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Microalgae are increasingly playing a significant role in many areas of research and development. Recent studies have demonstrated their ability to aid wound healing by their ability to generate oxygen, aiding the healing process. Bearing this in mind, the capability to spray/spin deposit microalgae in suspension (solution) or compartmentalize living microalgae within architectures such as fibers/scaffolds and beads, would have significance as healing mechanisms for addressing a wide range of wounds. Reconstructing microalgae-bearing architectures as either scaffolds or beads could be generated via electric field (bio-electrospraying and cell electrospinning) and non-electric field (aerodynamically assisted bio-jetting/threading) driven technologies. However, before studying the biomechanical properties of the generated living architectures, the microalgae exposed to these techniques must be interrogated from a molecular level upward first, to establish these techniques, have no negative effects brought on the processed microalgae. Therefore these studies, demonstrate the ability of both these jetting and threading technologies to directly handle living microalgae, in suspension or within a polymeric suspension, safely, and form algae-bearing architectures such as beads and fibers/scaffolds.
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Viral infection of farmed fish and shellfish represents a major issue within the aquaculture industry. One potential control strategy involves RNA interference of viral gene expression through the oral delivery of specific double-stranded RNA (dsRNA). In previous work, we have shown that recombinant dsRNA can be produced in the chloroplast of the edible microalga Chlamydomonas reinhardtii and used to control disease in shrimp. Here, we report a significant improvement in antiviral dsRNA production and its use to protect shrimp against white spot syndrome virus (WSSV). A new strategy for dsRNA synthesis was developed that uses two convergent copies of the endogenous rrnS promoter to drive high-level transcription of both strands of the WSSV gene element in the chloroplast. Quantitative RT-PCR indicated that ~119 ng dsRNA was produced per liter of culture of the transgenic microalga. This represents an ~10-fold increase in dsRNA relative to our previous report. The engineered alga was assessed for its ability to prevent WSSV infection when fed to shrimp larvae prior to a challenge with the virus. The survival of shrimp given feed supplemented with dried alga containing the dsRNA was significantly enhanced (~69% survival) relative to a negative control (<10% survival). The findings suggest that this new dsRNA production platform could be employed as a low-cost, low-tech control method for aquaculture.
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'Marker-free' strategies for creating transgenic microorganisms avoid the issue of potential transmission of antibiotic resistance genes to other microorganisms. An already-established strategy for engineering the chloroplast genome (=plastome) of the green microalga Chlamydomonas reinhardtii involves the restoration of photosynthetic function using a recipient strain carrying a plastome mutation in a key photosynthesis gene. Selection for transformant colonies is carried out on minimal media, such that only those cells in which the mutated gene has been replaced with a wild-type copy carried on the transgenic DNA are capable of phototrophic growth. However, this approach can suffer from issues of efficiency due to the slow growth of C. reinhardtii on minimal media and the slow die-back of the untransformed lawn of cells when using mutant strains with a limited photosensitivity phenotype. Furthermore, such phototrophic rescue has tended to rely on existing mutants that are not necessarily ideal for transformation and targeted transgene insertion: Mutants carrying point mutations can easily revert, and those with deletions that do not extend to the intended transgene insertion site can give rise to a sub-population of rescued lines that lack the transgene. In order to improve and accelerate the transformation pipeline for C. reinhardtii, we have created a novel recipient line, HNT6, carrying an engineered deletion in exon 3 of psaA, which encodes one of the core subunits of photosystem I (PSI). Such PSI mutants are highly light-sensitive allowing faster recovery of transformant colonies by selecting for light-tolerance on acetate-containing media, rather than phototrophic growth on minimal media. The deletion extends to a site upstream of psaA-3 that serves as a neutral locus for transgene insertion, thereby ensuring that all of the recovered colonies are transformants containing the transgene. We demonstrate the application of HNT6 using a luciferase reporter.
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Polyethylene terephthalate hydrolases (PETases) are a newly discovered and industrially important class of enzymes that catalyze the enzymatic degradation of polyethylene terephatalate (PET), one of the most abundant plastics in the world. The greater enzymatic efficiencies of PETases compared to close relatives from the cutinase and lipase families have resulted in increasing research interest. Despite this, further characterization of PETases is essential, particularly regarding their possible activity against other kinds of plastic. In this study, we exploited for the first time the use of the microalgal chloroplast for more sustainable synthesis of a PETase enzyme. A photosynthetic-restoration strategy was used to generate a marker-free transformant line of the green microalga Chlamydomonas reinhardtii in which the PETase from Ideonella sakaiensis was constitutively expressed in the chloroplast. Subsequently, the activity of the PETase against both PET and post-consumer plastics was investigated via atomic force microscopy, revealing evidence of degradation of the plastics.
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Chlamydomonas reinhardtii , Microalgas , Humanos , Microalgas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Plásticos , Hidrolases/metabolismo , Polietilenotereftalatos , Cloroplastos/metabolismoRESUMO
Microalgae are promising host organisms for the production of encapsulated recombinant proteins such as vaccines. However, bottlenecks in bioprocess development, such as the drying stage, need to be addressed to ensure feasibility at scale. In this study, we investigated the potential of spray drying to produce a recombinant vaccine in microalgae. A transformant line of Chlamydomonas reinhardtii carrying a subunit vaccine against salmonid alphavirus was created via chloroplast engineering. The integrity of the recombinant protein after spray drying and its stability after 27 months storage at -80 °C, +4 °C and room temperature were assessed by immunoblotting. The protein withstood spray drying without significant losses. Long-term storage at +4 °C and room temperature resulted in 50% and 92% degradation, respectively. Optimizing spray drying and storage conditions should minimize degradation and favour short-term storage at positive temperatures. Using data on yield and productivity, the economics of spray drying- and freeze drying-based bioprocesses were compared. The drying stage corresponded to 41% of the total production cost. Process optimization, genetic engineering and new market strategies were identified as potential targets for cost reduction. Overall, this study successfully demonstrates the suitability of spray drying as a process option for recombinant protein production in microalgae at the industrial scale.
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Terpenoids are a diverse class of naturally occurring compounds consisting of more than 50,000 structurally different molecules and are found in all living organisms. Many terpenoid compounds, in particular those isolated from plants, have applications in various commercial sectors including medicine, agriculture and cosmetics. However, these high value terpenoids are produced in relatively small quantities in their natural hosts and their chemical synthesis for large scale production is costly and complicated. Therefore, there is much focus on producing these compounds in novel biological hosts using metabolic engineering technologies. As a photosynthetic system, the unicellular green alga C. reinhardtii is of particular interest as the most well-studied model alga with well-established molecular tools for genetic manipulation. However, the direct manipulation of terpenoid biosynthetic pathways in C. reinhardtii necessitates a thorough understanding of the basic terpenoid metabolism. To gain a better understanding of the methylerythritol phosphate (MEP) pathway that leads to terpenoid biosynthesis in the chloroplast of C. reinhardtii, hence this study has investigated the effect of over-expressing 1-deoxy-d-xylulose-5-phosphate synthase (DXS) on plastidic downstream terpenoids. We produced marker-free chloroplast transformants of C. reinhardtii lines that express an additional cyanobacterial gene for DXS. The analysis of terpenoid content for the transgenic line demonstrates that overexpressing DXS resulted in a two-fold decrease in the chlorophyll levels while carotenoid levels showed variable changes: zeaxanthin and antherxanthin levels increased several-fold, lutein levels dropped to approximately half, but ß-carotene and violaxanthin did not show a significant change.
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Acid mine drainage (AMD) is a global issue and causes harmful environmental impacts. AMD has high acidity and contains a high concentration of heavy metals and metalloids, making it toxic to plants, animals, and humans. Traditional treatments for AMD have been widely used for a long time. Nevertheless, some limitations, such as low efficacy and secondary contamination, have led them to be replaced by other methods such as bio-based AMD treatments. This study reviewed three bio-based treatment methods using algae, biochar, and bacteria that can be used separately and potentially in combination for effective and sustainable AMD treatment to identify the removal mechanisms and essential parameters affecting AMD treatment. All bio-based methods, when applied as a single process and in combination (e.g. algae-biochar and algae-bacteria), were identified as effective treatments for AMD. Also, all these bio-based methods were found to be affected by some parameters (e.g. pH, temperature, biomass concentration and initial metal concentration) when removing heavy metals from AMD. However, we did not identify any research focusing on the combination of algae-biochar-bacteria as a consortium for AMD treatment. Therefore, due to the excellent performance in AMD treatment of algae, biochar and bacteria and the potential synergism among them, this review provides new insight and discusses the feasibility of a combination of algae-biochar-bacteria for AMD treatment.
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Metais Pesados , Mineração , Ácidos , Animais , Bactérias , Carvão VegetalRESUMO
The chloroplast represents an attractive compartment for light-driven biosynthesis of recombinant products, and advanced synthetic biology tools are available for engineering the chloroplast genome ( = plastome) of several algal and plant species. However, producing commercial lines will likely require several plastome manipulations. This presents issues with respect to selectable markers, since there are a limited number available, they can be used only once in a serial engineering strategy, and it is undesirable to retain marker genes for antibiotic resistance in the final transplastome. To address these problems, we have designed a rapid iterative selection system, known as CpPosNeg, for the green microalga Chlamydomonas reinhardtii that allows creation of marker-free transformants starting from wild-type strains. The system employs a dual marker encoding a fusion protein of E. coli aminoglycoside adenyltransferase (AadA: conferring spectinomycin resistance) and a variant of E. coli cytosine deaminase (CodA: conferring sensitivity to 5-fluorocytosine). Initial selection on spectinomycin allows stable transformants to be established and driven to homoplasmy. Subsequent selection on 5-fluorocytosine results in rapid loss of the dual marker through intramolecular recombination between the 3'UTR of the marker and the 3'UTR of the introduced transgene. We demonstrate the versatility of the CpPosNeg system by serial introduction of reporter genes into the plastome.
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Chlamydomonas reinhardtii , Chlamydomonas , Regiões 3' não Traduzidas , Aminoglicosídeos , Biomarcadores/metabolismo , Chlamydomonas/genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Escherichia coli/genética , Flucitosina/metabolismo , Espectinomicina/metabolismo , Transformação GenéticaRESUMO
Establishing the first human presence on Mars will be the most technically challenging undertaking yet in the exploration beyond our planet. The remoteness of Mars from Earth, the inhospitable surface conditions including low atmospheric pressure and cold temperatures, and the need for basic resources including water, pose a formidable challenge to this endeavour. The intersection of multiple disciplines will be required to provide solutions for temporary and eventually permanent Martian habitation. This review considers the role cyanobacteria and eukaryotic microalgae (collectively referred to here as 'microalgae') may have in supporting missions to the red planet. The current research using these microorganisms in biological life support systems is discussed, with a systematic analysis of their usage in each system conducted. The potential of microalgae to provide astronauts with oxygen, food, bio-polymers and pharmaceuticals is considered. An overview of microalgal experiments in space missions across the last 60 years is presented, and the research exploring the technical challenges of cultivation on Mars is discussed. From these findings, an argument for culturing microalgae in subterranean bioreactors is proposed. Finally, future synthetic biology approaches for enhancing the cyanobacterial/microalgal role in supporting human deep-space exploration are presented. We show that microalgae hold significant promise for providing solutions to many problems faced by the first Martian settlers, however these can only be realised with significant infrastructure and a reliable power source.
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Cianobactérias , Marte , Microalgas , Voo Espacial , Meio Ambiente Extraterreno , HumanosRESUMO
Sustainable and economically viable support for an ever-increasing global population requires a paradigm shift in agricultural productivity, including the application of biotechnology to generate future crop plants. Current genetic engineering approaches aimed at enhancing the photosynthetic efficiency or composition of the harvested tissues involve relatively simple manipulations of endogenous metabolism. However, radical rewiring of central metabolism using new-to-nature pathways, so-called "synthetic metabolism", may be needed to really bring about significant step changes. In many cases, this will require re-programming the metabolism of the chloroplast, or other plastids in non-green tissues, through a combination of chloroplast and nuclear engineering. However, current technologies for sophisticated chloroplast engineering ("transplastomics") of plants are limited to just a handful of species. Moreover, the testing of metabolic rewiring in the chloroplast of plant models is often impractical given their obligate phototrophy, the extended time needed to create stable non-chimeric transplastomic lines, and the technical challenges associated with regeneration of whole plants. In contrast, the unicellular green alga, Chlamydomonas reinhardtii is a facultative heterotroph that allows for extensive modification of chloroplast function, including non-photosynthetic designs. Moreover, chloroplast engineering in C. reinhardtii is facile, with the ability to generate novel lines in a matter of weeks, and a well-defined molecular toolbox allows for rapid iterations of the "Design-Build-Test-Learn" (DBTL) cycle of modern synthetic biology approaches. The recent development of combinatorial DNA assembly pipelines for designing and building transgene clusters, simple methods for marker-free delivery of these clusters into the chloroplast genome, and the pre-existing wealth of knowledge regarding chloroplast gene expression and regulation in C. reinhardtii further adds to the versatility of transplastomics using this organism. Herein, we review the inherent advantages of the algal chloroplast as a simple and tractable testbed for metabolic engineering designs, which could then be implemented in higher plants.
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Eukaryotic organisms such as plants are unable to utilise nitrogen gas (N2) directly as a source of this essential element and are dependent either on its biological conversion to ammonium by diazotrophic prokaryotes, or its supply as chemically synthesised nitrate fertiliser. The idea of genetically engineering crops with the capacity to fix N2 by introduction of the bacterial nitrogenase enzyme has long been discussed. However, the expression of an active nitrogenase must overcome several major challenges: the coordinated expression of multiple genes to assemble an enzyme complex containing several different metal cluster co-factors; the supply of sufficient ATP and reductant to the enzyme; the enzyme's sensitivity to oxygen; and the intracellular accumulation of ammonium. The chloroplast of plant cells represents an attractive location for nitrogenase expression, but engineering the organelle's genome is not yet feasible in most crop species. However, the unicellular green alga Chlamydomonas reinhardtii represents a simple model for photosynthetic eukaryotes with a genetically tractable chloroplast. In this review, we discuss the main advantages, and limitations, of this microalga as a testbed for producing such a complex multi-subunit enzyme. Furthermore, we suggest that a minimal set of six transgenes are necessary for chloroplast-localised synthesis of an 'Fe-only' nitrogenase, and from this set we demonstrate the stable expression and accumulation of the homocitrate synthase, NifV, under aerobic conditions. Arguably, further studies in C. reinhardtii aimed at testing expression and function of the full gene set would provide the groundwork for a concerted future effort to create nitrogen-fixing crops.
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Chlamydomonas reinhardtii/crescimento & desenvolvimento , Cloroplastos/metabolismo , Engenharia Genética/métodos , Nitrogenase/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Genoma de Cloroplastos , Fixação de Nitrogênio , Nitrogenase/metabolismo , Fotossíntese , Biologia SintéticaRESUMO
The application of microfluidic technologies to microalgal research is particularly appealing since these approaches allow the precise control of the extracellular environment and offer a high-throughput approach to studying dynamic cellular processes. To expand the portfolio of applications, here we present a droplet-based microfluidic method for analysis and screening of Phaeodactylum tricornutum and Nannochloropsis gaditana, which can be integrated into a genetic transformation workflow. Following encapsulation of single cells in picolitre-sized droplets, fluorescence signals arising from each cell can be used to assess its phenotypic state. In this work, the chlorophyll fluorescence intensity of each cell was quantified and used to identify populations of P. tricornutum cells grown in different light conditions. Further, individual P. tricornutum or N. gaditana cells engineered to express green fluorescent protein were distinguished and sorted from wild-type cells. This has been exploited as a rapid screen for transformed cells within a population, bypassing a major bottleneck in algal transformation workflows and offering an alternative strategy for the identification of genetically modified strains.
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The availability of routine methods for the genetic engineering of the chloroplast genome of Chlamydomonas reinhardtii is allowing researchers to explore the use of this microalga as a phototrophic cell platform for synthesis of high value recombinant proteins and metabolites. However, the established method for delivering transforming DNA into the algal chloroplast involves microparticle bombardment using an expensive "gene gun". Furthermore, selection of transformant lines most commonly involves the use of a bacterial antibiotic resistance gene. In this chapter, we describe a simple and cheap delivery method in which cell-DNA suspensions are agitated with glass beads: a method that is more commonly used for nuclear transformation of Chlamydomonas. We also describe the use of plasmid expression vectors that target transgenes to a neutral site within the chloroplast genome between psbH and trnE2, and employ psbH as the selectable marker-thereby avoiding issues of unwanted antibiotic resistance genes in the resulting transgenic lines. Finally, we highlight a feature in our latest vectors in which the presence of a novel tRNA gene on the plasmid results in recognition within the chloroplast of UGA stop codons in transgenes as tryptophan codons. This feature simplifies the cloning of transgenes that are normally toxic to E. coli, serves as a biocontainment strategy restricting the functional escape of transgenes from the algal chloroplast to environmental microorganisms, and offers a simple system of temperature-regulated translation of transgenes.
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Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Engenharia Genética/métodos , Plantas Geneticamente Modificadas/genética , Transformação Genética , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Vetores Genéticos , Genoma de Cloroplastos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , TransgenesRESUMO
The chloroplast of microalgae such as Chlamydomonas reinhardtii represents an attractive chassis for light-driven production of novel recombinant proteins and metabolites. Methods for the introduction and expression of transgenes in the chloroplast genome (=plastome) of C. reinhardtii are well-established and over 100 different proteins have been successfully produced. However, in almost all reported cases the complexity of the genetic engineering is low, and typically involves introduction into the plastome of just a single transgene together with a selectable marker. In order to exploit fully the potential of the algal chassis it is necessary to establish methods for multigenic engineering in which many transgenes can be stably incorporated into the plastome. This would allow the synthesis of multi-subunit proteins and the introduction into the chloroplast of whole new metabolic pathways. In this short communication we report a proof-of-concept study involving both a combinatorial and serial approach, with the goal of synthesizing five different test proteins in the C. reinhardtii chloroplast. Analysis of the various transgenic lines confirmed the successful integration of the transgenes and accumulation of the gene products. However, the work also highlights an issue of genetic instability when using the same untranslated region for each of the transgenes. Our findings therefore help to define appropriate strategies for robust multigenic engineering of the algal chloroplast.
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Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Genoma de Cloroplastos , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Engenharia Genética , Redes e Vias Metabólicas , Microalgas/genética , Microalgas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Edible microalgae have potential as low-cost cell factories for the production and oral delivery of recombinant proteins such as vaccines, anti-bacterials and gut-active enzymes that are beneficial to farmed animals including livestock, poultry and fish. However, a major economic and technical problem associated with large-scale cultivation of microalgae, even in closed photobioreactors, is invasion by contaminating microorganisms. Avoiding this requires costly media sterilisation, aseptic techniques during set-up and implementation of 'crop-protection' strategies during cultivation. Here, we report a strain improvement approach in which the chloroplast of Chlamydomonas reinhardtii is engineered to allow oxidation of phosphite to its bio-available form: phosphate. We have designed a synthetic version of the bacterial gene (ptxD)-encoding phosphite oxidoreductase such that it is highly expressed in the chloroplast but has a TrpâOpal codon reassignment for bio-containment of the transgene. Under mixotrophic conditions, the growth rate of the engineered alga is unaffected when phosphate is replaced with phosphite in the medium. Furthermore, under non-sterile conditions, growth of contaminating microorganisms is severely impeded in phosphite medium. This, therefore, offers the possibility of producing algal biomass under non-sterile conditions. The ptxD gene can also serve as a dominant marker for genetic engineering of any C. reinhardtii strain, thereby avoiding the use of antibiotic resistance genes as markers and allowing the 'retro-fitting' of existing engineered strains. As a proof of concept, we demonstrate the application of our ptxD technology to a strain expressing a subunit vaccine targeting a major viral pathogen of farmed fish.
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Biotecnologia/métodos , Chlamydomonas reinhardtii/enzimologia , Cloroplastos/enzimologia , Oxirredutases/metabolismo , Fosfatos/metabolismo , Fosfitos/metabolismo , Proteínas Recombinantes/metabolismo , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Meios de Cultura/química , Descontaminação/métodos , Engenharia Metabólica/métodos , Oxirredutases/genética , Proteínas Recombinantes/genéticaRESUMO
The green microalga Chlamydomonas reinhardtii is under development as a production host for recombinant proteins and whole-cell therapeutics. In particular, the cell wall-reduced strain TN72 is used as a model organism for protein expression and algal synthetic biology. However, the bioprocessing characteristics of TN72 and other C. reinhardtii strains have yet to be examined. Here we use a TN72 strain expressing a protein-based antibiotic (Pal) to study the scale-up of cell harvest and product recovery. Cell harvest was examined with 100L cultures in two intermittent-discharge continuous-flow disc-stack centrifuges at flow rates of 150-250 L.h-1, as well as with an ultra scale-down (USD) mimic of the centrifuges. Solids recovery exceeded 99.5% and the loss of product to the supernatant was below 2-3%. TN72 is intact following the high shear conditions of the feed zone, however discharge from both disc-stack centrifuges resulted in full cell breakage and in the case of Pal, partial degradation in the subsequent hours. We demonstrated that shake flask cultivation and the USD centrifuge technique can be used to predict the pilot-scale clarification efficiency and product release at the centrifuge inlet for TN72, but not the cell breakage on discharge. This study outlines a number of challenges for scale-up of recombinant protein production in the microalgal host in particular for whole cell therapeutics, but also opportunities for the bioprocessing of intracellular products from TN72.
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Coral reefs are some of the most important and ecologically diverse marine environments. At the base of the reef ecosystem are dinoflagellate algae, which live symbiotically within coral cells. Efforts to understand the relationship between alga and coral have been greatly hampered by the lack of an appropriate dinoflagellate genetic transformation technology. By making use of the plasmid-like fragmented chloroplast genome, we have introduced novel genetic material into the dinoflagellate chloroplast genome. We have shown that the introduced genes are expressed and confer the expected phenotypes. Genetically modified cultures have been grown for 1 year with subculturing, maintaining the introduced genes and phenotypes. This indicates that cells continue to divide after transformation and that the transformation is stable. This is the first report of stable chloroplast transformation in dinoflagellate algae.
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Cloroplastos/genética , Dinoflagellida/genética , Genética Microbiana/métodos , Transformação Genética , Dinoflagellida/crescimento & desenvolvimento , Expressão Gênica , Instabilidade Genômica , FenótipoRESUMO
BACKGROUND: The chloroplast of eukaryotic microalgae such as Chlamydomonas reinhardtii is a potential platform for metabolic engineering and the production of recombinant proteins. In industrial biotechnology, inducible expression is often used so that the translation or function of the heterologous protein does not interfere with biomass accumulation during the growth stage. However, the existing systems used in bacterial or fungal platforms do not transfer well to the microalgal chloroplast. We sought to develop a simple inducible expression system for the microalgal chloroplast, exploiting an unused stop codon (TGA) in the plastid genome. We have previously shown that this codon can be translated as tryptophan when we introduce into the chloroplast genome a trnWUCA gene encoding a plastidial transfer RNA with a modified anticodon sequence, UCA. RESULTS: A mutated version of our trnWUCA gene was developed that encodes a temperature-sensitive variant of the tRNA. This allows transgenes that have been modified to contain one or more internal TGA codons to be translated differentially according to the culture temperature, with a gradient of recombinant protein accumulation from 35 °C (low/off) to 15 °C (high). We have named this the CITRIC system, an acronym for cold-inducible translational readthrough in chloroplasts. The exact induction behaviour can be tailored by altering the number of TGA codons within the transgene. CONCLUSIONS: CITRIC adds to the suite of genetic engineering tools available for the microalgal chloroplast, allowing a greater degree of control over the timing of heterologous protein expression. It could also be used as a heat-repressible system for studying the function of essential native genes in the chloroplast. The genetic components of CITRIC are entirely plastid-based, so no engineering of the nuclear genome is required.
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Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Temperatura Baixa , Biossíntese de Proteínas , RNA de Transferência/metabolismo , Proteínas de Algas/metabolismo , Sequência de Bases , Chlamydomonas reinhardtii/genética , Códon/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas/genética , TransgenesRESUMO
Chlamydomonas reinhardtii is a model alga of increasing interest as a cell factory for the production of valuable compounds, including therapeutic proteins and bioactive metabolites. Expression of foreign genes in the chloroplast is particularly advantageous as: (i) accumulation of product in this sub-cellular compartment minimises potential toxicity to the rest of the cell; (ii) genes can integrate at specific loci of the chloroplast genome (plastome) by homologous recombination; (iii) the high ploidy of the plastome and the high-level expression of chloroplast genes can be exploited to achieve levels of recombinant protein as high as 5% total cell protein; (iv) the lack of any gene silencing mechanisms in the chloroplast ensures stable expression of transgenes. However, the generation of C. reinhardtii chloroplast transformants requires efficient methods of selection, and ideally methods for subsequent marker removal. Additionally, the use of reporter genes is critical to achieving a comprehensive understanding of gene expression, thereby informing experimental design for recombinant applications. This review discusses currently available selection and reporter systems for chloroplast engineering in C. reinhardtii, as well as those used for chloroplast engineering in higher plants and other microalgae, and looks to the future in terms of possible new markers and reporters that will further advance the C. reinhardtii chloroplast as an expression platform.