A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii

Bertalan, Ivo; Munder, Matthias C.; Weiß, Caroline; Kopf, Judith; Fischer, Dirk; Johanningmeier, Udo

doi:10.1016/j.jbiotec.2014.12.017

Cited by 36 publications

(27 citation statements)

References 38 publications

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“…As illustrated in Fig. 1, this results in marker-free transformants in which the only foreign DNA in the plastome is the gene of interest [27,28]. Expression of the gene is achieved by fusing the coding sequence to promoters and untranslated regions from highly expressed endogenous genes, such as the photosynthesis genes psaA and psbA.…”

Section: The Algal Chloroplast As a New Bio-factorymentioning

confidence: 99%

“…can be evaluated. We are now starting to see the application of synthetic biology principles to plastome engineering with the adoption of assembly standards such as Golden Gate and the creation of libraries of validated DNA parts that allow rapid one-step assembly of all the parts [27,56,57]. In the near future, we may see much more ambitious design strategies that involve extensive redesign of the plastome in silico such that large tracts of non-essential DNA are removed [58], essential endogenous genes are refactored into functional clusters [59] and multiple transgenes are engineered into different loci.…”

Section: Emerging Synthetic Biology Approachesmentioning

confidence: 99%

“…Currently, the promoter and 5¢UTR used to express transgenes are derived from endogenous photosynthetic genes. In some cases, expression levels can be improved by using the stronger promoter from the gene for the 16S ribosomal RNA fused to the 5¢UTR of a photosynthetic gene [27,61]. However, more often it is the performance of the 5¢UTR that is the bottleneck [62], with the efficiency of translation constrained by either the same feedback regulation that prevents the over-accumulation of individual photosynthetic subunits in the absence of their assembly partners (so-called 'control by epistasy of synthesis'), or by competition with the corresponding endogenous gene transcript for trans-acting factors that are required for transcript stability or translation, but are present in limiting concentration in the chloroplast [63].…”

Section: Emerging Synthetic Biology Approachesmentioning

confidence: 99%

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The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

2018

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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, some of the 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 paper 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.

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Section: The Algal Chloroplast As a New Bio-factorymentioning

confidence: 99%

Section: Emerging Synthetic Biology Approachesmentioning

confidence: 99%

Section: Emerging Synthetic Biology Approachesmentioning

confidence: 99%

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The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

2018

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“…Synthesized DNA fragments were assembled using a strategy involving Type IIS restriction enzyme, BsaI, (Bertalan et al, 2015) and ligated into pBR322 that had been digested with HindIII and AvaI to create the pBR322-aadA intermediate vector. The aadA cassette was then transferred to pUC19, resulting in YP4 (chloroplast Edit Plasmids are listed with the information on their core components in Table 1).…”

Section: Manuscript To Be Reviewedmentioning

confidence: 99%

Peer Review #2 of "Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts (v0.1)"

2020

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We present a new approach to edit both mitochondrial and chloroplast genomes. Organelles have been considered off-limits to CRISPR due to their impermeability to most RNA and DNA. This has prevented applications of Cas9/gRNA mediated genome editing in organelles while the tool has been widely used for engineering of nuclear DNA in a number of organisms in the last several years. To overcome the hurdle, we designed a new approach to enable organelle genome editing. The plasmids, designated "Edit Plasmids", were constructed with two expression cassettes, one for the expression of Cas9, codonoptimized for each organelle, under promoters specific to each organelle, and the other cassette for the expression of guide RNAs under another set of promoters specific to each organelle. In addition, Edit Plasmids were designed to carry the donor DNA for integration between two double-strand break sites induced by Cas9/gRNAs. Each donor DNA was flanked by the regions homologous to both ends of the integration site that were short enough to minimize spontaneous recombination events. Furthermore, the donor DNA was so modified that it did not carry functional gRNA target sites, allowing the stability of the integrated DNA without being excised by further Cas9/gRNAs activity. Edit Plasmids were introduced into organelles through microprojectile transformation. We confirmed donor DNA insertion at the target sites facilitated by homologous recombination only in the presence of Cas9/gRNA activity in yeast mitochondria and Chlamydomonas chloroplasts. We also showed that Edit Plasmids persist and replicate in mitochondria autonomously for several dozens of generations in the presence of the wild-type genomes. Finally, we did not find insertions and/or deletions (INDELs) at one of the Cas9 cleavage sites in chloroplasts, which are otherwise hallmarks of Cas9/gRNA-mediated NHEJ repair events in nuclear DNA. This is consistent with previous reports of the lack of NHEJ repair system in most bacteria, which are believed to be ancestors of organelles. This is the first demonstration of CRISPR-mediated genome editing in both mitochondria and chloroplasts in two distantly related organisms. The Edit Plasmid approach is expected to open the door to engineer organelle genomes of a wide range of organisms in a precise fashion.

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“…In most cases, the 3′-UTR seems less important in terms of gene expression regulation, and several different, e.g., the RBCS2, the PSAD, or the psbA 3′-UTRs, have been used successfully for nuclear or chloroplast gene expression, respectively (Bertalan et al 2015;Fischer and Rochaix 2001;Fuhrmann et al 2004). …”

Section: Intronsmentioning

confidence: 99%

Genetic tools and techniques for Chlamydomonas reinhardtii

Mussgnug

2015

Appl Microbiol Biotechnol

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The development of tools has always been a major driving force for the advancement of science. Optical microscopes were the first instruments that allowed discovery and descriptive studies of the subcellular features of microorganisms. Although optical and electron microscopes remained at the forefront of microbiological research tools since their inventions, the advent of molecular genetics brought about questions which had to be addressed with new "genetic tools". The unicellular green microalgal genus Chlamydomonas, especially the most prominent species C. reinhardtii, has become a frequently used model organism for many diverse fields of research and molecular genetic analyses of C. reinhardtii, as well as the available genetic tools and techniques, have become increasingly sophisticated throughout the last decades. The aim of this review is to provide an overview of the molecular key features of C. reinhardtii and summarize the progress related to the development of tools and techniques for genetic engineering of this organism, from pioneering DNA transformation experiments to state-of-the-art techniques for targeted nuclear genome editing and high-throughput screening approaches.

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A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii

Cited by 36 publications

References 38 publications

The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

Peer Review #2 of "Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts (v0.1)"

Genetic tools and techniques for Chlamydomonas reinhardtii

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