Red Alga Porphyridium Purpureum Research Articles

The red alga Porphyridium as a host for molecular farming: Efficient production of immunologically active hepatitis C virus glycoprotein

Microalgae are promising production platforms for the cost-effective production of recombinant proteins. We have recently established that the red alga Porphyridium purpureum provides superior transgene expression properties, due to the episomal maintenance of transformation vectors as multicopy plasmids in the nucleus. Here, we have explored the potential of Porphyridium to synthesize complex pharmaceutical proteins to high levels. Testing expression constructs for a candidate subunit vaccine against the hepatitis C virus (HCV), we show that the soluble HCV E2 glycoprotein can be produced in transgenic algal cultures to high levels. The antigen undergoes faithful posttranslational modification by N-glycosylation and is recognized by conformationally selective antibodies, suggesting that it adopts a proper antigenic conformation in the endoplasmic reticulum of red algal cells. We also report the experimental determination of the structure of the N-glycan moiety that is attached to glycosylated proteins in Porphyridium. Finally, we demonstrate the immunogenicity of the HCV antigen produced in red algae when administered by injection as pure protein or by feeding of algal biomass.

Optimized transgene expression in the red alga Porphyridium purpureum and efficient recombinant protein secretion into the culture medium

Microalgae represent a promising but yet underexplored production platform for biotechnology. The vast majority of studies on recombinant protein expression in algae have been conducted in a single species, the green alga Chlamydomonas reinhardtii. However, due to epigenetic silencing, transgene expression in Chlamydomonas is often inefficient. Here we have investigated parameters that govern efficient transgene expression in the red microalga Porphyridium purpureum. Porphyridium is unique in that the introduced transformation vectors are episomally maintained as autonomously replicating plasmids in the nucleus. We show that full codon optimization to the preferred codon usage in the Porphyridium genome confers superior transgene expression, not only at the level of protein accumulation, but also at the level of mRNA accumulation, indicating that high translation rates increase mRNA stability. Our optimized expression constructs resulted in YFP accumulation to unprecedented levels of up to 5% of the total soluble protein. We also designed expression cassettes that target foreign proteins to the secretory pathway and lead to efficient protein secretion into the culture medium, thus simplifying recombinant protein harvest and purification. Our study paves the way to the exploration of red microalgae as expression hosts in molecular farming for recombinant proteins and metabolites.

Pilot-scale cultivation of the red alga Porphyridium purpureum over a two-year period in a greenhouse

Microalgae can form renewable feedstock for many sectors. However, most microalgae commercially cultivated are difficult to grow in colder climates. To overcome this problem one could cultivate cold tolerant algae. One such alga is the red marine alga Porphyridium purpureum. It is currently often cultivated in bag systems in limited volumes. In this study we cultivated P. purpureum at pilot-scale in horizontal tubular reactors in a greenhouse. P. purpureum was grown in batch mode in a total volume of either 350, 500, 1000 or 1500 L. A total of 28 batches, spread over two years, were cultivated reaching a maximum dry weight of 1.94 g L−1. The growth was continuously monitored to calculate the productivities and growth rates. The P and N concentrations were measured frequently during four batches to calculate the P and N usage per gram biomass. To calculate CO2 fixation efficiencies the total CO2 consumption was monitored during 6 of the 28 batches. A maximum productivity and growth rate of respectively 0.195 g L−1 day−1 and 0.233 day−1 were achieved while the P and N usage varied from 3.2–6.19 and 14.5–36.6 mg g−1 biomass for P and N, respectively. Growth was supported between 10 °C and 30 °C, while temperatures above 34—35 °C proved to be detrimental. The CO2 efficiencies differed among the 6 tested periods with the highest utilization efficiency being 13.5% and the lowest a mere 1.5%. Further large-scale, continuous, tests would be needed to fully optimize the cultivation of P. purpureum in larger volumes.

In situ structure of the red algal phycobilisome-PSII-PSI-LHC megacomplex.

In oxygenic photosynthetic organisms, light energy is captured by antenna systems and transferred to photosystem II (PSII) and photosystem I (PSI) to drive photosynthesis1,2. The antenna systems of red algae consist of soluble phycobilisomes (PBSs) and transmembrane light-harvesting complexes (LHCs)3. Excitation energy transfer pathways from PBS to photosystems remain unclear owing to the lack of structural information. Here we present in situ structures of PBS-PSII-PSI-LHC megacomplexes from the red alga Porphyridium purpureum at near-atomic resolution using cryogenicelectron tomography and in situ single-particle analysis4, providing interaction details between PBS, PSII and PSI. The structures reveal several unidentified and incomplete proteins and their roles in the assembly of the megacomplex, as well as a huge and sophisticated pigment network. This work provides a solid structural basis for unravelling the mechanisms of PBS-PSII-PSI-LHC megacomplex assembly, efficient energy transfer from PBS to the two photosystems, and regulation of energy distribution between PSII and PSI.

Redshifting and Blueshifting of β82 Chromophores in the Phycocyanin Hexamer of Porphyridium purpureum Phycobilisomes Due to Linker Proteins.

Phycobilisomes in cyanobacteria and red algae are large protein complexes that absorb light and transfer energy for use in photosynthesis. The light energy absorbed by chromophores binding to phycobiliproteins in the peripheral rods can be funneled to the core through chromophores at very high efficiency. The molecular mechanism of excitation energy transfer within a phycobilisome is an example of a higher and unique function in a living organism. However, the mechanism underlying the high efficiency remains unclear. Thus, this study was carried out as a step to resolve this mechanism theoretically. The three-dimensional structure of phycobilisomes containing the linker proteins of the red alga Porphyridium purpureum was determined by cryoelectron microscopy at 2.82 Å resolution in 2020. Using these data, the absorption wavelength of each β82 chromophore in the phycocyanin hexamer located next to the core was calculated using quantum chemical treatment, considering the electric effect from its surrounding phycocyanin proteins and two linker proteins. In addition to unaffected chromophores, chromophores that were redshifted and blueshifted under the electrical influence of the two linker proteins were found. Namely, the chromophore serving as the energy sink in the rod was determined.

The evolution of pre-mRNA splicing and its machinery revealed by reduced extremophilic red algae.

The Cyanidiales are a group of mostly thermophilic and acidophilic red algae that thrive near volcanic vents. Despite their phylogenetic relationship, the reduced genomes of Cyanidioschyzon merolae and Galdieria sulphuraria are strikingly different with respect to pre-mRNA splicing, a ubiquitous eukaryotic feature. Introns are rare and spliceosomal machinery is extremely reduced in C.merolae, in contrast to G.sulphuraria. Previous studies also revealed divergent spliceosomes in the mesophilic red alga Porphyridium purpureum and the red algal derived plastid of Guillardia theta (Cryptophyta), along with unusually high levels of unspliced transcripts. To further examine the evolution of splicing in red algae, we compared C.merolae and G.sulphuraria, investigating splicing levels, intron position, intron sequence features, and the composition of the spliceosome. In addition to identifying 11 additional introns in C. merolae, our transcriptomic analysis also revealed typical eukaryotic splicing in G. sulphuraria, whereas most transcripts in C. merolae remain unspliced. The distribution of intron positions within their host genes was examined to provide insight into patterns of intron loss in red algae. We observed increasing variability of 5' splice sites and branch donor regions with increasing intron richness. We also found these relationships to be connected to reductions in and losses of corresponding parts of the spliceosome. Our findings highlight patterns of intron and spliceosome evolution in related red algae under the pressures of genome reduction.

Effects of harvesting on morphological and biochemical characteristics of microalgal biomass harvested by polyacrylamide addition, pH-induced flocculation, and centrifugation

The effects of microalgae harvesting methods on microalgal biomass quality were evaluated using three species namely the freshwater green alga Chlorella vulgaris, marine red alga Porphyridium purpureum and marine diatom Phaeodactylum tricornutum. Harvesting efficiencies of polyacrylamide addition, alkaline addition, and centrifugation ranged from 85 to 95, 59–92 and 100%, respectively, across these species. Morphology of the harvested cells (i.e. compromised cell walls) was significantly impacted by alkaline pH-induced flocculation for all three species. Over 50% of C. vulgaris cells were compromised with alkaline pH compared to < 10% with polyacrylamide and centrifugation. The metabolic profiles varied depending on harvesting methods. Species-specific decrease of certain metabolites was observed. These results suggest that the method of harvest can alter the metabolic profile of the biomass amongst the three harvesting methods, polyacrylamide addition showed higher harvesting efficiency with less compromised cells and higher retention of industry important biochemicals.

Photoprotective energy quenching in the red alga Porphyridium purpureum occurs at the core antenna of the photosystem II but not at its reaction center

Photosynthetic organisms have evolved light-harvesting antennae over time. In cyanobacteria, external phycobilisomes (PBSs) are the dominant antennae, whereas in green algae and higher plants, PBSs have been replaced by proteins of the Lhc family that are integrated in the membrane. Red algae represent an evolutionary intermediate between these two systems, as they employ both PBSs and membrane LHCR proteins as light-harvesting units. Understanding how red algae cope with light is not only interesting for biotechnological applications, but is also of evolutionary interest. For example, energy-dependent quenching (qE) is an essential photoprotective mechanism widely used by species from cyanobacteria to higher plants to avoid light damage; however, the quenching mechanism in red algae remains largely unexplored. Here, we used both pulse amplitude-modulated (PAM) and time-resolved chlorophyll fluorescence to characterize qE kinetics in the red alga Porphyridium purpureum. PAM traces confirmed that qE in P. purpureum is activated by a decrease in the thylakoid lumen pH, whereas time-resolved fluorescence results further revealed the quenching site and ultrafast quenching kinetics. We found that quenching exclusively takes place in the photosystem II (PSII) complexes and preferentially occurs at PSII’s core antenna rather than at its reaction center, with an overall quenching rate of 17.6 ± 3.0 ns−1. In conclusion, we propose that qE in red algae is not a reaction center type of quenching, and that there might be a membrane-bound protein that resembles PsbS of higher plants or LHCSR of green algae that senses low luminal pH and triggers qE in red algae.

Towards a general kinetic microalgae model: Extending a semi-deterministic green microalgae model for the cyanobacterium Arthrospira platensis and red alga Porphyridium purpureum

Mathematical models for microalgae and cyanobacteria are seldomly validated for different algal species, as such limiting their applicability. Therefore, in this research, a previously developed kinetic model describing the growth of the green microalgae species Chlorella vulgaris was used to simulate the growth of the cyanobacterium Arthrospira platensis and the red alga Porphyridium purpureum. Based on a global sensitivity analysis, the model parameter µmax,A was calibrated using respirometric-titrimetric data. Calibration yielded values of 5.76 ± 0.17 d-1, 2.06 ± 0.16 d-1 and 1.06 ± 0.09 d-1 for Chlorella vulgaris, Arthrospira platensis and Porphyridium purpureum, respectively. Model simulations revealed that the biological growth equations in this model are adequate. However, increased light intensities triggered a survival mechanism for Arthrospira platensis, which is currently not taken into account by the model, leading to bad model accuracy under these circumstances. Future work should address the most important survival mechanisms and include those in the model to widen its applicability.

Assessment of light distribution model for marine red microalga Porphyridium purpureum for sustainable production in photobioreactor

Microalgae can accumulate large amounts of nutrients from sustainable elements cycling and wastewater reuse. Light is a key factor determining productivity of algal biomass when recycling. However, light is unevenly distributed in photobioreactors due to light absorption and scatter by algal cells. Understanding the laws of light distribution is needed for optimizing the culture process. In this study, a light distribution model for red alga Porphyridium purpureum was used to optimize the cultivation process and improve culture performance by controlling light intensity. Compared to the Lambert-Beer model, the Hyperbolic law is more flexible and is applicable in a larger range of cell densities. In addition, the content of intracellular phycobiliprotein has a great influence on light distribution inside the photobioreactor while a higher biomass concentration reduces its impact. A maximum biomass concentration of 24.7 g/L along with 1.2 g/L/d cell growth rate was achieved by increasing incident light intensity in a stepwise fashion. The overall light conditions inside the photobioreactor during the culture process was also analyzed and discussed based on light distribution models. A volume-averaged light intensity regulated at ~50 μmol/m2/s along with an incident light intensity of less than 540 μmol/m2/s is an appropriate light condition for Porphyridium purpureum culture. The outcomes of light distribution model assessment and stepwise fashion regulation strategy can be applied to red microalgae in general, especially for high cell density cultivation. These findings provide an essential base for culture optimization, which can be used in future industrial applications of P. purpureum for water clean and sustainability of human nourishment production.

Molecular mechanism of arachidonic acid biosynthesis in Porphyridium purpureum promoted by nitrogen limitation.

The red alga Porphyridium purpureum has been known to produce polyunsaturated fatty acids, especially arachidonic acid (ARA), under stressful conditions. However, there is no consistent conclusion about the response of ARA in this alga to nitrogen (N) stress. Also, no research has been done to clearly elucidate the underlying molecular mechanisms of N stress. In this work, P. purpureum CoE1 was cultivated under nitrogen limitation conditions and the putative Δ5-desaturase related gene FADSD5 was isolated. The results showed that the fatty acids in P. purpureum CoE1 were significantly higher in the N limited cultures (54.3mgg-1) than in the N-replete cultures (45.3mgg-1) at the 18th day (t-test, p < 0.001), which was attributed to the upregulated abundance of the putative Δ5-desaturase related protein, Δ5-Des. The study also indicated that the expression of the putative Δ5-desaturase related gene, FADSD5, increased with cell growth, demonstrating considerable potentials for ARA biosynthesis in P. purpureum CoE1. These results might guide the direction in illuminating the biosynthetic pathway of fatty acids with molecular evidence and enable genetic modifications of P. purpureum CoE1 for enhancing the ARA accumulation.

Characterization of Pre-mRNA Splicing and Spliceosomal Machinery in Porphyridium purpureum and Evolutionary Implications for Red Algae.

Pre-mRNA splicing is a highly conserved eukaryotic process, but our understanding of it is limited by a historical focus on well-studied organisms such as humans and yeast. There is considerable diversity in mechanisms and components of pre-mRNA splicing, especially in lineages that have evolved under the pressures of genome reduction. The ancestor of red algae is thought to have undergone genome reduction prior to the lineage's radiation, resulting in overall gene and intron loss in extant groups. Previous studies on the extremophilic red alga Cyanidioschyzon merolae revealed an intron-sparse genome with a highly reduced spliceosome. To determine whether these features applied to other red algae, we investigated multiple aspects of pre-mRNA splicing in the mesophilic red alga Porphyridium purpureum. Through strand-specific RNA-Seq, we observed high levels of intron retention across a large number of its introns, and nearly half of the transcripts for these genes are not spliced at all. We also discovered a relationship between variability of 5' splice site sequences and levels of splicing. To further investigate the connections between intron retention and splicing machinery, we bioinformatically assembled the P.purpureum spliceosome, and biochemically verified the presence of snRNAs. While most other core spliceosomal components are present, our results suggest highly divergent or missing U1 snRNP proteins, despite the presence of an uncharacteristically long U1 snRNA. These unusual aspects highlight the diverse nature of pre-mRNA splicing that can be seen in lesser-studied eukaryotes, raising the importance of investigating fundamental eukaryotic processes outside of model organisms.

Structural basis of energy transfer in Porphyridium purpureum phycobilisome.

Photosynthetic organisms have developed various light-harvesting systems to adapt to their environments1. Phycobilisomes are large light-harvesting protein complexes found in cyanobacteria and red algae2-4, although how the energies of the chromophores within these complexes are modulated by their environment is unclear. Here we report the cryo-electron microscopy structure of a 14.7-megadalton phycobilisome with a hemiellipsoidal shape from the red alga Porphyridium purpureum. Within this complex we determine the structures of 706 protein subunits, including 528 phycoerythrin, 72 phycocyanin, 46 allophycocyanin and 60 linker proteins. In addition, 1,598 chromophores are resolved comprising 1,430 phycoerythrobilin, 48 phycourobilin and 120 phycocyanobilin molecules. The markedly improved resolution of our structure compared with that of the phycobilisome of Griffithsia pacifica5 enabled us to build an accurate atomic model of the P. purpureum phycobilisome system. The model reveals how the linker proteins affect the microenvironment of the chromophores, and suggests that interactions of the aromatic amino acids of the linker proteins with the chromophores may be a key factor in fine-tuning the energy states of the chromophores to ensure the efficient unidirectional transfer of energy.

Correction to: Induced cultivation pattern enhanced the phycoerythrin production in red alga Porphyridium purpureum.

The original version of the article unfortunately contained an error in Microalgae strain and culture medium section. Below is the corrected version.

Expansion of phycobilisome linker gene families in mesophilic red algae

The common ancestor of red algae (Rhodophyta) has undergone massive genome reduction, whereby 25% of the gene inventory has been lost, followed by its split into the species-poor extremophilic Cyanidiophytina and the broadly distributed mesophilic red algae. Success of the mesophile radiation is surprising given their highly reduced gene inventory. To address this latter issue, we combine an improved genome assembly from the unicellular red alga Porphyridium purpureum with a diverse collection of other algal genomes to reconstruct ancient endosymbiotic gene transfers (EGTs) and gene duplications. We find EGTs associated with the core photosynthetic machinery that may have played important roles in plastid establishment. More significant are the extensive duplications and diversification of nuclear gene families encoding phycobilisome linker proteins that stabilize light-harvesting functions. We speculate that the origin of these complex families in mesophilic red algae may have contributed to their adaptation to a diversity of light environments.

Rapid functional activation of a horizontally transferred eukaryotic gene in a bacterial genome in the absence of selection.

Horizontal gene transfer has occurred between organisms of all domains of life and contributed substantially to genome evolution in both prokaryotes and eukaryotes. Phylogenetic evidence suggests that eukaryotic genes horizontally transferred to bacteria provided useful new gene functions that improved metabolic plasticity and facilitated adaptation to new environments. How these eukaryotic genes evolved into functional bacterial genes is not known. Here, we have conducted a genetic screen to identify the mechanisms involved in functional activation of a eukaryotic gene after its transfer into a bacterial genome. We integrated a eukaryotic selectable marker gene cassette driven by expression elements from the red alga Porphyridium purpureum into the genome of Escherichia coli. Following growth under non-selective conditions, gene activation events were indentified by antibiotic selection. We show that gene activation in the bacterial recipient occurs at high frequency and involves two major types of spontaneous mutations: deletion and gene amplification. We further show that both mechanisms result in promoter capture and are frequently triggered by microhomology-mediated recombination. Our data suggest that horizontally transferred genes have a high probability of acquiring functionality, resulting in their maintenance if they confer a selective advantage.

Replication of bacterial plasmids in the nucleus of the red alga Porphyridium purpureum

Rhodophytes (red algae) are a diverse group of algae with great ecological and economic importance. However, tools for post-genomic research on red algae are still largely lacking. Here, we report the development of an efficient genetic transformation system for the model rhodophyte Porphyridium purpureum. We show that transgenes can be expressed to unprecedented levels of up to 5% of the total soluble protein. Surprisingly, the transgenic DNA is maintained episomally, as extrachromosomal high-copy number plasmid. The bacterial replication origin confers replication in the algal nucleus, thus providing an intriguing example of a prokaryotic replication origin functioning in a eukaryotic system. The extended presence of bacterial episomal elements may provide an evolutionary explanation for the frequent natural occurrence of extrachromosomal plasmids in red algae, and may also have contributed to the high rate of horizontal gene transfer from bacteria to the nuclear genome of Porphyridium purpureum and other rhodophytes.

Adaptive abilities of microscopic red alga &lt;i&gt;Porphyridium purpureum&lt;/i&gt; (strain PP-AB11) under change of salinity in the medium

Adaptive abilities of the microscopic red alga Porphyridium purpureum (strain PP-AB11, isolated from the northwestern Japan Sea) are investigated experimentally by its repeated cultivation under various water salinity. Under the salinity of 8 ‰, the growth of P. purpureum was inhibited (0.1 division/day) during the first four days though there were no morphological differences of the cells as compared with those in the control (mean cell size was 6.3±1.2 µm in both cases); the cell density increased to 85 % of the control value after 21 days exposure. Under the salinity of 4 ‰, the lag-phase was also 4 days but size of the cells increased (on average up to 7.8 ± 1.5 µm), the cell morphology changed including the cytoplasm retraction, and contraction and granulation of the chloroplasts; the cell density increased less (to 53 % of the control value) after 21 days exposure. Under the salinity of 2 ‰, changes of the cell morphology and density were the most prominent: density of viable cells decreased to 6 % of the control value after 4-days exposure, size of the cells increased on average up to 8.7 ± 1.6 µm due to hydration, granulation was noticed in the chloroplasts, and the cytoplasm became dense. Under the salinity of 0 ‰, all the cells died after 4 days. Being cultivated repeatedly, the alga was better adapted to low salinity: after 21-days exposure the cell density was 97 % of the control value under salinity of 8 ‰ and 84 % of the control value under salinity of 4 ‰, with the growth rate 0.5 divisions/day and without visible morphological differences of the cells as compared with those in the control. It was adapted even to salinity of 2 ‰: the cells morphology was restored though the growth rate after 21-days exposition was still lower than in the control because of serious deterioration of the cells. The growth rate and cell morphology did not significantly differ from the control after reinoculation of the culture, previously adapted to salinity of 2 ‰, into the medium with salinity of 32 ‰.

Recent mobility of plastid encoded group II introns and twintrons in five strains of the unicellular red alga Porphyridium.

Group II introns are closely linked to eukaryote evolution because nuclear spliceosomal introns and the small RNAs associated with the spliceosome are thought to trace their ancient origins to these mobile elements. Therefore, elucidating how group II introns move, and how they lose mobility can potentially shed light on fundamental aspects of eukaryote biology. To this end, we studied five strains of the unicellular red alga Porphyridium purpureum that surprisingly contain 42 group II introns in their plastid genomes. We focused on a subset of these introns that encode mobility-conferring intron-encoded proteins (IEPs) and found them to be distributed among the strains in a lineage-specific manner. The reverse transcriptase and maturase domains were present in all lineages but the DNA endonuclease domain was deleted in vertically inherited introns, demonstrating a key step in the loss of mobility. P. purpureum plastid intron RNAs had a classic group IIB secondary structure despite variability in the DIII and DVI domains. We report for the first time the presence of twintrons (introns-within-introns, derived from the same mobile element) in Rhodophyta. The P. purpureum IEPs and their mobile introns provide a valuable model for the study of mobile retroelements in eukaryotes and offer promise for biotechnological applications.

The development of Porphyridium purpureum (Bory de Saint-Vincent) Drew et Ross, 1965 (Rhodophyta) from Amursky Bay, Sea of Japan, in a laboratory culture

The growth of the microscopic red alga Porphyridium purpureum was experimentally studied in different culture media and at various levels of water salinity. The ability of this species to recover after cryopreservation was analyzed. It was revealed that the growth rate and generation time of P. purpureum during the exponential growth phase did not significantly differ between cultures on f medium and those on Goldberg’s medium. The species has been found to withstand salinity variations ranging from 8 to 32 vol %. Cryopreservation of P. purpureum cells using three-step freezing with trehalose as a cryoprotectant showed that the dynamics of cell density after thawing insignificantly differed from the control ones.