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.

Inorganic salt starvation improves the polysaccharide production and CO2 fixation by Porphyridium purpureum.

The microalgae industry shows a promising future in the production of high-value products such as pigments, phycoerythrin, polyunsaturated fatty acids, and polysaccharides. It was found that polysaccharides have high biomedical value (such as antiviral, antibacterial, antitumor, antioxidative) and industrial application prospects (such as antioxidants). This study aimed to improve the polysaccharides accumulation of Porphyridium purpureum CoE1, which was effectuated by inorganic salt starvation strategy whilst supplying rich carbon dioxide. At a culturing temperature of 25 °C, the highest polysaccharide content (2.89 g/L) was achieved in 50% artificial seawater on the 12th day. This accounted for approximately 37.29% of the dry biomass, signifying a 25.3% increase in polysaccharide production compared to the culture in 100% artificial seawater. Subsequently, separation, purification and characterization of polysaccharides produced were conducted. Furthermore, the assessment of CO2 fixation capacity during the cultivation of P. purpureum CoE1 was conducted in a 10 L photobioreactor. This indicated that the strain exhibited an excellent CO2 fixation capacity of 1.66 g CO2/g biomass/d. This study proposed an efficient and feasible approach that not only increasing the yield of polysaccharides by P. purpureum CoE1, but also fixing CO2 with a high rate, which showed great potential in the microalgae industry and Bio-Energy with Carbon Capture and Storage.

Effects of Silver Nanoparticles on the Red Microalga Porphyridium purpureum CNMN-AR-02, Cultivated on Two Nutrient Media.

The purpose of this study was to examine the influence of 10 and 20 nm nanoparticles (AgNPs) on the growth and biochemical composition of microalga Porphyridium purpureum CNMN-AR-02 in two media which differ by the total amount of mineral salts (MM1 with 33.02 g/L and MM2 with 21.65 g/L). Spectrophotometric methods were used to estimate the amount of biomass and its biochemical composition. This study provides evidence of both stimulatory and inhibitory effects of AgNPs on different parameters depending on the concentration, size, and composition of the nutrient medium. In relation to the mineral medium, AgNPs exhibited various effects on the content of proteins (an increase up to 20.5% in MM2 and a decrease up to 36.8% in MM1), carbohydrates (a decrease up to 35.8% in MM1 and 39.6% in MM2), phycobiliproteins (an increase up to 15.7% in MM2 and 56.8% in MM1), lipids (an increase up to 197% in MM1 and no changes found in MM2), antioxidant activity (a decrease in both media). The composition of the cultivation medium has been revealed as one of the factors influencing the involvement of nanoparticles in the biosynthetic activity of microalgae.

Impacts of microplastic and seawater acidification on unicellular red algae: Growth response, photosynthesis, antioxidant enzymes, and extracellular polymer substances.

Microplastics (MPs) pollution and seawater acidification have increasingly become huge threats to the ocean ecosystem. Their impacts on microalgae are of great importance, since microalgae are the main primary producers and play a critical role in marine ecosystems. However, the impact of microplastics and acidification on unicellular red algae, which have a unique phycobiliprotein antenna system, remains unclear. Therefore, the impacts of polystyrene-MPs alone and the combined effects of MPs and seawater acidification on the typical unicellular marine red algae Porphyridium purpureum were investigated in the current study. The result showed that, under normal seawater condition, microalgae densities were increased by 17.75-41.67 % compared to the control when microalgae were exposed to small-sized MPs (0.1 µm) at concentrations of 5-100 mg L-1. In addition, the photosystem II and antioxidant enzyme system were not subjected to negative effects. The large-sized MPs (1 µm) boosted microalgae growth at a low concentration of MPs (5 mg L-1). However, it was observed that microalgae growth was significantly inhibited when MPs concentration increased up to 50 and 100 mg L-1, accompanied by the remarkably reduced Fv/Fm value and the elevated levels of SOD, CAT enzymes, phycoerythrin (PE), and extracellular polysaccharide (EPS). Compared to the normal seawater condition, microalgae densities were enhanced by 52.11-332.56 % under seawater acidification, depending on MPs sizes and concentrations, due to the formed CO2-enrichment condition and appropriate pH range. PE content in microalgal cells was significantly enhanced, but SOD and CAT activities as well as EPS content markedly decreased under acidification conditions. Overall, the impacts of seawater acidification were more pronounced than MPs impacts on microalgae growth and physiological responses. These findings will contribute to a substantial understanding of the effects of MPs on marine unicellular red microalgae, especially in future seawater acidification scenarios.

Green and efficient method to acquire high-value phycobiliprotein from microalgal biomass involving deep eutectic solvent-based ultrasound-assisted extraction.

Phycoerythrin (PE) is a phycobiliprotein holding great potential as a high-value food colorant and medicine. Deep eutectic solvent (DES)-based ultrasound-assisted extraction (UAE) was applied to extract B-PE by disrupting the resistant polysaccharide cell wall of Porphyridium purpureum. The solubility of cell wall monomers in 31 DESs was predicted using COSMO-RS. Five glycerol-based DESs were tested for extraction, all of which showed significantly higher B-PE yields by up to 13.5 folds than water. The DES-dependent B-PE extraction efficiencies were proposedly associated with different cell disrupting capabilities and protein stabilizing effects of DESs. The DES-based UAE method could be considered green according to a metric assessment tool, AGREEprep. The crude extract containing DES was further subjected to aqueous two-phase system, two-step ammonium sulfate precipitation, and ultrafiltration processes. The final purified B-PE had a PE purity ratio of 3.60 and a PC purity ratio of 0.08, comparable to the purity of commercial products.

Maximizing Polysaccharides and Phycoerythrin in Porphyridium purpureum via the Addition of Exogenous Compounds: A Response-Surface-Methodology Approach.

Phycoerythrin and polysaccharides have significant commercial value in medicine, cosmetics, and food industries due to their excellent bioactive functions. To maximize the production of biomass, phycoerythrin, and polysaccharides in Porphyridium purpureum, culture media were supplemented with calcium gluconate (CG), magnesium gluconate (MG) and polypeptides (BT), and their optimal amounts were determined using the response surface methodology (RSM) based on three single-factor experiments. The optimal concentrations of CG, MG, and BT were determined to be 4, 12, and 2 g L-1, respectively. The RSM-based models indicated that biomass and phycoerythrin production were significantly affected only by MG and BT, respectively. However, polysaccharide production was significantly affected by the interactions between CG and BT and those between MG and BT, with no significant effect from BT alone. Using the optimized culture conditions, the maximum biomass (5.97 g L-1), phycoerythrin (102.95 mg L-1), and polysaccharide (1.42 g L-1) concentrations met and even surpassed the model-predicted maximums. After optimization, biomass, phycoerythrin, and polysaccharides concentrations increased by 132.3%, 27.97%, and 136.67%, respectively, compared to the control. Overall, this study establishes a strong foundation for the highly efficient production of phycoerythrin and polysaccharides using P. purpureum.

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.

Reconstruction of Long-Chain Polyunsaturated Acid Synthesis Pathways in Marine Red Microalga Porphyridium cruentum Using Lipidomics and Transcriptomics.

The marine red microalga Porphyridium can simultaneously synthesize long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (C20:5, EPA) and arachidonic acid (C20:4, ARA). However, the distribution and synthesis pathways of EPA and ARA in Porphyridium are not clearly understood. In this study, Porphyridium cruentum CCALA 415 was cultured in nitrogen-replete and nitrogen-limited conditions. Fatty acid content determination, transcriptomic, and lipidomic analyses were used to investigate the synthesis of ARA and EPA. The results show that membrane lipids were the main components of lipids, while storage lipids were present in a small proportion in CCALA 415. Nitrogen limitation enhanced the synthesis of storage lipids and ω6 fatty acids while inhibiting the synthesis of membrane lipids and ω3 fatty acids. A total of 217 glycerolipid molecular species were identified, and the most abundant species included monogalactosyldiglyceride (C16:0/C20:5) (MGDG) and phosphatidylcholine (C16:0/C20:4) (PC). ARA was mainly distributed in PC, and EPA was mainly distributed in MGDG. Among all the fatty acid desaturases (FADs), the expressions of Δ5FAD, Δ6FAD, Δ9FAD, and Δ12FAD were up-regulated, whereas those of Δ15FAD and Δ17FAD were down-regulated. Based on these results, only a small proportion of EPA was synthesized through the ω3 pathway, while the majority of EPA was synthesized through the ω6 pathway. ARA synthesized in the ER was likely shuttled into the chloroplast by DAG and was converted into EPA by Δ17FAD.

Enhanced growth and metabolite production from a novel strain of Porphyridium sp.

Microalgae are capable of generating numerous metabolites that possess notable biological activities and hold substantial promise for various industrial applications. Nevertheless, the taxonomic diversity of these photosynthetic microorganisms has not received thorough investigation. Using the 18S rRNA encoding gene, a recently discovered strain originating from the Tunisian coast (the governorate of Mahdia) was identified as a member of the Porphyridium genus. The growth response as well as the metabolite accumulation of Porphyridium sp. to different culture media (Pm, F/2, and Hemerick) was investigated over a period of 52 days. The highest biomass production was recorded with Pm medium (2 × 107 cell/mL). The apparent growth rates (µ) and the doubling time (Dt) were about 0.081 day-1 and 12.34 days, respectively. The highest chlorophyll a (0.678 ± 0.005 pg/cell), total carotenoids (0.18 ± 0.003 pg/cell), phycoerythrin (3.88 ± 0.003 pg/cell), and proteins (14.58 ± 0.35 pg/cell) contents were observed with F/2 medium. Cultivating Porphyridium sp. in both F/2 and Hemerick media yielded similar levels of starch accumulation. The Hemerick medium has proven to be the most suitable for the production of lipids (2.23% DW) and exopolysaccharides (5.41 ± 0.56 pg/cell).