Selectable Markers and Reporter Genes for Engineering the Chloroplast of Chlamydomonas reinhardtii (original) (raw)
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Production of therapeutic proteins in the chloroplast of Chlamydomonas reinhardtii
AMB Express, 2014
Chloroplast transformation in the photosynthetic alga Chlamydomonas reinhardtii has been used to explore the potential to use it as an inexpensive and easily scalable system for the production of therapeutic recombinant proteins. Diverse proteins, such as bacterial and viral antigens, antibodies and, immunotoxins have been successfully expressed in the chloroplast using endogenous and chimeric promoter sequences. In some cases, proteins have accumulated to high level, demonstrating that this technology could compete with current production platforms. This review focuses on the works that have engineered the chloroplast of C. reinhardtii with the aim of producing recombinant proteins intended for therapeutical use in humans or animals.
Multigenic engineering of the chloroplast genome in the green alga Chlamydomonas reinhardtii
Microbiology, 2020
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...
Molecular and General Genetics MGG, 2000
Reverse-genetic studies of chloroplast genes in the green alga Chlamydomonas reinhardtii have been hampered by the paucity of suitable selectable markers for chloroplast transformation. We have constructed a series of vectors for the targeted insertion and expression of foreign genes in the Chlamydomonas chloroplast genome. Using these vectors we have developed a novel selectable marker based on the bacterial gene aphA-6, which encodes an aminoglycoside phosphotransferase. The aphA-6 marker allows direct selection for transformants on medium containing either kanamycin or amikacin. The marker can be used to inactivate or modify speci®c chloroplast genes, and can be used as a reporter of gene expression. The availability of this marker now makes possible the serial transformation of the chloroplast genome of Chlamydomonas.
Production of Recombinant Proteins in the Chloroplast of the Green Alga Chlamydomonas reinhardtii
Methods in Molecular Biology, 2016
Chloroplast transformation in the green algae Chlamydomonas reinhardtii can be used for the production of valuable recombinant proteins. Here, we describe chloroplast transformation of C. reinhardtii followed by protein detection. Genes of interest integrate stably by homologous recombination into the chloroplast genome following introduction by particle bombardment. Genes are inherited and expressed in lines recovered after selection in the presence of an antibiotic. Recombinant proteins can be detected by conventional techniques like immunoblotting and purifi ed from liquid cultures.
Biotechnology and Genetic Engineering Reviews, 2016
Microalgae, also called microphytes, are a vast group of microscopic photosynthetic organisms living in aquatic ecosystems. Microalgae have attracted the attention of biotechnology industry as a platform for extracting natural products with high commercial value. During last decades, microalgae have been also used as cost-effective and easily scalable platform for the production of recombinant proteins with medical and industrial applications. Most progress in this field has been made with Chlamydomonas reinhardtii as a model organism mainly because of its simple life cycle, well-established genetics and ease of cultivation. However, due to the scarcity of existing infrastructure for commercial production and processing together with relatively low product yields, no recombinant products from C. reinhardtii have gained approval for commercial production and most of them are still in research and development. In this review, we focus on the chloroplast of C. reinhardtii as an algal recombinant expression platform and compare its advantages and disadvantages to other currently used expression systems. We then discuss the strategies for engineering the chloroplast of C. reinhardtii to produce recombinant cells and present a comprehensive overview of works that have used this platform for the expression of high-value products.
The algal chloroplast as a synthetic biology platform for production of therapeutic proteins
Microbiology, 2018
The chloroplast of Chlamydomonas reinhardtii and other microalgae represents an attractive new platform for the synthesis of recombinant therapeutics using synthetic biology (synbio) approaches. Transgenes can be designed in silico, assembled from validated DNA parts, and inserted at precise and predetermined locations within the chloroplast genome to give stable synthesis of a desired recombinant protein. Numerous recent examples of different therapeutic proteins produced successfully in the C. reinhardtii chloroplast highlight the potential of this green alga as a simple, low-cost and benign host. Furthermore, features of the alga may offer additional advantages over more-established microbial, mammalian or plant-based systems. These include efficient folding and accumulation of the product in the chloroplast; a lack of contaminating toxins or infectious agents; reduced downstream processing requirements; the possibility to make complex therapeutics such as immunotoxins, and the opportunity to use the whole alga as a low-cost oral vaccine. In this article we review the current status of algal chloroplast engineering with respect to therapeutic proteins. We also consider future advances in synbio tools, together with improvements to recipient strains, which will allow the design of bespoke strains with high levels of productivity.
Plant Molecular Biology, 2018
Key message Two intercistronic regions were identified as functional intercistronic expression elements (IEE) for the simultaneous expression of aphA-6 and gfp in a synthetic operon in the chloroplast of C. reinhardtii. Abstract Chlamydomonas reinhardtii, a biflagellate photosynthetic microalga, has been widely used in basic and applied science. Already three decades ago, Chlamydomonas had its chloroplast genome transformed and to this day constitutes the only alga routinely used in transplastomic technology. Despite the fact that over a 100 foreign genes have been expressed from the chloroplast genome, little has been done to address the challenge of expressing multiple genes in the form of operons, a development that is needed and crucial to push forward metabolic engineering and synthetic biology in this organism. Here, we studied five intercistronic regions and investigated if they can be used as intercistronic expression elements (IEE) in synthetic operons to drive the expression of foreign genes in the chloroplast of C. reinhardtii. The intercistronic regions were those from the psbB-psbT, psbN-psbH, psaC-petL, petL-trnN and tscA-chlN chloroplast operons, and the foreign genes were the aminoglycoside 3′-phosphotransferase (aphA-6), which confers resistance to kanamycin, and the green fluorescent protein gene (gfp). While all the intercistronic regions yielded lines that were resistant to kanamycin, only two (obtained with intercistronic regions from psbN-psbH and tscA-chlN) were identified as functional IEEs, yielding lines in which the second cistron (gfp) was translated and generated GFP. The IEEs we have identified could be useful for the stacking of genes for metabolic engineering or synthetic biology circuits in the chloroplast of C. reinhardtii.
Genetic Engineering of Algal Chloroplasts: Progress and Prospects
Физиология растений, 2013
The last few years has seen an ever increasing interest in the exploitation of microalgae as recom binant platforms for the synthesis of novel bioproducts. These could be biofuel molecules, speciality enzymes, nutraceuticals, or therapeutic proteins, such as antibodies, hormones, and vaccines. This exploitation requires the development of new genetic engineering technologies for those fast growing, robust species suited for intensive commercial cultivation in bioreactor systems. In particular, there is a need for routine methods for the genetic manipulation of the chloroplast genome, for two reasons: firstly, the chloroplast genetic system is well suited to the targeted insertion into the genome and high level expression of foreign genes; secondly, the organelle is the site of numerous biosynthetic pathways and therefore represents the obvious "chassis," on which to bolt new metabolic pathways that divert the carbon fixed by photosynthesis into novel hydrocarbons, pigments, etc. Stable transformation of the algal chloroplast was first demonstrated in 1988, using the model chlorophyte, Chlamydomonas reinhardtii. Since that time, tremendous advances have been made in the development of sophisticated tools for engineering this particular species, and efforts to transfer this technology to other commercially attractive species are starting to bear fruit. In this article, we review the current field of algal chloroplast transgenics and consider the prospects for the future.
Engineering the Chloroplast Encoded Proteins of Chlamydomonas
Photosynthesis Research, 2004
Over a decade ago (1988), John Boynton and colleagues successfully transformed the chloroplast genome of Chlamydomonas for the first time by complementation of a chloroplast deletion mutant. Since the first demonstration of chloroplast transformation the function and structure of many chloroplast encoded subunits of the photosynthetic apparatus has been characterized by site-directed mutagenesis. With the completion of the sequencing of the Chlamydomonas chloroplast genome the genetic tools are now in hand to characterize structure-function relationships for each of the chloroplast-encoded proteins of the photosynthetic apparatus.