Reinhardtii Chloroplast Genome Research Articles

Heterologous production of labdane-type diterpenes in the green alga Chlamydomonas reinhardtii

Labdane diterpenes (LDs), and especially sclareol, are important feedstocks for the pharmaceutical and cosmetic industries, and therefore several lines of research have led to their heterologous production in non-photosynthetic microbes and higher plants. The potential of microalgae as bioreactors of natural products has been established for a variety of bioactive metabolites, including terpenes. In this work, a codon optimized sequence encoding a key plant labdane-type diterpene (LD) cyclase, copal-8-ol diphosphate synthase from Cistus creticus (CcCLS), was introduced into the chloroplast genome of Chlamydomonas reinhardtii. Of 49 transplastomic algal lines, 12 produced variable amounts of four LD compounds, namely ent-manoyl oxide, sclareol, labda-13-ene-8α,15-diol and ent-13-epi-manoyl oxide. The total LD concentrations measured in the transplastomic lines reached 1.172 ± 0.05 μg/mg cell DW for the highest overall producer, while the highest yield for sclareol was 0.038 ± 0.001 μg/mg cell DW. Thus, transplastomic expression of a key plant labdane diterpene cyclase in the C. reinhardtii chloroplast genome enabled the production of important plant-specific LD compounds.

Chloroplast engineering of Chlamydomonas reinhardtii to use phosphite as phosphorus source

Phosphorus (P) is a key biological element and a limiting nutrient in aquatic and terrestrial environments. The vast majority of organisms can only uptake phosphorus in its most oxidized form, as phosphate (PO43−), whereas a few prokaryotic species can metabolize phosphorus in a more reduced state such as phosphite (PO33−). Recently, it has been shown that by expressing the ptxD gene, encoding a NAD-dependent phosphite dehydrogenase (also known as phosphonate dehydrogenase), in Chlamydomonas reinhardtii phosphite utilization can be enabled. However, this was done by transforming the nuclear genome, where gene silencing is frequent and random integration of transgenes can result in variable levels of gene expression and pleiotropic effects. The aim of this work was to investigate if phosphite assimilation in the eukaryotic algae Chlamydomonas reinhardtii can also be achieved by expressing a codon-optimized ptxD gene in the chloroplast. To do this, the ptxD gene was targeted and stably integrated into the psbA exon 5-5S rRNA intergenic region within the inverted repeats of the C. reinhardtii chloroplast genome. Integration was shown to occur in the targeted site and transplastomic lines were shown to be homoplasmic and to stably accumulate the NAD-dependent phosphite dehydrogenase PTXD, enabling the cells to use phosphite as the sole phosphorus source in a 0.1–5 mM concentration range. This work demonstrates that transplastomic lines of C. reinhardtii expressing the prokaryotic ptxD gene can effectively be cultivated in phosphite, opening new opportunities for microalgae cultivation.

Expression and membrane-targeting of an active plant cytochrome P450 in the chloroplast of the green alga Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii has potential as a cell factory for the production of recombinant proteins and other compounds, but mainstream adoption has been hindered by a scarcity of genetic tools and a need to identify products that can be generated in a cost-effective manner. A promising strategy is to use algal chloroplasts as a site for synthesis of high value bioactive compounds such as diterpenoids since these are derived from metabolic building blocks that occur naturally within the organelle. However, synthesis of these complex plant metabolites requires the introduction of membrane-associated enzymes including cytochrome P450 enzymes (P450s). Here, we show that a gene (CYP79A1) encoding a model P450 can be introduced into the C. reinhardtii chloroplast genome using a simple transformation system. The gene is stably expressed and the P450 is efficiently targeted into chloroplast membranes by means of its endogenous N-terminal anchor domain, where it is active and accounts for 0.4% of total cell protein. These results provide proof of concept for the introduction of diterpenoid synthesis pathways into the chloroplast of C. reinhardtii.

A Chlamydomonas-Derived Human Papillomavirus 16 E7 Vaccine Induces Specific Tumor Protection

BackgroundThe E7 protein of the Human Papillomavirus (HPV) type 16, being involved in malignant cellular transformation, represents a key antigen for developing therapeutic vaccines against HPV-related lesions and cancers. Recombinant production of this vaccine antigen in an active form and in compliance with good manufacturing practices (GMP) plays a crucial role for developing effective vaccines. E7-based therapeutic vaccines produced in plants have been shown to be active in tumor regression and protection in pre-clinical models. However, some drawbacks of in whole-plant vaccine production encouraged us to explore the production of the E7-based therapeutic vaccine in Chlamydomonas reinhardtii, an organism easy to grow and transform and fully amenable to GMP guidelines.Methodology/Principal FindingsAn expression cassette encoding E7GGG, a mutated, attenuated form of the E7 oncoprotein, alone or as a fusion with affinity tags (His6 or FLAG), under the control of the C. reinhardtii chloroplast psbD 5′ UTR and the psbA 3′ UTR, was introduced into the C. reinhardtii chloroplast genome by homologous recombination. The protein was mostly soluble and reached 0.12% of total soluble proteins. Affinity purification was optimized and performed for both tagged forms. Induction of specific anti-E7 IgGs and E7-specific T-cell proliferation were detected in C57BL/6 mice vaccinated with total Chlamydomonas extract and with affinity-purified protein. High levels of tumor protection were achieved after challenge with a tumor cell line expressing the E7 protein.ConclusionsThe C. reinhardtii chloroplast is a suitable expression system for the production of the E7GGG protein, in a soluble, immunogenic form. The production in contained and sterile conditions highlights the potential of microalgae as alternative platforms for the production of vaccines for human uses.

Short dispersed repeats in the Chlamydomonas chloroplast genome are collocated with sites for mRNA 3' end formation.

The Chlamydomonas reinhardtii chloroplast genome possesses thousands of small dispersed repeats (SDRs), which are of unknown function. Here, we used the petA gene as a model to investigate the role of SDRs in mRNA 3' end formation. In wild-type cells, petA mRNA accumulated as a major 1.3-kb transcript, whose 3' end was mapped to the distal end of a predicted stem-loop structure. To determine whether this stem-loop was required for petA mRNA stability, a series of deletions was constructed. These deletion strains accumulated a variety of petA mRNAs, for which approximate 3' ends were deduced. These 3' ends were found to flank stem-loop structures, many of which were formed partially or completely from inverted copies of SDRs. All strains accumulated wild-type levels of cytochrome f, demonstrating that alternative 3' termini are compatible with efficient translation. The ability to form alternative mRNA termini using SDRs lends additional flexibility to the chloroplast gene expression apparatus and thus could confer an evolutionary advantage.

Establishment ofChlamydomonas reinhardtii chloroplast expression

An expression vector pAC III containing the chimeric gene HAV-VP3P1 and HCV-C gene has been constructed and transferred toC. reinhardtii by the biolistic method. The transformants have been identified by PCR, Southern-blotting, Northern-blotting and Western-blotting assays after selecting on resistant medium and incubating in the dark. The results show that the chimeric gene has replaced thechlL gene ofC. reinhardtii chloroplast genome and expressed correctly under the control of theC. reinhardtii chloroplast double promoters 5′chlL-5′atpA. The analysis of SDS-PAGE indicates that the expressed protein accounts for 5.31 % of total soluble protein.

Extraordinary features in the Chlamydomonas reinhardtii chloroplast genome: (1) rps2 as part of a large open reading frame; (2) a C. reinhardtii specific repeat sequence

We have determined the DNA sequence of the 3574-bp chloroplast DNA fragment of Chlamydomonas reinhardtii formed by the overlap of BamHI fragment 3 and EcoRI fragment 5. This sequence encodes most of rps18 and orf570, an unidentified open reading frame that contains a 150 amino acid domain with high homology to the N-terminal part of 30 S ribosomal protein S2 of other chloroplast, cyanobacterial and bacterial genomes. Between these two sequences lies a highly repetitive sequence element of 500 bp, that is composed of multiple direct and inverted repeat sequences that occur in rearranged, but highly conserved form in at least 36 locations in the C. reinhardtii chloroplast genome. Among the conserved repeat sequences in the C. reinhardtii chloroplast genome we identified the borders of the inverted repeats near atpB and rps4. This might indicate that the conserved sequence elements are remainders of gene rearrangements in the chloroplast genome that occurred by relocations of the inverted repeats.

The chloroplast atpA gene cluster in Chlamydomonas reinhardtii. Functional analysis of a polycistronic transcription unit.

Most chloroplast genes in vascular plants are organized into polycistronic transcription units, which generate a complex pattern of mono-, di-, and polycistronic transcripts. In contrast, most Chlamydomonas reinhardtii chloroplast transcripts characterized to date have been monocistronic. This paper describes the atpA gene cluster in the C. reinhardtii chloroplast genome, which includes the atpA, psbI, cemA, and atpH genes, encoding the alpha-subunit of the coupling-factor-1 (CF1) ATP synthase, a small photosystem II polypeptide, a chloroplast envelope membrane protein, and subunit III of the CF0 ATP synthase, respectively. We show that promoters precede the atpA, psbI, and atpH genes, but not the cemA gene, and that cemA mRNA is present only as part of di-, tri-, or tetracistronic transcripts. Deletions introduced into the gene cluster reveal, first, that CF1-alpha can be translated from di- or polycistronic transcripts, and, second, that substantial reductions in mRNA quantity have minimal effects on protein synthesis rates. We suggest that posttranscriptional mRNA processing is common in C. reinhardtii chloroplasts, permitting the expression of multiple genes from a single promoter.

Requirement for the H Phosphoprotein in Photosystem II of Chlamydomonas reinhardtii

To dissect the expression of the psbB gene cluster of the Chlamydomonas reinhardtii chloroplast genome and to assess the role of the photosystem II H-phosphoprotein (PSII-H) in the biogenesis and/or stabilization of PSII, an aadA gene cassette conferring spectinomycin resistance was employed for mutagenesis. Disruption of the gene cluster has no effect on the abundance of transcripts of the upstream psbB/T locus. Likewise, interruption of psbB/T and psbH with a strong transcriptional terminator from the rbcL gene does not influence transcript accumulation. Thus, psbB/T and psbH may be independently transcribed, and the latter gene seems to have its own promoter in C. reinhardtii. In the absence of PSII-H, translation and thylakoid insertion of chloroplast PSII core proteins is unaffected, but PSII proteins do not accumulate. Because the deletion mutant also exhibits PSII deficiency when dark-grown, the effect is unrelated to photoinhibition. Turnover of proteins B and C of PSII and the polypeptides PSII protein A and PSII protein D is faster than in wild-type cells but is much slower than that observed in other PSII-deficient mutants of C. reinhardtii, suggesting a peripheral location of PSII-H in PSII. The role of PSII-H on PSII assembly was examined by sucrose gradient fractionation of pulse-labeled thylakoids; the accumulation of high-molecular-weight forms of PSII is severely impaired in the psbH deletion mutant. Thus, a primary role of PSII-H may be to facilitate PSII assembly/stability through dimerization. PSII-H phosphorylation, which possibly occurs at two sites, may also be germane to its role in regulating PSII structure, stabilization, or activity.

Two copies of a DNA element, ?Wendy?, in the chloroplast chromosome of Chlamydomonas reinhardtii between rearranged gene clusters

We have characterized two copies of a 2.4 kb DNA element that we call 'Wendy', in the chloroplast chromosome of Chlamydomonas reinhardtii. The two copies of Wendy reside in different single-copy regions at opposite positions in the chloroplast genome. Like many mobile DNA elements, both copies of Wendy are bordered by inverted repeats and contain several additional degenerate copies of these repeat sequences in direct or inverted orientation. In addition, four basepairs are repeated in direct orientation. Two major open reading frames (ORFs) are predicted from the DNA sequence of Wendy I. These ORFs are co-transcribed from a promoter inside the element. The deduced amino acid sequence of the larger of these ORFs shares some weak similarities with sequence motifs of transposases and integrases of other mobile elements. Wendy II appears to be altered relative to Wendy I by point mutations and small deletions and insertions which destroy the ORFs. The leader sequence of the Wendy transcript is nearly identical with the leader sequence of the rbcL transcript of C. reinhardtii, but not of C. moewusii (where the complete Wendy was also undetectable). Furthermore, both copies of Wendy are bracketed by gene clusters that are separated in C. reinhardtii but are contiguous in C. moewusii where they exist in an inverted orientation compared with C. reinhardtii. Wendy was not found in any of the completely sequenced chloroplast genomes of rice, tobacco, pine, Euglena or Marchantia, nor in any other GenBank entry. Our results suggest that Wendy has invaded C. reinhardtii after divergence from other species. Subsequent Wendy-dependent illegitimate homologous or site-specific recombination events or both may have contributed to scrambling of the C. reinhardtii chloroplast genome relative to genomes of other species.

Expression of the Chlamydomonas reinhardtii chloroplast tRNA Glu gene in a homologous in vitro transcription system is independent of upstream promoter elements

Chloroplast tRNA Glu is a bifunctional molecule involved in both the early steps of chlorophyll synthesis and chloroplast protein biosynthesis. Recently the enzymes involved in these processes have been characterized from the green alga Chlamydomonas reinhardtii. In order to investigate whether transcription of the gene for the tRNA Glu cofactor would be a possible point of regulation for the biosynthesis of chlorophyll, a homologous in vitro transcription system for C. reinhardtii chloroplast RNA polymerase was developed. The enzymatic activity was partially purified by ion-exchange chromatography to separate it from nuclear RNA polymerases. The highest rate of synthesis was found at pH 7.9, 40 m m KCl, 9 m m MgCl 2 and with 25 μg plasmid DNA containing the chloroplast tRNA gene per milliliter. The activity was not sensitive to high amounts of α-amanitin (500 μg/ml) and rifampicin, but was clearly inhibited by heparin. This system was used to undertake a promoter analysis of one of the two identical tRNA Glu gene copies found in the C. reinhardtii chloroplast genome ( trnE1). The analyzed tRNA gene behaved like a single transcription unit driven by its own promoter. The transcript terminated in a run of four consecutive T residues downstream of the gene. The nucleotide sequence in the 5′ region of the gene revealed several potential promoter elements with homology to known chloroplast promoters of the “−10 and −35 region” and the “ Euglena promoter” types. Surprisingly, deletion of the complete 5′ region did not affect in vitro transcription, while partial deletions of the 5′ and 3′ coding region totally abolished transcription. This indicates the presence of an internal control region previously found for genes transcribed by nuclear RNA polymerase III. Protein binding studies with the coding region of trnE1 using gel retardation assays demonstrated the formation of two differently sized complexes. In vitro transcription of the tRNA Glu gene in extracts prepared from light and dark grown algae failed to demonstrate any significant influence of light on the transcription reaction.

Chloroplast ribosomal intron of Chlamydomonas reinhardtii: in vitro self-splicing, DNA endonuclease activity and in vivo mobility.

All chloroplast 23S ribosomal RNA genes of the unicellular alga Chlamydomonas reinhardtii contain an 888 bp group I intron with an internal open reading frame (ORF). A precursor RNA encompassing the intron with its 5' and 3' flanking sequences was shown to self-splice both during in vitro transcription and upon incubation of the isolated pre-RNA under self-splicing conditions. Expression of the internal ORF in Escherichia coli in the presence of a plasmid containing a cDNA corresponding to the intronless form of the 23S rRNA gene resulted in specific cleavage of the cDNA at or close to the exon junction sequence. To test whether this ORF-encoded double-strand DNA endonuclease is involved in intron mobility in vivo, the same ribosomal cDNA was stably integrated into the C. reinhardtii chloroplast genome using particle gun mediated transformation. All the transformants with the cDNA integrated at the expected site in the chloroplast genome had the intron precisely inserted at the artificial exon junction site. These experiments demonstrate that the chloroplast ribosomal intron of C. reinhardtii behaves as a ribozyme in vitro and also as a mobile genetic element in vivo provided a target site is present.

Cotranscription of the wild-type chloroplast atpE gene encoding the CF1/CF0 epsilon subunit with the 3′ half of the rps7 gene in Chlamydomonas reinhardtii and characterization of frameshift mutations in atpE

We have characterized two independently isolated point mutants in Chlamydomonas reinhardtii, ac-u-a-1-15 and FUD 17, mapping to the chloroplast ac-u-a locus which corresponds to the atpE gene. Both mutants have a single A:T base pair deletion in a sequence of 6 A:T base pairs at nucleotide positions 102 to 107. This causes a frameshift, altering the coding sequence for the next 8 amino acids and creating a termination codon at amino acid position 44, 98 amino acids from the C-terminus of the protein. Assembly of the ATP synthase is impaired in the mutants; less than 5% of the wild-type level of alpha and beta subunits and no gamma or epsilon subunits are associated with thylakoid membranes of the mutants. The genes encoding the beta and epsilon subunits of the chloroplast ATP synthase from C. reinhardtii are not cotranscribed, in contrast to all other photosynthetic organisms examined to date. Four transcripts, of approximately 1.7, 2.9, 3.3 and 7.0 x 10(3) nucleotides (nt), are found for the atpE gene. S1 nuclease mapping of the 1.7 x 10(3) nt transcript shows that the atpE gene message is preceded by a leader of about 1250 nt. DNA sequence analysis of this region revealed a 159 bp open reading frame corresponding to the 3' half of the rps7 gene, encoding the S7 protein of the small subunit of the chloroplast ribosome. Only the 5' portion of this gene is located in the opposite unique sequence region of the C. reinhardtii chloroplast genome where the rps7 gene was previously mapped by heterologous hybridization.