Over-expression of a cyanobacterial gene for 1-deoxy-d-xylulose-5-phosphate synthase in the chloroplast of Chlamydomonas reinhardtii perturbs chlorophyll: carotenoid ratios

2.1 Algal strains, media and culture conditions

C. reinhardtii TN72 was used as a recipient line to generate the chloroplast transformant lines named T1.dxs, T2.dxs, T3.Δdxs, T4.Δdxs and Control. TN72 is a photosynthetic mutant of the cell wall deficient strain cw15 (mt+), with the loss of photosynthesis due to disruption of the chloroplast gene psbH with the aadA marker (Wannathong et al., 2016). The selection of transformant lines is therefore based on restoring a functional psbH gene and the ability to grow photosynthetically on acetate-free medium – i.e. high salt minimal (HSM) medium with 2% agar – at a light intensity of 50 µE m⁻² s⁻¹. All transformant lines were then maintained mixotrophically on Tris-acetate phosphate (TAP) medium with 2% agar under dim light conditions of approximately 5–10 µE m⁻² s⁻¹ at 20 °C (Harris, 2009). Transformant lines were cultured in liquid TAP medium shaking at 120 rpm under continuous illumination of 50–100 µE m⁻² s⁻¹ at 25 °C, as a standard growth conditions and for western blot analysis. When required, the growth was monitored by measuring the optical density at 750 nm with a spectrophotometer. For growth tests, the transformant lines were cultured in TAP medium in 1 L Erlenmeyer flasks at 100–200 µE m⁻² s⁻¹, 120 rpm shaking and 25 °C in an Algem® photobioreactor (Algenuity, Stewartby, UK) and the cell growth was monitored in situ by direct measurement of optical density at 740 nm.

2.2 Plasmid construction

The dxs coding sequence (locus tag SYNGTS_0514) was amplified from Synechocystis sp. PCC 6803 genomic DNA using primers 5′-CCAGCTCTTCTATGCACATCAGCGAACTGAC-3′ and 5′-GTCAGGCATGCTTATTAAGCGTAATCTGGAACATCGTATGGGTAACTAACTCCAGGAGCGACAAC-3′, and Phusion Polymerase (NEB). The PCR added an HA tag sequence to the 3′ end of the coding sequence and flanked the PCR product with SapI and SphI sites (underlined). The product was cloned between the SapI and SphI sites in the chloroplast expression vector pSRSapI, which has been described previously (Wannathong et al., 2016), to make plasmid pRY134a. Another construct, pRY134aΔdxs, was made by deleting a 141 bp region of dxs between two NcoI sites. This leads to a 47 amino acid in-frame deletion in DXS. All cloning was carried out using chemically competent E. coli DH5α according to standard protocols (Sambrook and Russell, 2001). Restriction enzymes and T4 DNA ligase were purchased from NEB and ThermoFisher.

2.3 C. reinhardtii chloroplast transformation

The chloroplast transformation method used was adapted from the original glass bead method described by Kindle et al., (1991), with some modifications. TN72 was grown in 500 ml TAP to mid-log phase (with a cell density of 1–2 × 10⁶ cells ml⁻¹) under dim light of approximately 5–10 µE m⁻² s⁻¹. The cells were harvested by centrifugation at 5000 rpm for 5 min at 16 °C and then re-suspended in fresh TAP to a concentration of 2 × 10⁸ cells ml⁻¹. Aliquots of the cell suspension were pipetted into test tubes containing ∼0.3 g of glass beads (acid washed, 425–600 μm diameter, Sigma). 10 μg of plasmid DNA was added to each tube containing 300 μl of cell suspension and then agitated for 15 s at maximum speed using a Vortex genie-2. Then 3 ml of molten 0.5% (w/v) HSM agar at 42 °C was added to each tube and immediately the mixture was poured onto 2% HSM (w/v) agar plates. The plates were incubated under a light intensity of approximately 50 µE m⁻² s⁻¹ at 25 °C for 3–4 weeks, with the selection based on restoration of photosynthesis, i.e. the ability to grow autotrophically on HSM agar plates. The putative transformant colonies were picked and re-streaked three times on HSM agar plates to ensure homoplasmy. To confirm transgene integration and homoplasmic of the chloroplast genome, genomic DNA was extracted using the Chelex 100 method as described by Werner and Mergenhagen (1998) and 2 µl of the DNA extract used in a PCR reaction. PCR analysis was carried out using a combination of three different primers (Table S1) and Phusion Polymerase (NEB) according to the manufacturer’s instructions. PCR products were analysed on 1% agarose gels and the size of PCR products were estimated by reference to a GeneRuler DNA Ladder Mix (Thermo Scientific).

2.4 Western blot analysis

To prepare protein extracts, C. reinhardtii transformant lines were grown to mid-log phase in a shaking incubator and the optical density of cultures at 750 nm was measured using a spectrophotometer. The C. reinhardtii cultures were then harvested by centrifugation at 5800g for 5 min and the pellets re-suspended in 0.8 M Tris.HCl pH 8.3, 0.2 M sorbitol and 1% (v/v) β-mercaptoethanol to equal cell densities. Samples were boiled for 3 min in the presence of 10% SDS (w/v), centrifuged at 21,000g for 2 min and the resulting supernatant was loaded onto a 15% acrylamide SDS-polyacrylamide gel (Laemmli, 1970). Protein separation was carried out in cold electrophoresis buffer (0.25 M Tris, 1.92 M glycine, 1% (w/v) SDS pH 8.3) for 90 min at 150 V. The proteins were transferred to a Hybond ECL nitrocellulose membrane (GE Healthcare) using a Trans-blot semi-dry transfer cell (Bio-Rad) at constant voltage of 19 V for an hour. Following the transfer, the membrane was blocked overnight in 5 % (w/v) skimmed milk powder in TBS-T (20 mM Tris base, pH adjusted to 7.4 with 5 M HCl, 137 mM NaCl, 0.1 % (v/v) Tween) at 4 °C shaking at 50 rpm. The membrane was rinsed briefly with TBS-T, followed by two rounds of 15 min washes in TBS-T while shaking at 50 rpm. Diluted rabbit anti-HA antibody (Sigma-Aldrich product H6908, 1:2000 in TBS-T + 0.5% milk) was used as a primary antibody and the membrane was treated with primary antibody for 1 h at room temperature with shaking at 50 rpm. The membrane was rinsed several times briefly and the membrane was then washed four times in TBS-T for 10 min. The membrane was incubated with secondary antibody ECL anti-rabbit IgG horseradish peroxidase-linked antibody (GE Healthcare product NA934, 1:5000 in TBS-T + 0.5% milk) for 1 h at room temperature with shaking at 50 rpm and the membrane was washed as for the primary antibody. The proteins were detected by the ECL method using a luminescence substrate detection kit according to the manufacturer's instructions (SuperSignal West Pico Chemiluminescent substrate, supplied by Pierce).

2.5 HPLC analysis

Pigments were extracted from 10 mg of freeze-dried biomass by agitation with 25 ml methanol. The separation and chromatographic analysis of pigments were performed on a Merck Hitachi HPLC equipped with a diode-array detector as described by Young and co-workers (Young et al., 1997), using a RP-18 column and a flow rate of 1 ml min⁻¹. The mobile phase consisted of: solvent A, ethyl acetate; solvent B acetonitrile/water (9:1, v/v) and the gradient programme applied was: 0–16 min 0–60%A; 16–30 min 60%A; 30–35 min 100%A. Injection volume was 100 µl. Pigments detection was carried out at 450 nm. Pigments standards were supplied by SIGMA or DHI (Hoersholm, Denmark).

3.1 Generation of marker-free transgenic lines of Chlamydomonas reinhardtii expressing a heterologous dxs gene in the chloroplast

Green microalgae such as C. reinhardtii contains only the plastidic MEP pathway for terpenoid biosynthesis (Lohr et al., 2012). A simplified illustration of the pathway is shown in Fig. 1. DXS catalyses the first biosynthetic step of this pathway and has been reported to be a rate limiting step for the production of the terpenoid precursors IPP and DMAPP in bacteria and plants (Estévez et al., 2001; Gong et al., 2006; Heider et al., 2014). To investigate the effect of overexpressing this enzyme on downstream terpenoid metabolites in C. reinhardtii, a plasmid (pRY134a) was constructed containing the dxs gene from the cyanobacterium Synechocystis sp. PCC6803 under the control of the C. reinhardtii psaA exon 1 promoter and 5′ untranslated region (Fig. 2a). The coding sequence of a C-terminal human influenza hemagglutinin (HA) epitope tag was added to the dxs gene to facilitate the detection of the DXS protein. The dxs gene from Synechocystis is more suitable for heterologous expression in the chloroplast than the nuclear-encoded native copy from C. reinhardtii. This is because the GC content and codon bias of the two genomes of C. reinhardtii are markedly different: the nuclear genome has a GC content of 64% and a bias for GC-rich codons whereas the chloroplast genome has a value of 34% and an AT-rich codon bias (Grossman et al., 2003). Specifically, the C. reinhardtii dxs coding sequence is ∼ 65% GC, whereas that of the Synechocystis dxs is ∼50%. The dxs coding sequence was amplified from Synechocystis genomic DNA and cloned into the expression vector pSRSapI to make plasmid pRY134a (Fig. 2a).

Close

Fig. 1

Overview of terpenoid biosynthetic pathway in the chloroplast of C. reinhardtii. The plastidic MEP pathway begins with the condensation of pyruvate and D-glyceraldehyde 3-phosphate (G3P) leading to the formation of isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). The condensation of DMAPP with one or more IPP units leads to the formation of the following direct precursors; geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP). The introduced additional copy of the gene is indicated by +, whereas the potential flux increase of the corresponding IPP and DMAPP is presented by ↑↑. The subsequent effect on downstream metabolites to be investigated is indicated by??. Dashed arrows indicate a number of steps are involved but are not shown. Abbreviations: DXS, 1-deoxy-d-xylulose-5-phosphate synthase; DXP, 1-deoxy-d-xylulose 5-phosphate; IDI, isopentenyl diphosphate isomerase; SPPs solanesyl diphosphate synthase; PSY, phytoene synthase and GGR, geranylgeranyl reductase.

Export to PPT

Close

Fig. 2

Analysis of partial deletion of dxs in pRY134aΔdxs plasmid. (A) The schematic representation of pRY134a, a cloning vector for introducing a cyanobacterial dxs into the Chlamydomonas chloroplast genome under the control of the psaA exon 1 promoter and the rbcL 3′UTR. The coding sequence of a nine residue haemagglutinin epitope (HA tag) was added to the 3′ end of the dxs gene before the stop codon so that the DXS protein could be detected using anti-HA antibody. The HA tag is indicated by an asterisk. The region of dxs that was deleted in pRY134aΔdxs is between NcoI restriction sites as shown. The flanking regions required for homologous recombination within the chloroplast genome is indicated as LHRR and RHRR, and the intact psbH acts as a selectable marker. The rest of the pRY134a plasmid is depicted as a dashed black line. TrnE2 is abbreviated as E2. (B) Test digestion showing the successful deletion of 141 bp in dxs gene resulting in fragments of the expected sizes, 4599 bp and 3488 bp, as indicated by red box. The lanes 1, 2, 3, and 4 represents different colonies tested for digestion. (C) Sanger sequencing chromatograph confirms the deletion of 141 bp in dxs for clone number 2. The dashed lines indicate the deleted region within the coding sequence of dxs.

Export to PPT

High levels of prenyl phosphate precursors (DMAPP/IPP/FPP) have been reported to be toxic in E. coli cells (Martin et al., 2003; Sivy et al., 2011). To investigate the possibility that over-expression of the heterologous dxs in the chloroplast may have similar deleterious effects on C. reinhardtii cells a non-functional version of dxs was created by deleting 141 bp of the coding sequence within plasmid pRY134a. The deletion was achieved by digesting pRY134a with NcoI at two sites to drop out the 141 bp region, and then re-ligating the plasmid. The resulting plasmid, pRY134aΔdxs, was checked by test digestion resulting in the expected fragment sizes, 4599 bp and 3488 bp (Fig. 2b). DNA sequencing confirmed the successful deletion of 141 bp in the gene (Fig. 2c).

The pRY134a and pRY134aΔdxs plasmids were used to transform C. reinhardtii TN72 (a non-photosynthetic psbH mutant) by agitating the cells with glass beads in the presence of the DNA. The expression cassette in both plasmids is flanked by homologous sequence that contains an intact copy of psbH, so homologous recombination into the chloroplast genome results in restoration of functional psbH and integration of the transgene, and allows the selection of restored photosynthetic transformants on minimal medium in the light (see Fig. S1 for transformation strategy to generate marker-less transgenic lines). Putative transformant colonies appeared after four weeks of incubation. Independent transformant lines named T1.dxs and T2.dxs were obtained using pRY134a, while the colonies named T3.Δdxs and T4.Δdxs were obtained using pRY134aΔdxs. These lines were re-streaked for a further three rounds of phototrophic selection on minimal medium to ensure the homoplasmic state of the chloroplast genome since C. reinhardtii has approximately 80 copies per cell. The integration of the transgene and homoplasmy of the genome in all transgenic lines were then examined by PCR using a specific set of three primers, as illustrated in Fig. 3. Successful transgene integration in all transgenic lines was confirmed by the presence of a 1134 bp band, while the homoplasmic state was indicated by the absence of the 880 bp band that arises from the untransformed copies of the parental plastome. A control strain (C) was included from previous studies and this strain was generated by transforming the TN72 strain with the pSRSapI plasmid but lacking a transgene (Young and Purton, 2014).

Close

Fig. 3

Three primer PCR to confirm transgene integration and homoplasmy of transformants. Left panel: schematic representation of PCR based method to confirm the integration of GOI and homoplasmy of transformants. PCR was performed using three primers in one reaction (F1, R1 and R2). The first primer pair (F1 and R1) amplifies part of the recipient line TN72 plastome, resulting in an 880 bp band, whereas the second primer pair (F1 and R2) amplifies part of the transformant plastome, resulting in a 1134 bp band. The successful integration of the gene of interest is indicated by the presence of the 1134 bp band and the homoplasmy is indicated by the absence of the 880 bp. The C. reinhardtii lines are TN72 recipient strain, T.dxs strains containing the dxs gene, T.Δdxs strains containing the mutant dxs gene and an empty-vector transformant as a control (C). TF is an abbreviation of transformant. Right panel: PCR confirming homoplasmic integration of the transgene into the chloroplast genome.

Export to PPT

3.2 Accumulation of the transgenic DXS protein in C. reinhardtii

To demonstrate the accumulation of the transgenic DXS protein, crude cell extracts were prepared from C. reinhardtii lines and analysed by western blot using antibodies against the HA-tag. The C. reinhardtii lines used were T1.dxs and T2.dxs for intact DXS protein, T3.Δdxs and T4.Δdxs for mutant DXS protein and C as a negative control. The DXS protein with an HA tag has an expected size of 70.4 kDa, and as shown in Fig. 4, there is a detectable level of the tagged DXS protein in the extracts of both T.dxs lines. For the T.Δdxs lines, the accumulation of the smaller mutant DXS protein was not detected at 65 kDa (Fig. 4). The deletion does not cause a frameshift in the coding sequence (Fig. S2), but might result in conformational changes of the protein structure that make it more vulnerable to degradation.

Close

Fig. 4

Expression of the cyanobacterial dxs gene in the C. reinhardtii lines demonstrated by western blot analysis of equalised lysate from T.dxs strains containing an intact cyanobacterial dxs gene, T.Δdxs strains containing the mutant dxs gene and C strain as a negative control (i.e. TN72 transformed with pSRSapI, an empty vector). Analysis with anti-HA antibody shows expression of dxs by a strong band running at an apparent mass of 70.4 kDa in cell lines T1.dxs (T1) and T2.dxs (T2) but not in T3.Δdxs (T3) and T4.Δdxs (T4), or in the negative control line (C). The immunoblot was visualised by ECL with one-minute exposure. Protein size was determined using the PageRuler^TM Prestained Protein ladder (Thermo Scientific).

Export to PPT

3.3 Overexpression of dxs does not affect C. reinhardtii growth

As high levels of prenyl phosphate precursors (DMAPP/IPP/FPP) were reported to be toxic in transgenic E. coli cells (Martin et al., 2003; Sivy et al., 2011), the growth of the transgenic lines was investigated using an Algem® photobioreactor system. As illustrated in Fig. 5, the growth of lines T1.dxs and T2.dxs was not inhibited compared to both the empty-vector control and T.Δdxs lines. Indeed, the growth rates of all four we very similar, and superior to that of the control; possibly because of an unrelated mutation that has arisen in our stock of the control strain. The results demonstrate that the presence of a cyanobacterial DXS protein in addition to the native one does not have a deleterious effect on growth.

Close

Fig. 5

Growth curves of C. reinhardtii cell lines with and without the dxs gene from Synechocystis 6803. All cultures were grown in TAP medium using the Algem® system and the optical density was measured at 740 nm. The growth conditions were 25 °C, 200 µE light, and continuous shaking at 120 rpm. Error bars represents ± standard deviation of triplicate cultures.

Export to PPT

3.4 Analysis of terpenoid profile of the genetically engineered C. reinhardtii

In order to determine the impact of expressing an additional dxs gene on terpenoid content, the quantity of plastidic terpenoids –i.e. carotenoids– such as violaxanthin, lutein and β-carotene was measured in the T1.dxs and compared with the levels found in the control line. Carotenoids were chosen because they encompass a large group of compounds derived from geranylgeranyl diphosphate precursor and represent one of the major product classes derived from the MEP pathway. The analysis of the T1.dxs line showed an overall increase in the levels of plastidic carotenoids such as β-carotene, zeaxanthin, antheraxanthin and violaxanthin (Fig. 6 and Fig. S3 for terpenoid profile). However, since the carotenoid levels are normalized to the chlorophyll content in the samples, then it is possible that the observed percentages changes are affected by changes in total chlorophyll in the transgenic line. This is possible since chlorophylls are tetrapyrrole molecules possessing a phytol ‘tail’ composed of a 20-carbon diterpene alcohol derived from GGPP. We therefore compared chlorophyll content in the control and transgenic lines, and indeed identified a significant change in chlorophyll levels when expressed per gram of algal dry weight. As shown in Fig. 6, there is a significant reduction by two folds in the levels of both chlorophyll a and b in the transgenic line – to 54% and 47% of control levels, respectively. Such a reduction in the chlorophyll levels is unexpected since the overexpression of dxs is presumed to increase the precursor pool of GGPP and this precursor pool is shared by carotenoid, phytol and plastoquinone pathways. In addition, several studies in plants reported that the overexpression of dxs resulted in an enhanced accumulation of both carotenoids and total chlorophyll content (Estévez et al., 2001; Munoz-Bertomeu et al., 2006). Nevertheless, this observation allows us to normalize the carotenoid changes to dry weight by dividing the transgenic values by approximately two-fold, and reveals that β-carotene and violaxanthin do not show a significant change in the DXS transformant, whereas zeaxanthin and antherxanthin levels have increased several-fold and lutein levels have dropped to approximately half.

Close

Fig. 6

Terpenoid analysis of the C. reinhardtii lines. The effect of expressing an additional dxs gene on carotenoid level (left panel) and chlorophyll level (right panel). Carotenoid and chlorophyll levels were determined by HPLC analysis. Abbreviations are Chl: chlorophyll and DW: dry weight.

Export to PPT