Abstract
BackgroundThe 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.ResultsA 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.ConclusionsCITRIC 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.
Highlights
The chloroplast of eukaryotic microalgae such as Chlamydomonas reinhardtii is a potential platform for metabolic engineering and the production of recombinant proteins
In this paper we demonstrate a new inducible expression system for the C. reinhardtii chloroplast that is based on a method that we developed earlier for preventing transgene expression during cloning and for biocontainment
Variant 1 of tRNATrp‐UCA allows temperature‐dependent readthrough of internal UGA codons We designed and tested four variants of the trnWUCAgene in order to determine whether they would produce a temperature-dependent tRNA in the chloroplast that was still able to recognise internal UGA codons within the transcript of a transgene at a lower temperature, but not at a higher temperature
Summary
The chloroplast of eukaryotic microalgae such as Chlamydomonas reinhardtii is a potential platform for metabolic engineering and the production of recombinant proteins. A wide range of proteins have been expressed in the C. reinhardtii chloroplast, as recently reviewed [4, 6,7,8]. These include complex multi-domain immunotoxins for cancer therapy [9], oral vaccine candidates for aquaculture [10], mosquitocidal proteins [11] and a cellulose-hydrolyzing enzyme [12]. This expression platform is capable of producing proteins with disulphide bonds or phosphorylation [13]
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