mM, the amplitude of peak current significantly dropped to approximately ~60%. Titration experiments revealed that the ratio of the rate constant of H+ uptake (kH) to that of K+ uptake (kK) is >108. Compared to the WT, the G263W/N61P mutant exhibited a decrease of approximately 40-fold in kH/kK. Previous studies focused on transforming NaR into K+ pumps have primarily targeted the intracellular ion uptake region of Krokinobacter eikastus rhodopsin 2 (KR2) to enhance K+ uptake. However, our results demonstrate that the naturally occurring WT NdR2 is capable of intracellular K+ uptake without requiring structural modifications on the intracellular region. This discovery provides diverse options for future K+ pump designs. Furthermore, we propose a novel photocurrent-based approach to evaluate K+ uptake, which can serve as a reference for similar studies on other ion pumps. In conclusion, our research not only provides new insights into the mechanism of K+ uptake but also offers a valuable point of reference for the development of optogenetic tools and other applications in this field.

Multistep conformational changes leading to the gate opening of light-driven sodium pump rhodopsin.

Membrane transport proteins require a gating mechanism that opens and closes the substrate transport pathway to carry out unidirectional transport. The "gating" involves large conformational changes and is achieved via multistep reactions. However, these elementary steps have not been clarified for most transporters due to the difficulty of detecting the individual steps. Here, we propose these steps for the gate opening of the bacterial Na+ pump rhodopsin, which outwardly pumps Na+ upon illumination. We herein solved an asymmetric dimer structure of Na+ pump rhodopsin from the bacterium Indibacter alkaliphilus. In one protomer, the Arg108 sidechain is oriented toward the protein center and appears to block a Na+ release pathway to the extracellular (EC) medium. In the other protomer, however, this sidechain swings to the EC side and then opens the release pathway. Assuming that the latter protomer mimics the Na+-releasing intermediate, we examined the mechanism for the swing motion of the Arg108 sidechain. On the EC surface of the first protomer, there is a characteristic cluster consisting of Glu10, Glu159, and Arg242 residues connecting three helices. In contrast, this cluster is disrupted in the second protomer. Our experimental results suggested that this disruption is a key process. The cluster disruption induces the outward movement of the Glu159-Arg242 pair and simultaneously rotates the seventh transmembrane helix. This rotation resultantly opens a space for the swing motion of the Arg108 sidechain. Thus, cluster disruption might occur during the photoreaction and then trigger sequential conformation changes leading to the gate-open state.

A mid-infrared laser microscope for the time-resolved study of light-induced protein conformational changes.

We have developed a confocal laser microscope operating in the mid-infrared range for the study of light-sensitive proteins, such as rhodopsins. The microscope features a co-aligned infrared and visible illumination path for the selective excitation and probing of proteins located in the IR focus only. An external-cavity tunable quantum cascade laser provides a wavelength tuning range (5.80-6.35 µm or 1570-1724 cm-1) suitable for studying protein conformational changes as a function of time delay after visible light excitation with a pulsed LED. Using cryogen-free detectors, the relative changes in the infrared absorption of rhodopsin thin films around 10-4 have been observed with a time resolution down to 30 ms. The measured full-width at half maximum of the Airy disk at λ = 6.08 µm in transmission mode with a confocal arrangement of apertures is 6.6 µm or 1.1λ. Dark-adapted sample replacement at the beginning of each photocycle is then enabled by exchanging the illuminated thin-film location with the microscope mapping stage synchronized to data acquisition and LED excitation and by averaging hundreds of time traces acquired in different nearby locations within a homogeneous film area. We demonstrate that this instrument provides crucial advantages for time-resolved IR studies of rhodopsin thin films with a slow photocycle. Time-resolved studies of inhomogeneous samples may also be possible with the presented instrument.

Improved production of ß-carotene in light-powered Escherichia coli by co-expression of Gloeobacter rhodopsin expression.

BACKGROUND: Providing sufficient and usable energy for the cell factory has long been a heated issue in biosynthesis as solar energy has never been rooted out from the strategy for improvement, and turning Escherichia coli (E. coli) into a phototrophic host has multiple captivating qualities for biosynthesis. In this study, ß-carotene was a stable compound for production in E. coli with the expression of four enzymes (CrtE, CrtB, CrtI, CrtY) for production due to its light-harvesting feature as an antenna pigment and as an antioxidant and important precursor for human health. The expression of Gloeobacter rhodopsin (GR) in microbial organisms was proved to have potential for application. RESULTS: The expression of fusion protein, GR-GFP, in E. coli showed visible GFP signal under fluorescent microscopy, and its in vivo proton pumping activity signal can be detected in induced photocurrent by electrodes on the chip under intervals of illumination. To assess the phototrophic synthesis ability of the host strain compared to wild-type and vector control strain in chemostat batch with illumination, the expression of red fluorescent protein (RFP) as a target protein showed its yield improvement in protein assay and also reflected its early dominance in RFP fluorescence signal during the incubation, whereas the synthesis of ß-carotene also showed yield increase by 1.36-fold and 2.32-fold compared with its wildtype and vector control strain. To investigate the effect of GR-GFP on E. coli, the growth of the host showed early rise into the exponential phase compared to the vector control strain and glucose turnover rate was elevated in increased glucose intake rate and upregulation of ATP-related genes in glycolysis (PtsG, Pgk, Pyk). CONCLUSION: We reported the first-time potential application of GR in the form of fusion protein GR-GFP. Expression of GR-GFP in E. coli improved the production of ß-carotene and RFP. Our work provides a strain of E. coli harboring phototrophic metabolism, thus paving path to a more sustainable and scalable production of biosynthesis.