CITRIC: cold-inducible translational readthrough in the chloroplast of Chlamydomonas reinhardtii using a novel temperature-sensitive transfer RNA

Abstract

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 trnW_UCA gene encoding a plastidial transfer RNA with a modified anticodon sequence, UCA.

Results: A mutated version of our trnW_UCA 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.

Keywords: Chloroplast; Inducible expression; Industrial biotechnology; Microalgae; Opal suppression; Premature termination codon; tRNA.

Fig. 1

Designs for testing different tRNA^Trp-UCA variants for temperature-sensitive behaviour. a Representation of the tRNA secondary structure with four variant versions annotated. Note the UCA anticodon (for UGA recognition) at the bottom of the structure. In chloroplasts, the 3′ CCA is added post-transcriptionally. Chemical modifications are not shown. b Construct design. Only the region that integrates into the chloroplast genome is shown; flanks are for homologous recombination into the genome. Each plasmid also contains an ampicillin resistance gene for selection during cloning in E. coli

Fig. 2

Analysis of the ability of the four tRNA variants to translate a test protein, CrCD, at different growth temperatures. Six C. reinhardtii cell lines, all containing the CD/2* gene but with different tRNA variants, were grown for 72 h at 20, 25, 30 or 35 °C. Crude extracts were equalised according to the culture optical density at 750 nm and subjected to SDS-PAGE and Western blotting using an anti-HA antibody to detect CrCD protein. See Additional file 1: Table S2 for culture conditions

Fig. 3

Growth test demonstrating that the introduction of tCI into the chloroplast genome allows the temperature-dependent translation of functional CrCD protein from the CD/2* gene. Liquid cultures grown at 35 °C were spotted onto TAP agar containing no drug (left panel) or 2 mg/ml 5-fluorocytosine (5-FC; right panel) then incubated at different temperatures for 10 days. Three C. reinhardtii cell lines were used, all containing the CD/2* gene. In the cell line with no additional tRNA gene (−), CD/2* is not translated so the cells grow on 5-FC. In the cell line with trnW_UCA encoding the constitutive version of the tRNA (+), CD/2* is translated at all growth temperatures and prevents growth on 5-FC. In the cell line with tCI, CrCD accumulation and subsequent growth inhibition depend on the temperature

Fig. 4

Investigation of CrCD protein accumulation in C. reinhardtii tCI cell lines upon varying the number of internal TGA codons and/or induction temperature. a Cell lines with 2, 4 or 6 internal TGA codons in the CD gene were pre-cultured at 30 °C then shifted to 18, 25 or 30 °C for induction. CrCD protein accumulation was measured by anti-HA immunoblot after 72 h. Equal culture volumes were used for each immunoblot sample, then anti-HA band intensity values were divided by the optical density (750 nm) of that culture to plot relative ‘per cell’ CrCD. Each bar shows the mean value for two cultures. b The CD/4* cell line was grown in four identical flask cultures at 35 °C to a high cell density (OD₇₅₀ = 2.1) then induced at 15, 18, 20 or 25 °C using Algem photobioreactors. CrCD accumulation was followed for 72 h after induction. Samples were equalised according to optical density at 750 nm and analysed by SDS-PAGE and anti-HA immunoblotting. Error bars (on all data points) show ± SD for two samples taken from the same flask at each timepoint

Fig. 5

Cold-inducible synthesis of cis-abienol synthase, TPS4. a Design of chloroplast expression constructs. The TPS4 genes include a C-terminal HA tag for detection. b Immunoblot using anti-HA antibodies to detect TPS4 protein in C. reinhardtii tCI cell lines induced at 18 °C. Samples were equalised according to the culture optical density at 750 nm before loading. The first three lanes show a constitutive TPS4 control cell line with no internal TGA codons. c Anti-HA immunoblot to compare induction temperatures of 18 and 25 °C in C. reinhardtii. Samples were equalised according to the culture optical density at 750 nm before loading. A strain synthesising CrCD (right lane) is included as a negative control for the TPS4 band. d Quantification of the band intensities in part c, showing the TPS4 induction time-course at two temperatures. e Anti-HA immunoblot to detect TPS4 protein in E. coli DH5α containing various TPS4 chloroplast expression plasmids. Total protein stain for this blot is shown in Additional file 1: Figure S6. The incubation temperature is given above each lane

Fig. 6

Plasmids available to introduce the CITRIC system into the C. reinhardtii chloroplast. The induction system needs only the tCI tRNA gene, plus a gene of interest containing internal TGA codons. The ampicillin resistance gene (ampR) is for selection in E. coli and is outside the region that integrates into the chloroplast genome. a pWUCA3, b pWUCA4