RESUMO
Advancing chloroplast genetic engineering in Chlamydomonas reinhardtii remains challenging, decades after its first successful transformation. This study introduces the development of a chloroplast-optimized mNeonGreen fluorescent reporter, enabling in vivo observation through a sixfold increase in fluorescence via context-aware construct engineering. Our research highlights the influence of transcriptional readthrough and antisense mRNA pairing on post-transcriptional regulation, pointing to novel strategies for optimizing heterologous gene expression. We further demonstrate the applicability of these insights using an accessible experimentation system using glass-bead transformation and reestablishment of photosynthesis using psbH mutants, focusing on the mitigation of transcriptional readthrough effects. By characterizing heterologous expression using regulatory elements such as PrrnS, 5'atpA, and 3' rbcL in a sense-transcriptional context, we further documented up to twofold improvement in fluorescence levels. Our findings contribute new tools for molecular biology research in the chloroplast and evidence fundamental gene regulation processes that could enable the development of more effective chloroplast engineering strategies. This work not only paves the way for more efficient genetic engineering of chloroplasts but also deepens our understanding of the regulatory mechanisms at play.
Assuntos
Chlamydomonas reinhardtii , Cloroplastos , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas , Transcrição Gênica , Genes Reporter , Fotossíntese/genética , RNA Antissenso/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismoRESUMO
The marine environment is a rich reservoir of diverse biological entities, many of which possess unique properties that are of immense value to biotechnological applications. One such example is the red fluorescent protein derived from the coral Discosoma sp. This protein, encoded by the DsRed gene, has been the subject of extensive research due to its potential applications in various fields. In the study, a variant of the red fluorescent protein was generated through random mutagenesis using the DsRed2 gene as a template. The process employed error-prone PCR (epPCR) to introduce random mutations, leading to the isolation of twelve gene variants. Among these, one variant stood out due to its unique spectral properties, exhibiting dual fluorescence emission at both 480 nm (green) and 550 nm (red). This novel variant was expressed in both Escherichia coli and zebrafish (Danio rerio) muscle, confirming the dual fluorescence emission in both model systems. One of the immediate applications of this novel protein variant is in ornamental aquaculture. The dual fluorescence can serve as a unique marker or trait, enhancing the aesthetic appeal of aquatic species in ornamental settings.
Assuntos
Antozoários , Proteína Vermelha Fluorescente , Animais , Fluorescência , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Antozoários/genética , Antozoários/metabolismo , Biotecnologia , Proteínas de Fluorescência VerdeRESUMO
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.
Assuntos
Bactérias , Proteínas Luminescentes/metabolismo , Bactérias/metabolismoRESUMO
Bioluminescence in Diptera is found in the Keroplatidae family, within Arachnocampininae and Keroplatinae subfamilies, with reported occurrences in Oceania, Eurasia, and Americas. Larvae of Orfelia fultoni, which inhabit stream banks in the Appalachian Mountains, emit the bluest bioluminescence among insects, using it for prey attraction, similarly to Arachnocampa spp. Although bioluminescence has a similar prey attraction function, the systems of Arachonocampininae and Keroplatinae subfamilies are morphologically/biochemically distinct, indicating different evolutionary origins. To identify the possible coding genes associated with physiological control, ecological adaptations, and origin/evolution of bioluminescence in the Keroplatinae subfamily, we performed the RNA-Seq analysis of O. fultoni larvae during day and night and compared it with the transcriptomes of Arachnocampa luminosa, and reanalyzed the previously published proteomic data of O. fultoni against the RNA-Seq dataset. The abundance of chaperones/heat-shock and hexamerin gene products at night and in luciferase enriched fractions supports their possible association and participation in bioluminescence. The low diversity of copies/families of opsins indicate a simpler visual system in O. fultoni. Noteworthy, gene products associated with silk protein biosynthesis in Orfelia were more similar to Lepidoptera than to the Arachnocampa, indicating that, similarly to the bioluminescent systems, at some point, the biochemical apparatus for web construction may have evolved independently in Orfelia and Arachnocampa.
Assuntos
Adaptação Fisiológica , Dípteros/genética , Proteínas de Insetos/metabolismo , Luminescência , Proteínas Luminescentes/metabolismo , RNA-Seq/métodos , Transcriptoma/efeitos da radiação , Animais , Dípteros/efeitos da radiação , Ecossistema , Proteínas de Insetos/genética , Luz , Proteínas Luminescentes/genética , Proteoma/análiseRESUMO
Eukaryotic cells are complex systems compartmentalized in membrane-bound organelles. Visualization of organellar electrical activity in living cells requires both a suitable reporter and non-invasive imaging at high spatiotemporal resolution. Here we present hVoSorg, an optical method to monitor changes in the membrane potential of subcellular membranes. This method takes advantage of a FRET pair consisting of a membrane-bound voltage-insensitive fluorescent donor and a non-fluorescent voltage-dependent acceptor that rapidly moves across the membrane in response to changes in polarity. Compared to the currently available techniques, hVoSorg has advantages including simple and precise subcellular targeting, the ability to record from individual organelles, and the potential for optical multiplexing of organellar activity.
Assuntos
Técnicas Biossensoriais , Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Potenciais da Membrana , Microscopia de Fluorescência , Imagem Óptica , Animais , Retículo Endoplasmático/metabolismo , Transferência Ressonante de Energia de Fluorescência , Genes Reporter , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células MCF-7 , Optogenética , Células PC12 , RatosRESUMO
Biotechnology advances have allowed bacteria, yeasts, plants, mammalian and insect cells to function as heterologous protein expression systems. Recently, microalgae have gained attention as an innovative platform for recombinant protein production, due to low culture media cost, compared to traditional systems, as well as the fact that microalgae such as Chlamydomonas reinhardtii are considered safe (GRAS) by the Food and Drug Administration (FDA). Previous studies showed that recombinant protein production in traditional platforms by semicontinuous process increased biomass and bio product productivity, when compared to batch process. As there is a lack of studies on semicontinuous process for recombinant protein production in microalgae, the production of recombinant mCherry fluorescent protein was evaluated by semicontinuous cultivation of Chlamydomonas reinhardtii in bubble column photobioreactor. This semicontinuous cultivation process was evaluated in the following conditions: 20%, 40%, and 60% culture portion withdrawal. The highest culture withdrawal percentage (60%) provided the best results, as an up to 161% increase in mCherry productivity (454.5 RFU h-1 - Relative Fluorescence Unit h-1 ), in comparison to batch cultivation (174.0 RFU h-1 ) of the same strain. All cultivations were carried out for 13 days, at pH 7, temperature 25°C and, by semicontinuous process, two culture withdrawals were taken during the cultivations. Throughout the production cycles, it was possible to obtain biomass concentration up to 1.36 g L-1 .
Assuntos
Técnicas de Cultura de Células/métodos , Chlamydomonas reinhardtii/metabolismo , Meios de Cultura/metabolismo , Substâncias Luminescentes/metabolismo , Proteínas Luminescentes/biossíntese , Fotobiorreatores/normas , Proteínas Recombinantes/biossíntese , Biomassa , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Vermelha FluorescenteRESUMO
The polyphosphoinositides (PPIn) are central regulatory lipids that direct membrane function in eukaryotic cells. Understanding how their synthesis is regulated is crucial to revealing these lipids' role in health and disease. PPIn are derived from the major structural lipid, phosphatidylinositol (PI). However, although the distribution of most PPIn has been characterized, the subcellular localization of PI available for PPIn synthesis is not known. Here, we used several orthogonal approaches to map the subcellular distribution of PI, including localizing exogenous fluorescent PI, as well as detecting lipid conversion products of endogenous PI after acute chemogenetic activation of PI-specific phospholipase and 4-kinase. We report that PI is broadly distributed throughout intracellular membrane compartments. However, there is a surprising lack of PI in the plasma membrane compared with the PPIn. These experiments implicate regulation of PI supply to the plasma membrane, as opposed to regulation of PPIn-kinases, as crucial to the control of PPIn synthesis and function at the PM.
Assuntos
Membrana Celular/metabolismo , Membranas Intracelulares/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Células COS , Chlorocebus aethiops , Diglicerídeos/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
For long time, studies on ectomycorrhiza (ECM) have been limited by inefficient expression of fluorescent proteins (FPs) in the fungal partner. To convert this situation, we have evaluated the basic requirements of FP expression in the model ECM homobasidiomycete Laccaria bicolor and established eGFP and mCherry as functional FP markers. Comparison of intron-containing and intronless FP-expression cassettes confirmed that intron-processing is indispensable for efficient FP expression in Laccaria. Nuclear FP localization was obtained via in-frame fusion of FPs between the intron-containing genomic gene sequences of Laccaria histone H2B, while cytosolic FP expression was produced by incorporating the intron-containing 5' fragment of the glyceraldehyde-3-phosphate dehydrogenase encoding gene. In addition, we have characterized the consensus Kozak sequence of strongly expressed genes in Laccaria and demonstrated its boosting effect on transgene mRNA accumulation. Based on these results, an Agrobacterium-mediated transformation compatible plasmid set was designed for easy use of FPs in Laccaria. The four cloning plasmids presented here allow fast and highly flexible construction of C-terminal in-frame fusions between the sequences of interest and the two FPs, expressed either from the endogenous gene promoter, allowing thus evaluation of the native regulation modes of the gene under study, or alternatively, from the constitutive Agaricus bisporus gpdII promoter for enhanced cellular protein localization assays. The molecular tools described here for cell-biological studies in Laccaria can also be exploited in studies of other biotrophic or saprotrophic basidiomycete species susceptible to genetic transformation.
Assuntos
Proteínas de Fluorescência Verde/genética , Laccaria/genética , Proteínas Luminescentes/genética , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Agrobacterium/genética , Basidiomycota/genética , Núcleo Celular/genética , Citosol/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Histonas/genética , Laccaria/metabolismo , Proteínas Luminescentes/metabolismo , Microrganismos Geneticamente Modificados , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Transformação Genética , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: Marine sessile organisms display a color palette that is the result of the expression of fluorescent and non-fluorescent proteins. Fluorescent proteins have uncovered transcriptional regulation, subcellular localization of proteins, and the fate of cells during development. Chromoproteins have received less attention until recent years as bioreporters. Here, we studied the properties of aeBlue, a a 25.91 kDa protein from the anemone Actinia equina. OBJECTIVE: To assess the properties of aeBlue chromoprotein under different physicochemical conditions. METHODS: In this article, during the purification of aeBlue we uncovered that it suffered a color shift when frozen. We studied the color shift by different temperature incubation and physicochemical conditions and light spectroscopy. To assess the possible structural changes in the protein, circular dichroism analysis, size exclusion chromatography and native PAGE was performed. RESULTS: We uncover that aeBlue chromoprotein, when expressed from a synthetic construct in Escherichia coli, showed a temperature dependent color shift. Protein purified at 4 °C by metal affinity chromatography exhibited a pinkish color and shifts back at higher temperatures to its intense blue color. Circular dichroism analysis revealed that the structure in the pink form of the protein has reduced secondary structure at 4 °C, but at 35 °C and higher, the structure shifts to a native conformation and Far UV- vis CD spectra revealed the shift in an aromatic residue of the chromophore. Also, the chromophore retains its properties in a wide range of conditions (pH, denaturants, reducing and oxidants agents). Quaternary structure is also maintained as a tetrameric conformation as shown by native gel and size exclusion chromatography. CONCLUSION: Our results suggest that the chromophore position in aeBlue is shifted from its native position rendering the pink color and the process to return it to its native blue conformation is temperature dependent.
Assuntos
Corantes/química , Proteínas Luminescentes/química , Pigmentos Biológicos/química , Proteínas/química , Anêmonas-do-Mar/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cor , Corantes/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Luz , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Oxirredução , Pigmentos Biológicos/metabolismo , Conformação Proteica , Desnaturação Proteica , Proteínas/metabolismo , Espectrofotometria , TemperaturaRESUMO
How the unique luciferase of Phrixothrix hirtus (PxRE) railroad worm catalyzes the emission of red bioluminescence using the same luciferin of fireflies, remains a mystery. Although PxRE luciferase is a very attractive tool for bioanalysis and bioimaging in hemoglobin rich tissues, it displays lower quantum yield (15%) when compared to green emitting luciferases (>40%). To identify which parts of PxRE luciferin binding site (LBS) determine bioluminescence color, and to develop brighter and more red-shifted emitting luciferases, we compared the effects of site-directed mutagenesis and of larger 6'-substituted aminoluciferin analogues (6'-morpholino- and 6'-pyrrolidinyl-LH) on the bioluminescence properties of PxRE and green-yellow emitting beetle luciferases. The effects of mutations in the benzothiazolyl and thiazolyl parts of PxRE LBS on the KM and catalytic efficiencies, indicated their importance for luciferin binding and catalysis. However, the absence of effects on the bioluminescence spectrum indicated a less interactive LBS in PxRE during light emission. Mutations at the bottom of LBS of PxRE blue-shifted the spectra and increased catalytic efficiency, suggesting that lack of interactions of this part of LBS with excited oxyluciferin phenolate underlie red light emission. The much higher bioluminescence activity and red-shifted spectra of PxRE luciferase with 6'-morpholino- (634 nm) and 6'-pyrrolidinyl-luciferins (644 nm), when compared to other beetle luciferases, revealed a larger luciferin phenolate binding pocket. The size and orientation of the side-chains of L/I/H348 are critical for amino-analogues accommodation and modulate bioluminescence color, affecting the interactions and mobility of excited oxyluciferin phenolate. The PxRE luciferase and 6'-aminoluciferins provide potential far-red combinations for bioimaging applications.