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Dunaliella salina has received much attention as a cell factory for the industrial production of the well-known antioxidant β-carotene, but basic studies on the genetic background of the ROS signaling that mediates the biosynthesis and massive accumulation of β-carotene are still lacking. In this study, we show that the second stage of β-carotene accumulation in D. salina is mediated by ROS production by NADPH oxidase homologs. Photosystem II quickly lost its function when exposed to high light (HL), and almost no photosynthesis occurred even after 24 h. Interestingly, when ROS production was blocked in PSII, accumulated β-carotene content was less than the control by 24 h. On the other hand, inhibition of ROS production by NADPH oxidase was a similar accumulation of β-carotene until 24 h, but lower than the control after 24 h. We identified two NADPH oxidase homologs in D. salina and used quantitative PCR to show that the expression of these genes was closely linked to β-carotene biosynthesis in the second stage. These results suggest that NADPH oxidase maintains β-carotene synthesis by generating ROS signals instead of the photosynthetic system which lost function due to HL stress. • β-carotene synthesis and cell growth in Dunaliella salina have an inverse correlation under high light conditions. • D. salina has two NADPH oxidase homologs that regulate β-carotene synthesis in the second stage via ROS signaling. • Genetic engineering of PSII has the potential to enable both stable growth and β-carotene synthesis under high light. [ABSTRACT FROM AUTHOR]

Phaeodactylum tricornutum is an extensively studied model diatom and a promising candidate for biochemical and biotechnological engineering aimed at increasing biomass production. Phosphoenolpyruvate carboxylase (PEPCase), which converts phosphoenolpyruvate (PEP) to oxaloacetate (OAA) using bicarbonate, is a key metabolic enzyme of the tricarboxylic acid (TCA) cycle and the C4 photosynthetic pathway. Two transgenic P. tricornutum lines overexpressing PtPEPCase1 (PtPEPC1, JGI protein ID: 27976) were constructed and their photosynthetic productivity, cell growth, and biomass were characterized. The levels of PtPEPC1 mRNAs in the two transgenic lines were increased by 2.3 and 11.2-fold and the amounts of the PEPCase protein by 1.3 and 2.3-fold, respectively. PtPEPC1 was targeted to mitochondria. Addition of bicarbonate to the P. tricornutum culture increased biomass of the transformants by about 12% compared to wild type, and their maximum specific growth rate in exponential phase was about 10% greater than that of wild type. The transformants also exhibited higher photosynthetic productivity. We conclude that overexpression of mitochondrial PtPEPC1 enhanced both photosynthetic productivity and TCA cycle activity in P. tricornutum , thereby enhancing biomass production in the presence of dissolved inorganic carbon. Our findings suggest that P. tricornutum overexpressing PtPEPC1 can be used to mitigate rising atmospheric CO 2 levels or for sustainable microalgal biomass production. [ABSTRACT FROM AUTHOR]

Chlamydomonas reinhardtii of the microalgal model species lacks phosphatidylcholine (PC), and PC is replaced by diacylglyceryl N , N , N -trimethylhomoserine (DGTS). DGTS is a betaine lipid that is placed in the endoplasmic reticulum (ER) and is synthesized by a single gene, BTA1 . In this study, we aimed to ascertain the turnover between membrane and neutral lipids via BTA1 knockdown transformants. Transgenic lines CrBta-hm13 and CrBta-hm31 were 80% and 60% downregulated in BTA1 gene expression levels, respectively. Both transformants had half the amount of DGTS, which coincided with decreased monogalactosyldiacylglycerol (MGDG), which was increased approximately threefold in neutral lipids. While the reduction of DGTS was shown to arise from inhibition of DGTS synthesis, decreased MGDG was affected by internal stress, such as ER stress, which was induced to have a decreased amount of DGTS in the ER. However, galactolipids, except for MGDG, and phospholipids in the transformants were maintained at similar levels. In the transformants, the molar proportion of C16:4 and C18:3(9,12,15), which are the major fatty acids of MGDG, was significantly increased in triacylglycerol (TAG) because of MGDG degradation. Thus, lipid turnover arising from the downregulation of DGTS and induction of ER stress caused a decrease in DGTS and MGDG, which generated a synergy effect on the accumulation of TAG. This study implies that genetic modification of a membrane lipid synthesis pathway could not only be a suitable approach to target accumulation of TAG, but could also suggest a mechanism for the lipid turnover between membrane lipids and neutral lipids. [ABSTRACT FROM AUTHOR]

Dunaliella salina is a unicellular halophilic green alga, which can survive even in saturated brine solutions (up to 5.0 M NaCl). In D. salina , the carbonic anhydrase (CA, EC 4.2.1.1) is essential for cells to acquire carbon and to cope with the low CO 2 environment under high salt conditions. For the first time, the present study describes the existence of eight genes coding for different types of D. salina CAs: five alpha-type (DsCAs) and three gamma-type (DsgCAs). Beta-type CAs appear to be lacking in D. salina , as they could not be found. Under either high salt or limited CO 2 condition, the CAs from D. salina showed different expression patterns, with phylogenetically close CAs exhibiting similar gene expression patterns. For the biological characterization of DsCA2b, which is a newly identified α-type CA, the enzyme was successfully produced as a soluble protein by truncating the membrane spanning regions at both ends (trDsCA2b). It was demonstrated that this truncated trDsCA2b version of the CA enzyme was active. Purified trDsCA2b clearly showed increased CA activity at increased NaCl concentrations of up to 3.0 M. Based on in silico analysis and our predicted 3D structure of DsCA2b, we propose that this enzyme is localized in the plasma membrane of cells and active on the extracellular side. Our results are in agreement with the hypothesis that the DsCAs are essential enzymes for carbon acquisition mechanism under salt stress and CO 2 stress in D. salina . [ABSTRACT FROM AUTHOR]

This increasing consumption of fossil fuels and emission of greenhouse gases causes climate change. Thus, the development of alternative energy sources such as biofuel is necessary. Microalgae have been spotlighted as renewable energy resources because the potential for producing biofuels from CO 2 . However, the unit cost for the production of algae-based biofuel must be reduced for commercialization compared to the petroleum. The optimization of culture conditions of microalgal strains is crucial to increase lipid production for economically viable biofuels. We rapidly analyzed the combined effect of various stress conditions (nitrogen starvation, temperature, pH, and salt concentration) on the lipid production in various strains using a multiplex microfluidic system, enabling multiple operations from cell culture to lipid extraction of different strains. We found the lipid productivity was enhanced by 25 to 54% under combinations of two stress condition compared to the single stress condition. However, the combination of more than three stress conditions reduced the lipid productivities of all microalgal strains because of more stressful environment to the cells compared to the combinations of two stress conditions. We further validated the synergistic effect of combined stress conditions in flask culture with the increases in lipid productivities by up to 106% compared to the single stress condition. We also observed that fatty acids composition, which influences the quality of algal biofuels, was changed according to the combination of stress conditions. In particular, C. protothecoides can be good candidate for production of high quality of biodiesel, because it has high CN, IV and low CFPP which is suitable for high quality of biodiesel. These results indicate that combination of multiple stress conditions can be efficient strategy for the optimization of microalgal cultivation to produce algal biofuels with high quality and economic feasibility. [ABSTRACT FROM AUTHOR]

Phaeodactylum tricornutum is a pennate model diatom whose entire genome has been sequenced and an expressed sequence tag database established. This study aimed at developing a genetic tool kit to modify the genetic traits of P. tricornutum . A new expression system containing the elongation factor 2 ( EF2 ) promoter fused to the luciferase gene was constructed, resulting in the pPhaT-EF2-Luc vector. This new vector was compared to the pPhaT-BP-Luc which harbors the fcpB promoter of P. tricornutum . After P. tricornutum transformation with these vectors, the presence of the exogenous luciferase gene inserted into the genomic DNA of the selected transformants was confirmed by Southern blot analysis. In the transformants, the fcpB promoter was activated dramatically upon 5 h of light exposure and its activity was reduced in the dark. However, the expression level of the Luc gene regulated by the EF2 promoter was not significantly affected by light and was higher than that of the Luc gene regulated by the fcpB promoter. Transformants harboring the pEF2::Luc cassette had a high level of luciferase protein expression and constant luciferase activity throughout the light/dark cycle. In addition, the EF2 promoter of P. tricornutum was able to drive the expression of a heterologous gene in the green alga Chlamydomonas reinhardtii . These results indicate that the P. tricornutum EF2 promoter can be used for the expression of target genes in genetically transformed P. tricornutum and C. reinhardtii . [ABSTRACT FROM AUTHOR]

Dunaliella salina is a model photosynthetic organism for studying osmoregulation. To further understand the molecular basis of short-term hyperosmotic-stress responses, gene expression was investigated by RNA-Seq of D. salina cells sampled at 15, 30, 60, and 120 min following exposure to salt and glycerol stress. De novo reconstruction of the transcriptome using Illumina paired-end reads generated 155,996 contigs, out of which 31,302 transcripts could be annotated by gene ontology, assigning them to 64 functional groups. Further functional analysis of the transcripts was performed using KEGG Enzyme Commission numbers. Out of a total of 4415 transcripts with more than two-fold differential expression, 479 transcripts were identified representing 414 enzyme commission numbers that were mapped to 120 KEGG pathways. The "Chromosome and associated proteins", "Transporters", "Cytoskeleton proteins", and 92 other KEGG pathways mapped for differentially expressed transcripts were common to salt stress and glycerol stress. In addition, differentially expressed transcripts mapped uniquely to 22 metabolic pathways for glycerol stress and 7 metabolic pathways mapped uniquely for salt stress. The transcripts mapping to core carbon metabolism within starch, glycolysis, Calvin cycle, and glycerol metabolic pathways were analyzed and 19 transcripts showed differential expression. Cluster analysis resulted in the identification of 939 transcripts, which showed similar expression trends. Among the fastest and most strongly induced genes, glyoxylate cycle and fatty acid desaturation-related genes implied the induction of photorespiration following rapid shifts in the osmolarity of the growth medium. These results provide novel insights for further analyses of physiological responses to short-term osmotic shock. • Transcriptomes of D. salina changed significantly within 120 min after osmotic stress. • Unique and common responding pathways of osmotic stresses were identified. • Transcript abundance of glycerol metabolism related pathways were investigated. • Transcripts with similar expression trends of two types of stress were identified. • Transcriptomes of different osmotic shocks were compared by multivariate analyses. [ABSTRACT FROM AUTHOR]

Life in high salinity environments poses challenges to cells in a variety of ways: maintenance of ion homeostasis and nutrient acquisition, often while concomitantly enduring saturating irradiances. Dunaliella salina has an exceptional ability to thrive even in saturated brine solutions. This ability has made it a model organism for studying responses to abiotic stress factors. Here we describe the occurrence of unique gene families, expansion of gene families, or gene losses that might be linked to osmoadaptive strategies. We discovered multiple unique genes coding for several of the homologous superfamily of the Ser-Thr-rich glycosyl-phosphatidyl-inositol-anchored membrane family and of the glycolipid 2-alpha-mannosyltransferase family, suggesting that such components on the cell surface are essential to life in high salt. Gene expansion was found in families that participate in sensing of abiotic stress and signal transduction in plants. One example is the patched family of the Sonic Hedgehog receptor proteins, supporting a previous hypothesis that plasma membrane sterols are important for sensing changes in salinities in D. salina. We also investigated genome-based capabilities regarding glycerol metabolism and present an extensive map for core carbon metabolism. We postulate that a second broader glycerol cycle exists that also connects to photorespiration, thus extending the previously described glycerol cycle. Further genome-based analysis of isoprenoid and carotenoid metabolism revealed duplications of genes for 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and phytoene synthase (PSY), with the second gene copy of each enzyme being clustered together. Moreover, we identified two genes predicted to code for a prokaryotic-type phytoene desaturase (CRTI), indicating that D. salina may have eukaryotic and prokaryotic elements comprising its carotenoid biosynthesis pathways. In brief, our genomic data provide the basis for further gene discoveries regarding sensing abiotic stress, the metabolism of this halophilic alga, and its potential in biotechnological applications. Unlabelled Image • Genes were identified coding for proteins potentially responsible for unique adaptations to life in hypersaline environments. • Discovery of genes coding for annexins. • Identification of expansion of the gene family coding for Sonic Hedgehog receptor-related proteins. • Discovery of a novel gene cluster coding for a 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and a phytoene synthase (PSY). • Detection of genes with eukaryotic gene structures predicted to code for the bacterial-type phytoene desaturases (CrtI). [ABSTRACT FROM AUTHOR]

The green alga Dunaliella has long been a model organism for studying abiotic stress responses, and its commercial use for β-carotene production is still being expanded. In the context of the special issue on "The Alga Dunaliella ", we present an overview of the research status of Dunaliella. We highlight some of the existing challenges and provide an outlook for future research. Importantly, the issue of Dunaliella taxonomy has not yet been resolved. As confirmation of the species delineation still awaits a concerted community effort, we propose that reference strains should be designated for some species. Due to the existing resources, we suggest this designation for D. salina be the strain CCAP19/18. In addition, we suggest as references for the halophilic D. viridis the type strain SAG 44.89 and for the marine D. tertiolecta the type strain CCAP19/6B. Such reference strains will be required to consolidate already-reported results and to enable novel, fundamental, and applied research questions to be addressed as well as to open the door to informed strain engineering. We highlight some novel developments that are still not widely known, such as the recent publication of the genome of D. salina. In spite of these challenges, many "omics" studies are increasing knowledge at the molecular level, and the concomitant refinement of the molecular toolbox for Dunaliella species is expected to provide an understanding of the metabolic networks, which will make metabolic engineering and biorefinery approaches commercially viable. • A brief review of the state of the art of Dunaliella research is presented. • A summary of systems biology research with Dunaliella algae is included. • A review of taxonomy issues within the genus of Dunaliella is presented. • Reference strains are proposed for several Dunaliella species. [ABSTRACT FROM AUTHOR]

Carotenoid pigments have been reported the bioactive substances having an antioxidant effect and anti-cancer effect. Recently, the industrial application of natural violaxanthin with anticancer properties has been expanding. Accordingly, in this study, lutein-deficient Chlorella vulgaris , a high producer of violaxanthin, was generated by chemical random mutagenesis. The lutein-deficient mutant was containing only 15.2% lutein compared to wild type. As a cause of this phenomenon, a single point mutation (A336V) on lycopene epsilon cyclase was identified. Consequently, the competitive metabolic pathway of α-branch of xanthophyll synthesis was elevated, causing a 3.18-fold increase in violaxanthin production, compared to that of the wild type. Additionally, the improvement of the growth rate of the mutant (1.3 fold higher than wild type) was observed that caused by enhancement of photosynthesis. As a result of optimization of violaxanthin production, the highest amount of violaxanthin production was achieved under 100 μmol photons m−2 s−1 with acetic acid as the carbon source. Consequently, 3.70 ± 0.45 mg/g violaxanthin, along with 0.45 ± 0.03 mg/g and 0.44 ± 0.02 mg/g of antheraxanthin and zeaxanthin were accumulated in the mutant respectively. These results demonstrate that lutein-deficient Chlorella vulgaris can produce violaxanthin at a concentration equivalent to the highest levels previously reported in marine microalgae and suggesting its potential as a new violaxanthin-producing natural host. • Lutein-deficient Chlorella vulgaris (CvLD) was generated by random mutagenesis. • A single point mutation (A336V) was identified on lycopene epsilon cyclase in CvLD. • Biomass productivity of CvLD was higher than wild type by 1.29 times. • Violaxanthin production of CvLD under optimal condition was 3.70 ± 0.45 mg-Vio/g-DCW. [ABSTRACT FROM AUTHOR]

Under nitrogen (N) starvation, certain microalgae increase carbon storage in the form of lipid droplets while also downregulating photosynthesis and eventually terminating growth. To improve lipid yield, we asked whether lipid droplets and N starvation responses can be induced without limiting growth or photosynthesis. In the chlorophyte Chlamydomonas reinhardtii , N starvation induces gametogenesis alongside lipid droplet accumulation, and gametogenesis has been observed in arginine-fed cultures wherein the arginine was provided as the sole N source. We therefore assessed whether arginine-fed cultures displayed N starvation responses other than gametogenesis in mixo- and phototrophic conditions, representing two primary modes of nutrition that may affect N starvation responses. We showed that arginine-fed cultures using two lab strains of C. reinhardtii supported normal mixotrophic growth, constitutively turned on N starvation-induced genes at the equivalent level to N-starved cells, and increased the triacylglycerol content of total fatty acids by 125–400% relative to ammonium-fed cultures. The lipid profile of triacylglycerol in these arginine-fed mixotrophic cultures exhibited 3 to 5.5-fold enrichment of saturated and monounsaturated fatty acids, a preferred characteristic of biodiesel precursors. Arginine-fed phototrophic cultures likewise turned on N starvation-induced genes, accumulated lipid droplets, and led to a 50% reduced growth rate per day while reaching a 3 to 6.5-fold more cell density at the stationary phase relative to ammonium-fed cultures. To test the applicability of our result to algae outside the green lineage, we conducted similar experiments with the diatom Phaeodactylum tricornutum , which also accumulated 2 to 7-fold more neutral lipids in arginine-fed phototrophic cultures without growth impairment relative to nitrate-fed cultures. We document a system wherein N starvation responses are induced without compromising photosynthesis or growth, thereby suited to the production of valuable chemicals and biofuel precursors without requiring stressors in microalgae. • Arginine catabolism leads to the activation of nitrogen starvation responses. • Arginine-fed cultures grow up to six times higher density than ammonium-fed cultures. • Triacylglycerol from arginine-fed culture is enriched in saturated and monounsaturated fatty acids. [ABSTRACT FROM AUTHOR]