Polar Lipids of Marine Microalgae Nannochloropsis oceanica and Chlorococcum amblystomatis Mitigate the LPS-Induced Pro-Inflammatory Response in Macrophages.

Microalgae are recognized as a relevant source of bioactive compounds. Among these bioactive products, lipids, mainly glycolipids, have been shown to present immunomodulatory properties with the potential to mitigate chronic inflammation. This study aimed to evaluate the anti-inflammatory effect of polar lipids isolated from Nannochloropsis oceanica and Chlorococcum amblystomatis. Three fractions enriched in (1) digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG), (2) monogalactosyldiacylglycerol (MGDG), and (3) diacylglyceryl-trimethylhomoserine (DGTS) and phospholipids (PL) were obtained from the total lipid extracts (TE) of N. oceanica and C. amblystomatis, and their anti-inflammatory effect was assessed by analyzing their capacity to counteract nitric oxide (NO) production and transcription of pro-inflammatory genes Nos2, Ptgs2, Tnfa, and Il1b in lipopolysaccharide (LPS)-activated macrophages. For both microalgae, TE and Fractions 1 and 3 strongly inhibited NO production, although to different extents. A strong reduction in the LPS-induced transcription of Nos2, Ptgs2, Tnfa, and Il1b was observed for N. oceanica and C. amblystomatis lipids. The most active fractions were the DGTS-and-PL-enriched fraction from N. oceanica and the DGDG-and-SQDG-enriched fraction from C. amblystomatis. Our results reveal that microalgae lipids have strong anti-inflammatory capacity and may be explored as functional ingredients or nutraceuticals, offering a natural solution to tackle chronic inflammation-associated diseases.

Restorative Effect of Microalgae Nannochloropsis oceanica Lipid Extract on Phospholipid Metabolism in Keratinocytes Exposed to UVB Radiation.

Ultraviolet B (UVB) radiation induces oxidative stress in skin cells, generating reactive oxygen species (ROS) and perturbing enzyme-mediated metabolism. This disruption is evidenced with elevated concentrations of metabolites that play important roles in the modulation of redox homeostasis and inflammatory responses. Thus, this research sought to determine the impacts of the lipid extract derived from the Nannochloropsis oceanica microalgae on phospholipid metabolic processes in keratinocytes subjected to UVB exposure. UVB-irradiated keratinocytes were treated with the microalgae extract. Subsequently, analyses were performed on cell lysates to ascertain the levels of phospholipid/free fatty acids (GC-FID), lipid peroxidation byproducts (GC-MS), and endocannabinoids/eicosanoids (LC-MS), as well as to measure the enzymatic activities linked with phospholipid metabolism, receptor expression, and total antioxidant status (spectrophotometric methods). The extract from N. oceanica microalgae, by diminishing the activities of enzymes involved in the synthesis of endocannabinoids and eicosanoids (PLA2/COX1/2/LOX), augmented the concentrations of anti-inflammatory and antioxidant polyunsaturated fatty acids (PUFAs), namely DHA and EPA. These concentrations are typically diminished due to UVB irradiation. As a consequence, there was a marked reduction in the levels of pro-inflammatory arachidonic acid (AA) and associated pro-inflammatory eicosanoids and endocannabinoids, as well as the expression of CB1/TRPV1 receptors. The microalgal extract also mitigated the increase in lipid peroxidation byproducts, specifically MDA in non-irradiated samples and 10-F4t-NeuroP in both control and post-UVB exposure. These findings indicate that the lipid extract derived from N. oceanica, by mitigating the deleterious impacts of UVB radiation on keratinocyte phospholipids, assumed a pivotal role in reinstating intracellular metabolic equilibrium.

Production of a viral surface protein in Nannochloropsis oceanica for fish vaccination against infectious pancreatic necrosis virus.

Nannochloropsis oceanica is a unicellular oleaginous microalga of emerging biotechnological interest with a sequenced, annotated genome, available transcriptomic and proteomic data, and well-established basic molecular tools for genetic engineering. To establish N. oceanica as a eukaryotic host for recombinant protein synthesis and develop molecular technology for vaccine production, we chose the viral surface protein 2 (VP2) of a pathogenic fish virus that causes infectious pancreatic necrosis as a model vaccine. Upon stable nuclear transformation of N. oceanica strain CCMP1779 with the codon-optimized VP2 gene, a Venus reporter fusion served to evaluate the strength of different endogenous promoters in transformant populations by qPCR and flow cytometry. The highest VP2 yields were achieved for the elongation factor promoter, with enhancer effects by its N-terminal leader sequence. Individual transformants differed in their production capability of reporter-free VP2 by orders of magnitude. When subjecting the best candidates to kinetic analyses of growth and VP2 production in photobioreactors, recombinant protein integrity was maintained until the early stationary growth phase, and a high yield of 4.4% VP2 of total soluble protein was achieved. The maximum yield correlated with multiple integrations of the expression vector into the nuclear genome. The results demonstrate that N. oceanica was successfully engineered to constitute a robust platform for high-level production of a model subunit vaccine. The molecular methodology established here can likely be adapted in a straightforward manner to the production of further vaccines in the same host, allowing their distribution to fish, vertebrates, or humans via a microalgae-containing diet. KEY POINTS: • We engineered N. oceanica for recombinant protein production. • The antigenic surface protein 2 of IPN virus could indeed be expressed in the host. • A high yield of 4.4% VP2 of total soluble protein was achieved in N. oceanica.

Characterization of Nannochloropsis oceanica Rose Bengal Mutants Sheds Light on Acclimation Mechanisms to High Light When Grown in Low Temperature.

A barrier to realizing Nannochloropsis oceanica's potential for omega-3 eicosapentaenoic acid (EPA) production is the disparity between conditions that are optimal for growth and those that are optimal for EPA biomass content. A case in point is temperature: higher content of polyunsaturated fatty acid, and especially EPA, is observed in low-temperature (LT) environments, where growth rates are often inhibited. We hypothesized that mutant strains of N. oceanica resistant to the singlet-oxygen photosensitizer Rose Bengal (RB) would withstand the oxidative stress conditions that prevail in the combined stressful environment of high light (HL; 250 µmol photons m-2 s-1) and LT (18°C). This growth environment caused the wild-type (WT) strain to experience a spike in lipid peroxidation and an inability to proliferate, whereas growth and homeostatic reactive oxygen species levels were observed in the mutant strains. We suggest that the mutant strains' success in this environment can be attributed to their truncated photosystem II antennas and their increased ability to diffuse energy in those antennas as heat (non-photosynthetic quenching). As a result, the mutant strains produced upward of four times more EPA than the WT strain in this HL-LT environment. The major plastidial lipid monogalactosyldiacylglycerol was a likely target for oxidative damage, contributing to the photosynthetic inhibition of the WT strain. A mutation in the NO10G01010.1 gene, causing a subunit of the 2-oxoisovalerate dehydrogenase E1 protein to become non-functional, was determined to be the likely source of tolerance in the RB113 mutant strain.

Development of a novel nannochloropsis strain with enhanced violaxanthin yield for large-scale production.

BACKGROUND: Nannochloropsis is a marine microalga that has been extensively studied. The major carotenoid produced by this group of microalgae is violaxanthin, which exhibits anti-inflammatory, anti-photoaging, and antiproliferative activities. Therefore, it has a wide range of potential applications. However, large-scale production of this pigment has not been much studied, thereby limiting its industrial application. RESULTS: To develop a novel strain producing high amount of violaxanthin, various Nannochloropsis species were isolated from seawater samples and their violaxanthin production potential were compared. Of the strains tested, N. oceanica WS-1 exhibited the highest violaxanthin productivity; to further enhance the violaxanthin yield of WS-1, we performed gamma-ray-mediated random mutagenesis followed by colorimetric screening. As a result, Mutant M1 was selected because of its significant higher violaxanthin content and biomass productivity than WS-1 (5.21 ± 0.33 mg g- 1 and 0.2101 g L- 1 d- 1, respectively). Subsequently, we employed a 10 L-scale bioreactor to confirm the large-scale production potential of M1, and the results indicated a 43.54 % increase in violaxanthin production compared with WS-1. In addition, comparative transcriptomic analysis performed under normal light condition identified possible mechanisms associated with remediating photo-inhibitory damage and other key responses in M1, which seemed to at least partially explain enhanced violaxanthin content and delayed growth. CONCLUSIONS: Nannochloropsis oceanica mutant (M1) with enhanced violaxanthin content was developed and its physiological characteristics were investigated. In addition, enhanced production of violaxanthin was demonstrated in the large-scale cultivation. Key transcriptomic responses that are seemingly associated with different physiological responses of M1 were elucidated under normal light condition, the details of which would guide ongoing efforts to further maximize the industrial potential of violaxanthin producing strains.

Ethanol Extraction of Polar Lipids from Nannochloropsis oceanica for Food, Feed, and Biotechnology Applications Evaluated Using Lipidomic Approaches.

Nannochloropsis oceanica can accumulate lipids and is a good source of polar lipids, which are emerging as new value-added compounds with high commercial value for the food, nutraceutical, and pharmaceutical industries. Some applications may limit the extraction solvents, such as food applications that require safe food-grade solvents, such as ethanol. However, the effect of using ethanol as an extraction solvent on the quality of the extracted polar lipidome, compared to other more traditional methods, is not yet well established. In this study, the polar lipid profile of N. oceanica extracts was obtained using different solvents, including chloroform/methanol (CM), dichloromethane/methanol (DM), dichloromethane/ethanol (DE), and ethanol (E), and evaluated by modern lipidomic methods using LC-MS/MS. Ultrasonic bath (E + USB)- and ultrasonic probe (E + USP)-assisted methodologies were implemented to increase the lipid extraction yields using ethanol. The polar lipid signature and antioxidant activity of DM, E + USB, and E + USP resemble conventional CM, demonstrating a similar extraction efficiency, while the DE and ethanol extracts were significantly different. Our results showed the impact of different extraction solvents in the polar lipid composition of the final extracts and demonstrated the feasibility of E + USB and E + USP as safe and food-grade sources of polar lipids, with the potential for high-added-value biotechnological applications.

Kinetics Growth and Recovery of Valuable Nutrients from Selangor Peat Swamp and Pristine Forest Soils Using Different Extraction Methods as Potential Microalgae Growth Enhancers.

Soil extracts are useful nutrients to enhance the growth of microalgae. Therefore, the present study attempts for the use of virgin soils from Peninsular Malaysia as growth enhancer. Soils collected from Raja Musa Forest Reserve (RMFR) and Ayer Hitam Forest Reserve (AHFR) were treated using different extraction methods. The total dissolved nitrogen (TDN), total dissolved phosphorus (TDP), and dissolved organic carbon (DOC) concentrations in the autoclave methods were relatively higher than natural extraction with up to 132.0 mg N/L, 10.7 mg P/L, and 2629 mg C/L, respectively for RMFR. The results of TDN, TDP, and DOC suggested that the best extraction methods are autoclaved at 121 °C twice with increasing 87%, 84%, and 95%, respectively. Chlorella vulgaris TRG 4C dominated the growth at 121 °C twice extraction method in the RMRF and AHRF samples, with increasing 54.3% and 14%, respectively. The specific growth rate (µ) of both microalgae were relatively higher, 0.23 d-1 in the Ayer Hitam Soil. This extract served well as a microalgal growth promoter, reducing the cost and the needs for synthetic medium. Mass production of microalgae as aquatic feed will be attempted eventually. The high recovery rate of nutrients has a huge potential to serve as a growth promoter for microalgae.

Identification of circadian rhythms in Nannochloropsis species using bioluminescence reporter lines.

Circadian clocks allow organisms to predict environmental changes caused by the rotation of the Earth. Although circadian rhythms are widespread among different taxa, the core components of circadian oscillators are not conserved and differ between bacteria, plants, animals and fungi. Stramenopiles are a large group of organisms in which circadian rhythms have been only poorly characterized and no clock components have been identified. We have investigated cell division and molecular rhythms in Nannochloropsis species. In the four strains tested, cell division occurred principally during the night period under diel conditions; however, these rhythms damped within 2-3 days after transfer to constant light. We developed firefly luciferase reporters for the long-term monitoring of in vivo transcriptional rhythms in two Nannochlropsis species, Nannochloropsis oceanica CCMP1779 and Nannochloropsis salina CCMP537. The reporter lines express anticipatory behavior under light/dark cycles and free-running bioluminescence rhythms with periods of ~21-31 h that damped within ~3-4 days under constant light. Using different entrainment regimes, we demonstrate that these rhythms are modulated by a circadian-type oscillator. In addition, the phase of free-running luminescence rhythms can be modulated pharmacologically using a CK1 ε/δ inhibitor, suggesting a role of this kinase in the Nannochloropsis clock. Together with the molecular and genomic tools available for Nannochloropsis species, these reporter lines represent an excellent system for future studies on the molecular mechanisms of stramenopile circadian oscillators.

Development of a constitutive and an auto-inducible high-yield expression system for recombinant protein production in the microalga Nannochloropsis oceanica.

Photoautotrophic microalgae offer a great potential as novel hosts for efficient recombinant protein production. Nannochloropsis oceanica produces an extraordinarily high content of polyunsaturated fatty acids, and its robust growth characteristics, published genome sequence and efficient nuclear transformation make N. oceanica a promising candidate for biotechnological applications. To establish a robust and flexible system for recombinant protein production, we cloned six endogenous, potentially constitutive or inducible promoters from N. oceanica strain CCMP1779 and investigated their strength using monomeric Venus as reporter gene. Microscopic pre-screening of individual transformants revealed that the promoters of elongation factor (EF), tubulin (TUB) and nitrate reductase (NR) enabled high reporter gene expression. Comparative quantitative analyses of transformant populations by flow cytometry and qRT-PCR demonstrated the highest Venus expression from the EF promoter and the NR promoter if extended by an N-terminal 14-amino acid leader sequence. The kinetics of reporter gene expression were analysed during photobioreactor cultivation, achieving Venus yields of 0.3% (for EF) and 4.9% (for NR::LS) of total soluble protein. Since inducible expression systems enable the production of toxic proteins, we developed an auto-induction medium for the NR promoter transformants. By switching the N source from ammonium to nitrate in the presence of low ammonium concentrations, the starting point of Venus induction could be fine-tuned and shifted towards exponential growth phase while maintaining high recombinant protein yields. Taken together, we demonstrate that a model recombinant protein can be produced robustly and at very high levels in N. oceanica not only under constitutive but also under auto-inducible cultivation conditions. KEY POINTS: • Nannochloropsis oceanica might serve as host for recombinant protein production. • Comparative promoter strength analyses were conducted for twelve different constructs. • Robust high-yield recombinant protein production was achieved under constitutive conditions. • The nitrate reductase promoter enabled protein production under auto-induction conditions.

Optimized methods of chromatin immunoprecipitation for profiling histone modifications in industrial microalgae Nannochloropsis spp.

Epigenetic factors such as histone modifications play integral roles in plant development and stress response, yet their implications in algae remain poorly understood. In the industrial oleaginous microalgae Nannochloropsis spp., the lack of an efficient methodology for chromatin immunoprecipitation (ChIP), which determines the specific genomic location of various histone modifications, has hindered probing the epigenetic basis of their photosynthetic carbon conversion and storage as oil. Here, a detailed ChIP protocol was developed for Nannochloropsis oceanica, which represents a reliable approach for the analysis of histone modifications, chromatin state, and transcription factor-binding sites at the epigenetic level. Using ChIP-qPCR, genes related to photosynthetic carbon fixation in this microalga were systematically assessed. Furthermore, a ChIP-Seq protocol was established and optimized, which generated a genome-wide profile of histone modification events, using histone mark H3K9Ac as an example. These results are the first step for appreciation of the chromatin landscape in industrial oleaginous microalgae and for epigenetics-based microalgal feedstock development.

The Oxygen quantum yield in diverse algae and cyanobacteria is controlled by partitioning of flux between linear and cyclic electron flow within photosystem II.

We have measured flash-induced oxygen quantum yields (O2-QYs) and primary charge separation (Chl variable fluorescence yield, Fv/Fm) in vivo among phylogenetically diverse microalgae and cyanobacteria. Higher O2-QYs can be attained in cells by releasing constraints on charge transfer at the Photosystem II (PSII) acceptor side by adding membrane-permeable benzoquinone (BQ) derivatives that oxidize plastosemiquinone QB(-) and QBH2. This method allows uncoupling PSII turnover from its natural regulation in living cells, without artifacts of isolating PSII complexes. This approach reveals different extents of regulation across species, controlled at the QB(-) acceptor site. Arthrospira maxima is confirmed as the most efficient PSII-WOC (water oxidizing complex) and exhibits the least regulation of flux. Thermosynechococcus elongatus exhibits an O2-QY of 30%, suggesting strong downregulation. WOC cycle simulations with the most accurate model (VZAD) show that a light-driven backward transition (net addition of an electron to the WOC, distinct from recombination) occurs in up to 25% of native PSIIs in the S2 and S3 states, while adding BQ prevents backward transitions and increases the lifetime of S2 and S3 by 10-fold. Backward transitions occur in PSIIs that have plastosemiquinone radicals in the QB site and are postulated to be physiologically regulated pathways for storing light energy as proton gradient through direct PSII-cyclic electron flow (PSII-CEF). PSII-CEF is independent of classical PSI/cyt-b6f-CEF and provides an alternative proton translocation pathway for energy conversion. PSII-CEF enables variable fluxes between linear and cyclic electron pathways, thus accommodating species-dependent needs for redox and ion-gradient energy sources powered by a single photosystem.

Pigment structure in the violaxanthin-chlorophyll-a-binding protein VCP.

Resonance Raman spectroscopy was used to evaluate pigment-binding site properties in the violaxanthin-chlorophyll-a-binding protein (VCP) from Nannochloropsis oceanica. The pigments bound to this antenna protein are chlorophyll-a, violaxanthin, and vaucheriaxanthin. The molecular structures of bound Chl-a molecules are discussed with respect to those of the plant antenna proteins LHCII and CP29, the crystal structures of which are known. We show that three populations of carotenoid molecules are bound by VCP, each of which is in an all-trans configuration. We assign the lower-energy absorption transition of each of these as follows. One violaxanthin population absorbs at 485 nm, while the second population is red-shifted and absorbs at 503 nm. The vaucheriaxanthin population absorbs at 525 nm, a position red-shifted by 2138 cm-1 as compared to isolated vaucheriaxanthin in n-hexane. The red-shifted violaxanthin is slightly less planar than the blue-absorbing one, as observed for the two central luteins in LHCII, and we suggest that these violaxanthins occupy the two equivalent binding sites in VCP at the centre of the cross-brace. The presence of a highly red-shifted vaucheriaxanthin in VCP is reminiscent of the situation of FCP, in which (even more) highly red-shifted populations of fucoxanthin are present. Tuning carotenoids to absorb in the green-yellow region of the visible spectrum appears to be a common evolutionary response to competition with other photosynthetic species in the aquatic environment.

Transcriptional coordination of physiological responses in Nannochloropsis oceanica CCMP1779 under light/dark cycles.

Nannochloropsis oceanica CCMP1779 is a marine unicellular stramenopile and an emerging reference species for basic research on oleogenic microalgae with biotechnological relevance. We investigated its physiology and transcriptome under light/dark cycles. We observed oscillations in lipid content and a predominance of cell division in the first half of the dark phase. Globally, more than 60% of the genes cycled in N. oceanica CCMP1779, with gene expression peaking at different times of the day. Interestingly, the phase of expression of genes involved in certain biological processes was conserved across photosynthetic lineages. Furthermore, in agreement with our physiological studies we found the processes of lipid metabolism and cell division enriched in cycling genes. For example, there was tight coordination of genes involved in the lower part of glycolysis, fatty acid synthesis and lipid production at dawn preceding lipid accumulation during the day. Our results suggest that diel lipid storage plays a key role for N. oceanica CCMP1779 growth under natural conditions making this alga a promising model to gain a basic mechanistic understanding of triacylglycerol production in photosynthetic cells. Our data will help the formulation of new hypotheses on the role of cyclic gene expression in cell growth and metabolism in Nannochloropsis.

Antagonistic roles of abscisic acid and cytokinin during response to nitrogen depletion in oleaginous microalga Nannochloropsis oceanica expand the evolutionary breadth of phytohormone function.

The origin of phytohormones is poorly understood, and their physiological roles in microalgae remain elusive. Genome comparison of photosynthetic autotrophic eukaryotes has revealed that the biosynthetic pathways of abscisic acid (ABA) and cytokinins (CKs) emerged in unicellular algae. While ABA and CK degradation mechanisms emerged broadly in algal lineages, complete vascular plant-type conjugation pathways emerged prior to the rise of Streptophyta. In microalgae, a complete set of proteins from the canonical ABA and CK sensing and signaling pathways is not essential, but individual components are present, suggesting stepwise recruitment of phytohormone signaling components. In the oleaginous eustigmatophyte Nannochloropsis oceanica IMET1, UHPLC-MS/MS detected a wide array of plant hormones, despite a phytohormone profile that is very distinct from that of flowering plants. Time-series transcriptional analysis during nitrogen depletion revealed activation of the ABA biosynthetic pathway and antagonistic transcription of CK biosynthetic genes. Correspondingly, the ABA level increases while the dominant bioactive CK forms decrease. Moreover, exogenous CKs stimulate cell-cycle progression while exogenous ABA acts as both an algal growth repressor and a positive regulator in response to stresses. The presence of such functional flowering plant-like phytohormone signaling systems in Nannochloropsis sp. suggests a much earlier origin of phytohormone biosynthesis and degradation than previously believed, and supports the presence in microalgae of as yet unknown conjugation and sensing/signaling systems that may be exploited for microalgal feedstock development.

Lipidomic analysis can distinguish between two morphologically similar strains of Nannochloropsis oceanica.

The two morphologically similar microalgae NMBluh014 and NMBluh-X belong to two different strains of Nannochloropsis oceanica. They possess obviously different feeding effects on bivalves, but are indistinguishable by 18S rRNA and morphological features. In this work, lipidomic analysis followed by principal component analysis and orthogonal projections to latent structures discriminant analysis provided a clear distinction between these strains. Metabolites that definitively contribute to the classification were selected as potential biomarkers. The most important difference in polar lipids were sulfoquinovosyldiacylglycerol (containing 18:1/16:0 and 18:3/16:0) and monogalactosyldiacylglycerol (containing 18:3/16:3 and 20:5/14:0), which were detected only in NMBluh-X. Additionally, an exhaustive qualitative and quantitative profiling of the neutral lipid triacylglycerol (TAG) in the two strains was carried out. The predominant species of TAG containing 16:1/16:1/16:1 acyl groups was detected only in NMBluh-X with a content of ~93.67 ± 11.85 nmol · mg(-1) dry algae at the onset of stationary phase. Meanwhile, TAG containing 16:0/16:0/16:0 was the main TAG in NMBluh014 with a content of 40.25 ± 3.92 nmol · mg(-1) . These results provided the most straightforward evidence for differentiating the two species. The metabolomic profiling indicated that NMBluh-X underwent significant chemical and physiological changes during the growth process, whereas NMBluh014 did not show such noticeable time-dependent metabolite change. This study is the first using Ultra Performance Liquid Chromatography coupled with Electrospray ionization-Quadrupole-Time of Flight Mass Spectrometry (UPLC-Q-TOF-MS) for lipidomic profiling with multivariate statistical analysis to explore lipidomic differences of plesiomorphous microalgae. Our results demonstrate that lipidomic profiling is a valid chemotaxonomic tool in the study of microalgal systematics.

Omega-3 fatty acid obtained from Nannochloropsis oceanica cultures grown under low urea protect against Abeta-induced neural damage.

Amyloid-beta (Abeta) protein is a key factor in the pathogenesis of Alzheimer's disease (AD). Moreover, it has been reported that oxidative stress is involved in the biochemical pathway by which Abeta can lead to neuronal dysfunction. Recently, docosahexaenoic acid (DHA; C22:6) and eicosapentaenoic acid (EPA; C20:5n-3) have been reported to protect against AD. However, these omega-3 fatty acids are frequently obtained from fish oil and may contain heavy metals. In this study, we utilized Nannochloropsis oceanica to produce omega-3 fatty acid. We observed that when urea levels (nitrogen source) were lowered from 2 to 0.2 g/L in Nannochloropsis oceanica cultures, EPA production increased. Moreover, EPA in Nannochloropsis oceanica effectively promoted antioxidant activity to counter the Abeta-induced oxidative stress in Neuro-2A cells. These results indicate that Nannochloropsis oceanica may be potentially used as a therapeutic agent or as a functional food that promotes protection against AD.

Transcriptomic Analysis Reveals the Effect of Urea on Metabolism of Nannochloropsis oceanica.

The eukaryotic microalga Nannochloropsis oceanica represents a promising bioresource for the production of biofuels and pharmaceuticals. Urea, a crucial nutrient for the photosynthetic N. oceanica, stimulates the accumulation of substances such as lipids, which influence growth and physiology. However, the specific mechanisms by which N. oceanica responds and adapts to urea addition remain unknown. High-throughput mRNA sequencing and differential gene expression analysis under control and urea-added conditions revealed significant metabolic changes. This involved the differential expression of 2104 genes, with 1354 being upregulated and 750 downregulated, resulting in the reprogramming of crucial pathways such as carbon and nitrogen metabolism, photosynthesis, and lipid metabolism. The results specifically showed that genes associated with photosynthesis in N. oceanica were significantly downregulated, particularly those related to light-harvesting proteins. Interestingly, urea absorption and transport may depend not only on specialized transport channels such as urease but also on alternative transport channels such as the ABC transporter family and the CLC protein family. In addition, urea caused specific changes in carbon and lipid metabolism. Genes associated with the Calvin cycle and carbon concentration mechanisms were significantly upregulated. In lipid metabolism, the expression of genes associated with lipases and polyunsaturated fatty acid synthesis was highly activated. Furthermore, the expression of several genes involved in the tricarboxylic acid cycle and folate metabolism was enhanced, making important contributions to energy supply and the synthesis and modification of genes and macromolecules. Our observations indicate that N. oceanica actively and dynamically regulates the redistribution of carbon and nitrogen after urea addition, providing references for further research on the effects of urea on N. oceanica.

Genome-wide adenine N6-methylation map reveals epigenomic regulation of lipid accumulation in Nannochloropsis.

Epigenetic marks on histones and DNA, such as DNA methylation at N6-adenine (6mA), play crucial roles in gene expression and genome maintenance, but their deposition and function in microalgae remain largely uncharacterized. Here, we report a genome-wide 6mA map for the model industrial oleaginous microalga Nannochloropsis oceanica produced by single-molecule real-time sequencing. Found in 0.1% of adenines, 6mA sites are mostly enriched at the AGGYV motif, more abundant in transposons and 3' untranslated regions, and associated with active transcription. Moreover, 6mA gradually increases in abundance along the direction of gene transcription and shows special positional enrichment near splicing donor and transcription termination sites. Highly expressed genes tend to show greater 6mA abundance in the gene body than do poorly expressed genes, indicating a positive interaction between 6mA and general transcription factors. Furthermore, knockout of the putative 6mA methylase NO08G00280 by genome editing leads to changes in methylation patterns that are correlated with changes in the expression of molybdenum cofactor, sulfate transporter, glycosyl transferase, and lipase genes that underlie reductions in biomass and oil productivity. By contrast, knockout of the candidate demethylase NO06G02500 results in increased 6mA levels and reduced growth. Unraveling the epigenomic players and their roles in biomass productivity and lipid metabolism lays a foundation for epigenetic engineering of industrial microalgae.

Effect of phosphorus limitation on Se uptake efficiency in the microalga Nannochloropsis oceanica.

Microalgae are considered an efficient accumulator and promising source of Se for feed additive purposes. This study aimed at investigating, for the first time, the effect of phosphorus limitation on Se accumulation and uptake efficiency in N.oceanica. A range of phosphorus concentrations (0-2470 µM) were tested in either the presence or absence of sodium selenite (0, 5, 30 µM). Se accumulation was increased up to 16-fold and Se uptake efficiency was increased up to 3.6-fold under phosphorus growth-limiting concentrations. N.oceanica was then cultivated in a 1.8L flat-panel photobioreactor in batch operation under two phosphorus growth-limiting concentrations (250 and 750 µM) where the accumulation of Se in the microalgal biomass, as well as its presence in the spent medium were analysed. This study is the first to investigate the effect of phosphorus limitation for increasing Se accumulation in microalgae, and to prevent the release of Se in wastewater.

Growth and fatty acid distribution over lipid classes in Nannochloropsis oceanica acclimated to different temperatures.

After light, temperature is the most relevant environmental parameter in outdoors cultivation of microalgae. Suboptimal and supraoptimal temperatures negatively impact growth and photosynthetic performance with a subsequent effect on lipid accumulation. It is generally recognised that lower temperatures trigger an increase in fatty acid desaturation while higher temperatures trigger the opposite reaction. The effect of temperature on lipid classes has been less studied in microalgae and in certain cases, the effect of light cannot be completely excluded. In this research, the effect of temperature on growth, photosynthesis, and lipid class accumulation in Nannochloropsis oceanica was studied at a fixed light gradient with a constant incident light intensity (670 µmol m-2 s-1). A turbidostat approach was used to achieve temperature acclimated cultures of Nannochloropsis oceanica. Optimal growth was found at 25-29°C, while growth was completely arrested at temperatures higher than 31°C and lower than 9°C. Acclimation to low temperatures triggered a decrease in absorption cross section and photosynthesis rates with a tipping point at 17°C. Reduced light absorption was correlated with a decrease in content of the plastid lipids monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol. The increase of diacylglyceryltrimethylhomo-serine content at lower temperatures indicated a relevant role of this lipid class in temperature tolerance. Triacylglycerol content increased at 17°C and decreased at 9°C emphasising a metabolic switch in stress response. Total and polar eicosapentaenoic acid content remained constant at 3.5 and 2.4% w/w, despite the fluctuating lipid contents. Results show an extensive mobilisation of eicosapentaenoic acid between polar lipids classes at 9°C to ensure cell survival under critical conditions.