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).

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 cryogenic electron 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.

Effect of specific irradiance on productivity and pigment and protein production of Porphyridium purpureum (Rhodophyta) semi-continuous culture.

Porphyridium purpureum is a promising microalga species due to the content of various valuable compounds. In this study, specific irradiance parameter, representing the amount of light energy per unit of microalgae biomass, was introduced. The growth characteristics and pigments and protein accumulation of P. purpureum culture were investigated under semi-continuous mode. Varying dilution rate and surface irradiance resulted in a specific irradiance of 0.2-6.7 W g-1. Using mathematical modeling, we determined the patterns of changes in biomass, pigments, protein content and productivity of P. purpureum culture depending on specific irradiance. The content of target compounds was maximized under the lowest level of specific irradiance (0.2-1.2 W g-1), while the highest productivity of this components was reached under 1.2-1.7 W g-1. Overall, lower irradiance levels were favorable for P. purpureum cultivation based on the energy consumption and production characteristics of this species.

Integrated green one-step strategy for concurrent recovery of phycobiliproteins and polyunsaturated fatty acids from wet Porphyridium biomass.

A novel and green one-step simultaneous extraction process of phycobiliproteins and polyunsaturated fatty acids (PUFAs) from wet Porphyridium biomass has been done and optimized by using three phase partitioning (TPP) process. Results showed that the coupling of ammonium sulfate and protein buoyancy-promoting t-butanol afforded the best TPP to extract phycobiliproteins and PUFAs in term of the extraction performance and cost-effectiveness. TPP process gave the best capability to simultaneously extract Porphyridium-derived phycobiliproteins and PUFAs in 20% ammonium sulfate, 0.5% biomass, and 1:0.5 slurry to t-butanol ratio at 100 rpm and 20 °C for 10 min of extraction time. Moreover, the established TPP system achieved excellent reproducibility in the extraction of Porphyridium biomass from different sources (Porphyridium cruentum and P. purpureum); and was successfully implemented in pilot-scale (20-L), indicating its industrial potential as a promising integrated approach to comprehensively exploit Porphyridium as a renewable bioresource for high-value bioproducts.

Exploring the Diversity of Red Microalgae for Exopolysaccharide Production.

Microalgae constitute a remarkable biological diversity but a limited number of them have been the object of study for their ability to produce exoplysaccharides (EPS). Among them, the red marine microalgae Porphyridium or Rhodella produce sulphated EPS, exhibiting some biological activities with potential interest in the pharmaceutical and cosmetic industries. EPS from Porphyridium and Rhodella being relatively similar in their composition, it has long been considered that all the red microalgae produced similar EPS and no attention was paid to other red microalgae. The objective of our work was then to explore the diversity of red microalgae for the production of EPS, focusing in this first step on the screening of the strains for their ability to produce EPS and preliminary structural characterization. The study was conducted with 11 microalgae strains belonging to the proteorhodophytina subphylum. All microalgae were able to produce EPS, released in the culture medium (strains belonging to Porphyridiophyceae and Rhodellophyceae classes) or remaining bound to the cells (strains from Stylonematophyceae class). The analysis of monosaccharides composition was found significantly different, with for instance high levels of glucuronic acids in the EPS from C. japonica and N. cyanea, but also strong differences in the sulphation degrees of polymers (between 1.2 and 28.7% eq. SO4).

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.

Transcriptomic survey reveals multiple adaptation mechanisms in response to nitrogen deprivation in marine Porphyridium cruentum.

Single-cell red microalga Porphyridium cruentum is potentially considered to be the bioresource for biofuel and pharmaceutical production. Nitrogen is a kind of nutrient component for photosynthetic P. cruentum. Meanwhile, nitrogen stress could induce to accumulate some substances such as lipid and phycoerythrin and affect its growth and physiology. However, how marine microalga Porphyridium cruentum respond and adapt to nitrogen starvation remains elusive. Here, acclimation of the metabolic reprogramming to changes in the nutrient environment was studied by high-throughput mRNA sequencing in the unicellular red alga P. cruentum. Firstly, to reveal transcriptional regulation, de novo transcriptome was assembled and 8,244 unigenes were annotated based on different database. Secondly, under nitrogen deprivation, 2100 unigenes displayed differential expression (1134 upregulation and 966 downregulation, respectively) and some pathways including carbon/nitrogen metabolism, photosynthesis, and lipid metabolism would be reprogrammed in P. cruentum. The result demonstrated that nitrate assimilation (with related unigenes of 8-493 fold upregulation) would be strengthen and photosynthesis (with related unigenes of 6-35 fold downregulation) be impaired under nitrogen deprivation. Importantly, compared to other green algae, red microalga P. cruentum presented a different expression pattern of lipid metabolism in response to nitrogen stress. These observations will also provide novel insight for understanding adaption mechanisms and potential targets for metabolic engineering and synthetic biology in P. cruentum.

Implementation of kLa-Based Strategy for Scaling Up Porphyridium purpureum (Red Marine Microalga) to Produce High-Value Phycoerythrin, Fatty Acids, and Proteins.

Porphyridium purpureum is a well-known Rhodophyta that recently has attracted enormous attention because of its capacity to produce many high-value metabolites such as the pigment phycoerythrin and several high-value fatty acids. Phycoerythrin is a fluorescent red protein-pigment commercially relevant with antioxidant, antimicrobial activity, and fluorescent properties. The volumetric mass transfer coefficient (kLa) was kept constant within the different scaling-up stages in the present study. This scaling-up strategy was sought to maintain phycoerythrin production and other high-value metabolites by Porphyridium purpureum, using hanging-bag photobioreactors. The kLa was monitored to ensure the appropriate mixing and CO2 diffusion in the entire culture during the scaling process (16, 80, and 400 L). Then, biomass concentration, proteins, fatty acids, carbohydrates, and phycoerythrin were determined in each step of the scaling-up process. The kLa at 16 L reached a level of 0.0052 s-1, while at 80 L, a value of 0.0024 s-1 was achieved. This work result indicated that at 400 L, 1.22 g L-1 of biomass was obtained, and total carbohydrates (117.24 mg L-1), proteins (240.63 mg L-1), and lipids (17.75% DW) were accumulated. Regarding fatty acids production, 46.03% palmitic, 8.03% linoleic, 22.67% arachidonic, and 2.55% eicosapentaenoic acid were identified, principally. The phycoerythrin production was 20.88 mg L-1 with a purity of 2.75, making it viable for food-related applications. The results of these experiments provide insight into the high-scale production of phycoerythrin via the cultivation of P. purpureum in an inexpensive and straightforward culture system.

Photobiological Effects on Biochemical Composition in Porphyridium cruentum (Rhodophyta) with a Biotechnological Application.

This study describes the relation of photosynthetic capacity, growth and biochemical compounds in the microalgae Porphyridium cruentum under saturated irradiance (200 µmol m-2  s-1 ) by white light (WL) and low-pressure sodium vapor lamps (SOX lamps-control) and supplemented by fluorescent lamps (FLs) with different light qualities (blue: λmax = 440 nm; green: λmax = 560 nm; and red: λmax = 660 nm). The maximum photosynthetic efficiency (Fv / Fm ) showed a positive correlation with the light quality by saturating light SOX in mixture with stimulating blue light than the white light (WL) at the harvest day (10 days). The production, that is maximal electron transport rate (ETRmax ), and energy dissipation, that is maximal nonphotochemical quenching (NPQmax ), had the same pattern throughout the time (3-6 days) being the values higher under white light (WL) compared with SOX and SOX plus supplemented different light qualities. Total protein levels increased significantly in the presence of SOX light, while phycoerythrin (B-PE) showed significant differences under SOX+ blue light. Arachidonic acid (ARA) was higher under SOX and SOX plus supplemented different light qualities than that under WL, whereas eicosapentaenoic acid (EPA) was the reverse. The high photomorphogenic potential by SOX light shows promising application for microalgal biotechnology.

Immunomodulatory, Antioxidant Activity and Cytotoxic Effect of Sulfated Polysaccharides from Porphyridium cruentum. (S.F.Gray) Nägeli.

Porphyridium cruentum is a unicellular microalga that can synthesize and secrete to the culture medium-high amounts of polysaccharides. In this study, the immunomodulatory, cytotoxic effect and antioxidant activity of the sulfated polysaccharides (PcSPs) were determinate. The PcSPs were precipitated with 2% Cetylpyridinium bromide hydrate and ethanol and purified by dialysis. The extract was lyophilized for its characterization by Fourier transform-Infrared (FT-IR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). The antioxidant activity of PcSPs were examined with assay 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and compared with that of the biomass, observing significant differences between the results obtained from the PcSPs and biomass. To determine their ability to induce cytokine production Tumor Necrosis Factor alpha (TNF-α) and interleukina-6 (IL-6), the immunomodulatory activity of the PcSPs has been evaluated. In the mouse macrophage cell line (RAW 264.7), PcSPs are potent inducers of IL-6 cytokines but mainly of TNF-α. The cytotoxic capacity of PcSPs was measured by the MTT colorimetric assay in colorectal carcinoma (HTC-116), human leukemia (U-937 and HL-60), breast cancer (MCF-7), lung cancer (NCI-H460) and human gingival fibroblasts (HGF-1) cell lines. The IC50 value of 2311.20 µg mL-1, 1676.74 µg mL-1, 1089.63 µg mL-1, 5498.14 µg mL-1 and 2861.49 µg mL-1 respectively in the tumor lines and 5022.55 µg mL-1 in gingival fibroblasts were obtained. Our study suggested that PcSPs from P. cruentum have a moderate immunomodulatory and cytotoxic effect. The results obtained indicate that the polysaccharides from P. cruentum are potent inducers of IL-6 cytokines and, most importantly, of TNF-α. PcSPs showed no evidence of antigenic activity or hypersensitivity when administered intraperitoneally in mice. Furthermore, the in vivo study revealed an improvement of local inflammatory response against stress in the peritoneum. These findings suggest that the PcSPs from P. cruentum might have potential as a valuable ingredient in nutraceutical products.

Scaffolding proteins guide the evolution of algal light harvesting antennas.

Photosynthetic organisms have developed diverse antennas composed of chromophorylated proteins to increase photon capture. Cryptophyte algae acquired their photosynthetic organelles (plastids) from a red alga by secondary endosymbiosis. Cryptophytes lost the primary red algal antenna, the red algal phycobilisome, replacing it with a unique antenna composed of αß protomers, where the ß subunit originates from the red algal phycobilisome. The origin of the cryptophyte antenna, particularly the unique α subunit, is unknown. Here we show that the cryptophyte antenna evolved from a complex between a red algal scaffolding protein and phycoerythrin ß. Published cryo-EM maps for two red algal phycobilisomes contain clusters of unmodelled density homologous to the cryptophyte-αß protomer. We modelled these densities, identifying a new family of scaffolding proteins related to red algal phycobilisome linker proteins that possess multiple copies of a cryptophyte-α-like domain. These domains bind to, and stabilise, a conserved hydrophobic surface on phycoerythrin ß, which is the same binding site for its primary partner in the red algal phycobilisome, phycoerythrin α. We propose that after endosymbiosis these scaffolding proteins outcompeted the primary binding partner of phycoerythrin ß, resulting in the demise of the red algal phycobilisome and emergence of the cryptophyte antenna.

Optimization of Exopolysaccharides Production by Porphyridium sordidum and Their Potential to Induce Defense Responses in Arabidopsis thaliana against Fusarium oxysporum.

Polysaccharides from marine algae are one novel source of plant defense elicitors for alternative and eco-friendly plant protection against phytopathogens. The effect of exopolysaccharides (EPS) produced by Porphyridium sordidum on elicitation of Arabidopsis thaliana defense responses against Fusarium oxysporum was evaluated. Firstly, in order to enhance EPS production, a Box-Behnken experimental design was carried out to optimize NaCl, NaNO3 and MgSO4 concentrations in the culture medium of microalgae. A maximum EPS production (2.45 g/L) higher than that of the control (0.7 g/L) was observed for 41.62 g/L NaCl, 0.63 g/L NaNO3 and 7.2 g/L MgSO4 concentrations. Structurally, the EPS contained mainly galactose, xylose and glucose. Secondly, the elicitor effect of EPS was evaluated by investigating the plant defense-related signaling pathways that include activation of Salicylic or Jasmonic Acid-dependent pathway genes. A solution of 2 mg/mL of EPS has led to the control of fungal growth by the plant. Results showed that EPS foliar application induced phenylalaline ammonia lyase and H2O2 accumulation. Expression profile analysis of the defense-related genes using qRT-PCR revealed the up-regulation of Superoxide dismutases (SOD), Peroxidase (POD), Pathogenesis-related protein 1 (PR-1) and Cytochrome P450 monooxyge-nase (CYP), while Catalase (CAT) and Plant defensin 1.2 (PDF1.2) were not induced. Results suggest that EPS may induce the elicitation of A. thaliana's defense response against F. oxysporum, activating the Salicylic Acid pathway.

Exopolysaccharide from Porphyridium cruentum (purpureum) is Not Toxic and Stimulates Immune Response against Vibriosis: The Assessment Using Zebrafish and White Shrimp Litopenaeus vannamei.

Exopolysaccharides, or extracellular polysaccharides (EPS, sPS), represent a valuable metabolite compound synthesized from red microalgae. It is a non-toxic natural agent and can be applied as an immunostimulant. The toxicity test of exopolysaccharides from Porphyridium has been done in vivo using zebrafish (Danio rerio) embryonic model, or the ZET (zebrafish embryotoxicity test). The administration of extracellular polysaccharides or exopolysaccharides (EPS) from microalgae Porphyridium cruentum (synonym: P. purpureum) to shrimps Litopenaeus vannamei was investigated to determine the effect of this immunostimulant on their non-specific immune response and to test if this compound can be used as a protective agent for shrimps in relation to Vibrio infection. For immune response, exopolysaccharides were given to shrimps via the immersion method on day 1 and booster on day 8. Shrimp hemocytes were taken on day 1 (EPS administration), day 7 (no treatment), day 8 (EPS booster) and day 9 (Vibrio infection) and tested for their immune response on each treatment. The result shows that the EPS is not toxic, as represented by the normal embryonic development and the mortality data. In the Pacific white shrimps, an increase in the values of all immune parameters was shown, in line with the increasing EPS concentration, except for the differential hemocyte count (DHC). In detail, an increase was noted in total hemocytes (THC) value, phagocytotic activity (PA) and respiratory burst (RB) in line with the EPS concentration increase. These results and other previous studies indicate that EPS from Porphyridium is safe, enhances immune parameters in shrimp rapidly, and has the ability to act as an immunostimulant or an immunomodulator. It is a good modulator for the non-specific immune cells of Pacific white shrimps, and it can be used as a preventive agent against vibriosis.

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 (54.3 mg g-1) than in the N-replete cultures (45.3 mg g-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.

Improvement of Unsaturated Fatty Acid Production from Porphyridium cruentum Using a Two-Phase Culture System in a Photobioreactor with Light-Emitting Diodes (LEDs).

In this study, the culture conditions for Porphyridium cruentum were optimized to obtain the maximum biomass and lipid productions. The eicosapentaenoic acid content was increased by pH optimization. P. cruentum was cultured with modified F/2 medium in 14-L photobioreactors using a two-phase culture system, in which the green (520 nm) and red (625 nm) light-emitting diodes (LEDs) were used during the first and second phases for biomass production and lipid production, respectively. Various parameters, including aeration rate, light intensity, photoperiod, and pH were optimized. The maximum biomass concentration of 0.91 g dcw/l was obtained with an aeration rate of 0.75 vvm, a light intensity of 300 µmol m-2s-1, and a photoperiod of 24:0 h. The maximum lipid production of 51.8% (w/w) was obtained with a light intensity of 400 µmol m-2s-1 and a photoperiod of 18:6 h. Additionally, the eicosapentaenoic acid and unsaturated fatty acid contents reached 30.6% to 56.2% at pH 6.0.

Light-diffractive patterning of Porphyridium purpureum.

Light guidance is a convenient and versatile way to control the positions of phototactic microorganisms. However, the illumination strategies require adaption to the respective organism. We report on the generation of structures composed of the gliding and exopolysaccharide-secreting algae Porphyridium purpureum via their photomovement. Light patterns from a two-dimensional computer-generated hologram were projected onto inoculated agar plates. The obtained pixelated algae patterns were evaluated with regard to the illuminated intensity, contrast and pixel size. Upper and lower thresholds for algae accumulation were determined, allowing to enhance future manipulation of phototactic microorganisms.

Optimal cultivation towards enhanced biomass and floridean starch production by Porphyridium marinum.

Optimal conditions for maximal biomass and starch production by the marine red microalgae Porphyridium marinum were investigated. Box-Behnken Design was used to model the effect of light intensity, NaNO3 concentration and salinity on the growth of microalgae but also on their starch and protein contents. These three factors increased biomass production by 13.6% in optimized conditions. A maximum starch production (140.21 µg·mL-1), 30.6% higher than that of the control, was attained at a light intensity of 100 µmol photons·m-2·s-1, a NaNO3 concentration of 1 g·L-1 and a NaCl concentration of 20 g·L-1. FT-IR spectroscopy was used to estimate the biochemical composition (carbohydrate accumulation) of P. marinum and revealed significant changes (P < 0.05) depending on culture conditions. FT-IR analysis highlighted also that the culture conditions leading to highest starch production by P. marinum corresponded to lowest sulfated polysaccharide and protein contents.

Growth and Biochemical Composition of Porphyridium purpureum SCS-02 under Different Nitrogen Concentrations.

Microalgae of the genus Porphyridium show great potential for large-scale commercial cultivation, as they accumulate large quantities of B-phycoerythrin (B-PE), long chain polyunsaturated fatty acids (LC-PUFAs) and exopolysaccharide (EPS). The present study aimed to adjust culture nitrogen concentrations to produce Porphyridium biomass rich in B-PE, LC-PUFAs and EPS. Porphyridium purpureum SCS-02 was cultured in ASW culture medium with low nitrogen supply (LN, 3.5 mM), medium nitrogen supply (MN, 5.9 mM) or high nitrogen supply (HN, 17.6 mM). HN significantly enhanced the accumulation of biomass, intracellular protein, B-PE and eicosapentaenoic acid. LN increased the intracellular carbohydrate and arachidonic acid content, and promoted the secretion of EPS. The total lipids content was almost unaffected by nitrogen concentration. Based on these results, a semi-continuous two-step process was proposed, which included the production of biomass rich in B-PE and LC-PUFAs with sufficient nitrogen, and induced EPS excretion with limited nitrogen and strong light.

Terpene Biosynthesis in Red Algae Is Catalyzed by Microbial Type But Not Typical Plant Terpene Synthases.

Red algae (Rhodophyta) and land plants belong to the monophyletic clade Archaeplastida, and taxa of both groups are rich producers of terpene secondary metabolites. The terpene carbon skeletons of land plants are made by two types of terpene synthases: typical plant terpene synthases and microbial-type terpene synthases (MTPSLs); however, terpene biosynthesis in red algae is poorly understood. By systematic sequence analysis of seven genomes and 34 transcriptomes of red algae, MTPSL homologs were identified within one genome and two transcriptomes, whereas no homolog of typical plant terpene synthase genes was found. Phylogenetic analysis showed that red algae MTPSLs group with bacterial terpene synthases. Analysis of the genome assembly and characterization of neighboring genes demonstrated red algal MTPSLs to be bona fide red algal genes and not microbial contaminants. MTPSL genes from Porphyridium purpureum and Erythrolobus australicus were characterized via heterologous expression in Escherichia coli and demonstrated to have sesquiterpene synthase activities. We detected a number of volatile sesquiterpenes in the headspace of P. purpureum and E. australicus cultures, most identical to the in vitro products of the respective MTPSLs. Expression of the MTPSL gene in P. purpureum was found to be induced by methyl jasmonate, suggesting a role for this gene in host defense. In summary, this study indicates that the formation of terpene carbon skeletons in red algae is carried out by MTPSLs that are phylogenetically unrelated to typical plant terpene synthases and most likely originated in Rhodophyta via horizontal gene transfer from bacteria.