Chlamydomonas reinhardtii chloroplasts as protein factories

Protein-based therapeutics are the fastest growing sector of drug development, mainly because of the high sensitivity and specificity of these molecules. Their high specificity leads to few side effects and excellent success rates in drug development. However, the inherent complexity of these molecules restricts their synthesis to living cells, making recombinant proteins expensive to produce. In addition to therapeutic uses, recombinant proteins also have a variety of industrial applications and are important research reagents. Eukaryotic algae offer the potential to produce high yields of recombinant proteins more rapidly and at much lower cost than traditional cell culture. Additionally, transgenic algae can be grown in complete containment, reducing any risk of environmental contamination. This system might also be used for the oral delivery of therapeutic proteins, as green algae are edible and do not contain endotoxins or human viral or prion contaminants.     

Photorespiratory ammonium assimilation in chloroplasts of Chlamydomonas reinhardtii

The effect of CO₂ concentration on the rate of photorespiratory ammonium excretion and on glutamine synthetase (GS) and carbonic anhydrase (CA) isoenzymes activities has been studied in Chlamydomonas reinhardtii cw-15 mutant (lacking cell wall) and in the high CO₂-requiring double mutant cia-3/cw-15 (lacking cell wall and chloroplastic carbonic anhydrase). In cw-15 cells, both the extracellular (CA_ext) and chloroplastic (CA_chl) CA activities increased after transferring cells from media bubbled with 5% CO₂ in air (v/v, high-C_i cells) to 0.03% CO₂ (low-C_i cells), whereas in cia-3/cw-15 cells only the CA_ext was induced after adaptation to low-C_i conditions and the CA_chl activity was negligible. During adaptation to low-C_i conditions in the presence of 1 mM of l-methionine-D,L-sulfoximine (MSX), a specific inhibitor of GS activity, both mutant strains excreted photorespiratory ammonium into nitrogen free medium. In addition, the ammonium excretion rate by cw-15 in the presence of MSX was lower in cells grown and kept at 5% CO₂ than in high-C_i cells adapted to 0.03% CO₂. The double mutant cia-3/cw-15 excreted photorespiratory ammonium at a higher rate than did cw-15. Total GS activity (GS-1 plus GS-2) increased during adaptation to 0.03% CO₂ in both strains of C. reinhardtii. However, only the activity GS-2, which is located in the chloroplast, increased during the adaptation to low CO₂, whereas the cytosolic GS-1 levels remained similar in high and low-C_i cells. We conclude that: (1) cia-3/cw-15 cells lack chloroplastic CA activity; (2) in C. reinhardtii photorespiratory ammonium is refixed in the chloroplasts through the GS-2/GOGAT cycle; and (3) chloroplastic GS-2 concentration changes in response to the variation of environmental CO₂ concentration.     

Double strand break-induced recombination in Chlamydomonas reinhardtii chloroplasts.

The mechanisms of chloroplast recombination are largely unknown. Using the chloroplast-encoded homing endonuclease I-CreI from Chlamydomonas reinhardtii, an experimental system is described that allows the study of double strand break (DSB)-induced recombination in chloroplasts. The I-CreI endonuclease is encoded by the chloroplast ribosomal group I intron of C.reinhardtii and cleaves specifically intronless copies of the large ribosomal RNA (23S) gene. To study DSB-induced recombination in chloroplast DNA, the genes encoding the I-CreI endonuclease were deleted and a target site for I-CreI, embedded in a cDNA of the 23S gene, was integrated at an ectopic location. Endonuclease function was transiently provided by mating the strains containing the recombination substrate to a wild-type strain. The outcome of DSB repair was analyzed in haploid progeny of these crosses. Interestingly, resolution of DSB repair strictly depended upon the relative orientation of the ectopic ribosomal cDNA and the adjacent copy of the 23S gene. Gene conversion was observed when the 23S cDNA and the neighbouring copy of the 23S gene were in opposite orientation, leading to mobilization of the intron to the 23S cDNA. In contrast, arrangement of the 23S cDNA in direct repeat orientation relative to the proximal 23S gene resulted in a deletion between the 23S cDNA and the 23S gene. These results demonstrate that C.reinhardtii chloroplasts have an efficient system for DSB repair and that homologous recombination is strongly stimulated by DSBs in chloroplast DNA.     

Shine-Dalgarno-like sequences are not required for translation of chloroplast mRNAs in Chlamydomonas reinhardtii chloroplasts or in Escherichia coli

Initiation of translation in Escherichia coli and related eubacteria involves well-defined interactions between a conserved Shine-Dalgarno (SD) sequence immediately upstream of the initiation codon in the mRNA leader and an equally conserved anti-SD sequence at the 3′ end of the 16S rRNA. SD-like sequences found in the leaders of many, but not all, mRNAs from cyanobacteria and chloroplasts are hypervariable in location, size, and base composition compared to those in E. coli, while anti-SD sequences in the respective 16S rRNAs remain highly conserved. We have examined the function of the SD-like sequences found in the leaders of four chloroplast genes of the green alga Chlamydomonas reinhardtii using replacement mutagenesis to eliminate complementarity with the anti-SD sequences and insertion of canonical SD sequences (GGAGG) at positions ?9 to ?5 relative to the initiation codon. Promoter-leader regions of the atpB, atpE, rps4, and rps7 genes representing the diversity of chloroplast SD-like sequences were fused to aadA and uidA reporter genes encoding spectinomycin resistance and GUS activity respectively. Analysis of chloroplast transformants of C. reinhardtii and transformants of E. coli carrying the wild-type and mutant reporter constructs revealed that mutagenic replacement of the putative SD sequences had no effect on the expression of either the aadA or uidA reporter genes. Chloroplast transformants with the canonical SD sequence also showed no differences in reporter gene expression, whereas expression of the reporter genes was increased by 10 to 30% in the E. coli transformants. Collectively our results suggest that even though SD-dependent initiation predominates in E. coli, this bacterium also has the capacity to initiate translation by an SD-independent mechanism. In contrast, plant chloroplasts, and very probably their cyanobacterial ancestors, appear to have adopted the SD-independent mechanism for translational initiation of most mRNAs.     

Thioredoxin activation of phosphoribulokinase in a bi-enzyme complex from Chlamydomonas reinhardtii chloroplasts

The activation of oxidized phosphoribulokinase either "free" or as part of a bi-enzyme complex by reduced thioredoxins during the enzyme reaction was studied. In the presence of reduced thioredoxin, the product of the reaction catalyzed by phosphoribulokinase within the bi-enzyme complex does not appear in a linear fashion. It follows a mono-exponential pattern that suggests a slow dissociation process of the bi-enzyme complex in the assay cuvette. A plot of the steady state of product appearance against thioredoxin concentration gave a sigmoid curve. On the basis of our experimental results, we propose a minimum model of the activation of phosphoribulokinase by reduced thioredoxin. Reduced thioredoxin may act on the phosphoribulokinase, either within the complex or in the dissociated metastable form. However, the time required to activate the enzyme as part of the complex is shorter (about 20 s) than that required to activate the dissociated form (about 10 min). This might be of physiological relevance, and we discuss the role of the interactions between phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase in the regulation of the Calvin cycle.     

Formation of the isocyclic ring of chlorophyll by isolated Chlamydomonas reinhardtii chloroplasts

Chlamydomonas reinhardtii chloroplasts catalyzed two sequential steps of Chl biosynthesis, S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase and Mg-protoporphyrin IX monomethyl ester oxidative cyclase. A double mutant strain of C. reinhardtii was constructed which has a cell wall deficiency and is unable to form chlorophyll in the dark. Dark-grown cells were disrupted with a BioNeb nebulizer under conditions which lysed the plasma membrane but not the chloroplast envelope. Chloroplasts were purified by Percoll density gradient centrifugation. The purified chloroplasts were used to define components required for the biosynthesis of Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) from Mg-protoporphyrin IX. Product formation requires the addition of Mg-protoporphyrin IX, the substrate for S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase which produces Mg-protoporphyrin IX monomethyl ester. The Mg-protoporphyrin IX monomethyl ester that is generated in situ is the substrate for Mg-protoporphyrin IX monomethyl ester oxidative cyclase. The reaction product was identified as Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) by excitation and emission spectrofluorometry and HPLC on ion-paired reverse-phase and polyethylene columns. Mg-2,4-divinylpheoporphyrin a ₅ formation by the coupled enzyme system required O₂ and was stimulated by the addition of NADP⁺, an NADPH regenerating system, and S-adenosyl-l-methionine. Product was formed at a relatively steady rate for at least 60 min.Abbreviations MgDVP Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) - SAM S-adenosyl-l-methionine     

Dynamics of post-translational modifications and protein stability in the stroma of Chlamydomonas reinhardtii chloroplasts

The proteome of any system is a dynamic entity dependent on the intracellular concentration of the entire set of expressed proteins. In turn, this whole protein concentration will be reliant on the stability/turnover of each protein as dictated by their relative rates of synthesis and degradation. In this study, we have investigated the dynamics of the stromal proteome in the model organism Chlamydomonas reinhardtii by characterizing the half-life of the whole set of proteins. 2-DE stromal proteins profiling was set up and coupled with MS analyses. These identifications featuring an average of 26% sequence coverage and eight non-redundant peptides per protein have been obtained for 600 independent samples related to 253 distinct spots. An interactive map of the global stromal proteome, of 274 distinct protein variants is now available on-line at http://www.isv.cnrs-gif.fr/gel2dv2/. N-α-terminal-Acetylation (NTA) was noticed to be the most frequently detectable post-translational modification, and new experimental data related to the chloroplastic transit peptide cleavage site was obtained. Using this data set supplemented with series of pulse-chase experiments, elements directing the relationship between half-life and N-termini were analyzed. Positive correlation between NTA and protein half-life suggests that NTA could contribute to protein stabilization in the stroma.     

Production of milk-derived bioactive peptides as precursor chimeric proteins in chloroplasts of Chlamydomonas reinhardtii

Bioactive peptides are considered high value-added ingredients in functional foods, and the main sources of these are milk, egg, plants, among others. A major limitation in their commercial use is the cost of production. This study deals with the design and production of a chimeric protein in chloroplasts of the microalgae Chlamydomonas reinhardtii to generate bioactive peptides of antihypertensive, opioid, antimicrobial, and hypocholesterolemic activities. A synthetic gene, designated as NCQ, coding for the selected chimeric protein, is transferred to C. reinhardtii using biolistic bombardment. Transplastomic transformants have been identified by PCR and Western blots following selection on a spectinomycin-containing medium. An ELISA quantification assay has revealed that the expressed NCQ protein accumulated at levels ranging between 0.16 and 2.4 % of total soluble protein. These findings demonstrate that chloroplasts of C. reinhardtii could serve as a robust expression platform for production of bioactive peptides.     

Antimycin A effect on the electron transport in chloroplasts of two Chlamydomonas reinhardtii strains

The effects of antimycin A on the redox state of plastoquinone and on electron donation to photosystem I (PS I) were studied in sulfur-deprived Chlamydomonas reinhardtii cells of the strains cc406 and 137c. We found that this reagent suppresses cyclic electron flow around PS I in the cc406 strain, whereas this inhibitory effect was completely absent in the 137c strain. In the latter strain, antimycin A induced rapid reduction of plastoquinone in the dark and considerably enhanced the rate of electron donation to P₇₀₀ ⁺ in the dark. Importantly, neither myxothiazol, an inhibitor of mitochondrial respiration, FCCP, a protonophore, nor propyl gallate, an inhibitor of the plastid terminal oxidase, induced such a strong effect like antimycin A. The results indicate that in the chloroplast of the 137c strain, antimycin A has a site of action outside of the machinery of cyclic electron flow.     

Gas exchange in intact isolated chloroplasts from Chlamydomonas reinhardtii during starch degradation in the dark

During starch degradation in intact isolated chloroplasts from Chlamydomonas reinhardtii gas exchange was studied with a mass spectrometer. Oxygen uptake by intact chloroplasts in the dark never exceeded 1.5% of the starch degradation rate [maximum 15 nmol O₂ (mg Chl)⁻¹ h⁻¹ consumed. 1 000 nmol glucose (mg Chl)⁻¹h⁻¹ degraded]. Evolution of CO₂ under aerobic conditions [9.8–28 nmol (mg Chl)⁻¹ h⁻¹] was stimulated by addition of 0.1–0.5 m M oxaloacetate [393–425 nmol CO₂ (mg Chl)⁻¹ h⁻¹]. Pyridoxal phosphate (5 m M ) inhibited starch degradation by more than 80%, but had no effect on O₂ uptake. Starch degradation rates and CO₂ evolution did not differ under acrobic and anaerobic conditions. Increasing P_i in the reaction medium from 0.5 m M to 5.0 m M stimulated starch degradation by 230 and 260% under aerobic and anaerobic conditions, respectively. A rapid autooxidation of reduced ferredoxin was observed in a reconstituted system consisting of purified Chlamydomonas ferredoxin, purified Chlamydomonas NADP-ferredoxin oxidoreductase (EC 1.6.7.1) and NADPH. Addition of isolated thylakoids from C. reinhardtii did not affect the rate of O₂ uptake. Our results clearly indicate the absence of any oxygen requirement during starch degradation in isolated chloroplasts.     

Characterization of an l-aspartate aminotransferase activity in Chlamydomonas reinhardtii

The isolation and characterization of an l -aspartate aminotransferase (AAT) activity (EC 2.6.1.1) in the unicellular green alga Chlamydomonas reinhardtii 6145c are reported for the first time. The enzyme transaminates aspartate with the 2-oxoglutarate-glutamate system, and exhibits maximum aminotransferase activity at pH 7.8 and 37°C. It has an Mr of 138 kDa, contains pyridoxal 5'-phosphate, and has a K_m apparent for oxalacetate of 0.55 m M and exhibits positive co-operativity with l -aspartate with an S_0.5 of 2.53 m M and a Hill coefficient of 1.57. In vivo, activity levels were affected by the carbon and nitrogen sources and by the change in the dark-light conditions. All these responses are interpreted in terms of a possible physiological regulation of AAT activity to keep the intracellular pools of glutamate and aspartate within margins compatible with environmental fluctuations.     

The in vivo cleavage of carotenoids into retinoids in Chlamydomonas reinhardtii

A variety of carotenoids have been incorporated in vivo intoa carotenoid mutant of the unicellular alga, Chlamydomonas reinhardtii.The mutant can synthesize retinal from exogenous carotenoids.In this way the first step in biosynthesis of retinoids, namelyhow ß-carotene is converted into retinal, has beenstudied. Since retinal forms with opsin the photoreceptor forphototaxis in Chlamydomonas, the action-spectral peaks of thephototaxis restored by carotenoid incorporation were used topredict the products formed by the enzyme that cleaves thesecarotenoids. The data suggest that the physiologically relevantcleavage of ß-carotene into retinal is central ratherthan excentric. When apocarotenoids are substrates, the enzymetargets the double bond located a constant distance away fromthe carbonyl group on the acyclic end and, consequently, retinalis not produced. Interestingly, dehydro-analogues containinga triple bond are preferentially cleaved and oxidized at thetriple bond relative to the corresponding analogue with onlydouble bonds. Key words: Chlamydomonas reinhardtii, Chlorophyceae, retinoid biosynthesis, carotenoid cleavage, action spectrum     

Overexpression of the sulfate transporter-encoding SULTR2 increases chromium accumulation in Chlamydomonas reinhardtii

Hexavalent chromium [Cr(Ⅵ)] is a highly toxic contaminant in aquatic systems, and microalgae represent promising bioremediators of metal-containing wastewater. However, the metal-binding capacity of algal cells is limited. Therefore, we improved the cellular Cr(Ⅵ) biosorption capacity of Chlamydomonas reinhardtii by overexpressing the sulfate transporter gene SULTR2. SULTR2 was predominantly located in the cytoplasm of the cell, and few proteins mobilized to the cell membrane as a Cr transporter under Cr stress conditions. Intracellular Cr accumulation was almost doubled in SULTR2-overexpressing transgenic strains after exposure to 30 μM K₂Cr₂O₇ for 4 d. Alginate-based immobilization increased the rate of Cr removal from 43.81% to 88.15% for SULTR2-overexpressing transgenic strains after exposure to 10 μM K₂Cr₂O₇ for 6 d. The immobilized cells also displayed a significant increase in nutrient removal efficiency compared to that of free-swimming cells. Therefore, SULTR2 overexpression in algae has a great potential for the bioremediation of Cr(Ⅵ)-containing wastewater.     

Insertion of new sequences into the catalytic domain of an enzyme

Activities of enzymes can be modified by the replacement of active-site amino acids with residues that strengthen specific interactions with substrates or that alter the specificity. The scope for engineered enzymes would be broadened if additional, new sequences could be inserted into a catalytic domain. Properly designed, these sequences could encode new ligand binding sites, be intermediates in the construction of chimeric enzymes, or alter the internal flexibility and "breathing" modes of the active-site region. As a first step toward this objective, we inserted oligopeptides of up to 14 amino acids into various locations within an 82 amino acid region of the adenylate synthesis domain of Escherichia coli methionyl-tRNA synthetase. These sites include ones that are flanked by sequences that are conserved between the proteins from E. coli and the yeast Saccharomyces cerevisiae and those that are essential for activity and stability. We found that all of the insertional mutants are stable and some have catalytic parameters for adenylate synthesis that are comparable to those of the wild-type enzyme. Thus, such an approach may provide for a variety of novel applications.     

Large arrays of tandemly repeated DNA sequences in the green alga Chlamydomonas reinhardtii

We describe the characterization of tandemly repeated DNA sequences, which resemble the satellite DNA sequences of multicellular eucaryotes, in the unicellular green alga Chlamydomonas reinhardtii. Restriction enzymes that cleave C. reinhardtii DNA relatively frequently produce a number of high molecular weight DNA fragments in addition to the bulk of low molecular weight DNA fragments. pTANC 1.5 contains a 1.5 kb Sau3A fragment cloned from one of these large bands. pTANC 1.5 hybridized to at least three large arrays (200 to 700 kb) of tandemly repeated DNA sequences in the cell-wall-deficient strain cw1.5. These arrays are composed of repeat units that are each cleaved once by BamHl into bands of 1.5, 1.9, 2.0 and 2.5 kb in size. The copy numbers of the 1.5, 1.9, 2.0 and 2.5 kb Bamhl bands vary between different C. reinhardtii strains. Chlamydomonas smithii and a number of C. reinhardtii strains are deficient in all four BamHl bands. Genetic analysis of wild-type strain 137c, which is deficient in the 2.0 kb BamHl band, indicates that the 1.5, 1.9 and 2.5 kb BamHl bands derive from at least five loci. The 1.5, 1.9 and 2.5 kb repeat units are not extensively interspersed with each other in strain 137c. Pulsed-field gel electrophoresis of intact C. reinhardtii chromosomes indicates that TANC arrays are present on more than one chromosome.