Multigenic engineering of the chloroplast genome in the green alga Chlamydomonas reinhardtii

Abstract

<jats:p>The chloroplast of microalgae such as <jats:italic>Chlamydomonas reinhardtii</jats:italic> 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 <jats:italic>C. reinhardtii</jats:italic> 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 <jats:italic>C. reinhardtii</jats:italic> 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.</jats:p>

The Chloroplast of Chlamydomonas reinhardtii as a Testbed for Engineering Nitrogen Fixation into Plants

“…SDS polyacrylamide gel electrophoresis was used to fractionate the cell lysates, which were then probed using an antibody against the HA epitope. Western blot analysis confirmed that the four colonies analysed produced the recombinant enzyme (Figure 4C), although levels were markedly lower than that seen for a control transformant engineered to make an HA-tagged serine protease (SplB) [54]. A transformed line lacking any transgenic coding sequence was used as a negative control.…”

“…Hence, the algal chloroplast is increasingly being recognised as a potential site for light-driven metabolic engineering [36]. Recent studies reporting the expression of multiple transgenes from the plastomes of both C. reinhardtii [53,54] and tobacco [43] demonstrate that complex genetic engineering, such as that required for the introduction of multi-step metabolic pathways or multi-subunit enzyme complexes, is feasible within the chloroplast. Currently, the main technical challenges for the successful expression of a set of nif genes in the C. reinhardtii chloroplast relate to the need for advanced synthetic biology (SynBio) tools: namely, (i) a need for a standardised DNA assembly method that would allow a rapid SynBio approach to the building and testing of multiple transgene configurations, and (ii) an increased number of validated regulatory DNA parts and inducible mechanisms for tuneable regulation of these transgenes.…”

“…Thus, we suggest that a minimal set of six genes, encoding the A. vinelandii structural subunits (anfHDGK) and the K. pneumoniae biosynthetic components (nifBV), could be used as a basis for investigating nitrogen fixation in eukaryotic algae (Figure 2B,C); although, including the NifU/NifS system might prove crucial for securing full activity of the enzyme. The expression of multiple transgenes in the chloroplast of C. reinhardtii is certainly achievable, and the six genes could be expressed either as separate transcriptional units [54] or as several small operons [53] with gene cluster inserts at several neutral loci within the plastome [65]. However, it will be critical to tune the strength of expression of each gene, with the aim of achieving the correct stoichiometric levels of each anf/nif gene product.…”

The Potential of Algal Biotechnology to Produce Antiviral Compounds and Biopharmaceuticals

“…As for the case of chloroplast expression, a series of vectors optimized with specific promoters and UTRs have been described [ 172 , 173 ]. A remarkable example is the use of photorestoration systems in which the use of selectable markers is avoided since the strain carries a mutation that abolish photosynthesis, which is restored upon the foreign DNA insertion that contains the functional gene [ 174 ]. Moreover, inducible expression systems have been developed for the chloroplast and constitute a promise for the field (especially when the target biopharmaceutical exerts toxic effects in the algae species used as host) [ 175 ] to separate the growth phase from the expression phase as requirement to maximize production.…”

Microbial Musings – June 2020

“…As these photosynthetic microbes contain chloroplasts, which have their own genomes, called the plastome, they can be manipulated to introduce foreign genes for overproduction and also to introduce enzymes that can alter chloroplast metabolism to produce new chemicals [8]. In this work the authors assemble multigene constructs and are able to move them in a single event onto the chloroplast genome, being able to detect synthesis of the products of all three genes they introduced at once [9]. While promising, they also note that care must be taken in designing the regulatory elements for each separate transcriptional unit and they see recombination between 3′UTRs when they are very similar.…”