The cadmium tolerance and bioaccumulation mechanism of Tetratostichococcus sp. P1: insight from transcriptomics analysis.

Cadmium (Cd) has become a severe issue in relatively low concentration and attracts expert attention due to its toxicity, accumulation, and biomagnification in living organisms. Cd does not have a biological role and causes serious health issues. Therefore, Cd pollutants should be reduced and removed from the environment. Microalgae have great potential for Cd absorption for waste treatment since they are more environmentally friendly than existing treatment methods and have strong metal sorption selectivity. This study evaluated the tolerance and ability of the microalga Tetratostichococcus sp. P1 to remove Cd ions under acidic conditions and reveal mechanisms based on transcriptomics analysis. The results showed that Tetratostichococcus sp. P1 had a high Cd tolerance that survived under the presence of Cd up to 100 µM, and IC50, the half-maximal inhibitory concentration value, was 57.0 µM, calculated from the change in growth rate based on the chlorophyll content. Long-term Cd exposure affected the algal morphology and photosynthetic pigments of the alga. Tetratostichococcus sp. P1 removed Cd with a maximum uptake of 1.55 mg g-1 dry weight. Transcriptomic analysis revealed the upregulation of the expression of genes related to metal binding, such as metallothionein. Group A, Group B transporters and glutathione, were also found upregulated. While the downregulation of the genes were related to photosynthesis, mitochondria electron transport, ABC-2 transporter, polysaccharide metabolic process, and cell division. This research is the first study on heavy metal bioremediation using Tetratostichococcus sp. P1 and provides a new potential microalga strain for heavy metal removal in wastewater.[Figure: see text]Abbreviations:BP: Biological process; bZIP: Basic Leucine Zipper; CC: Cellular component; ccc1: Ca (II)-sensitive cross complementary 1; Cd: Cadmium; CDF: Cation diffusion facilitator; Chl: Chlorophyll; CTR: Cu TRansporter families; DAGs: Directed acyclic graphs; DEGs: Differentially expressed genes; DVR: Divinyl chlorophyllide, an 8-vinyl-reductase; FPN: FerroportinN; FTIR: Fourier transform infrared; FTR: Fe TRansporter; GO: Gene Ontology; IC50: Growth half maximal inhibitory concentration; ICP: Inductively coupled plasma; MF: molecular function; NRAMPs: Natural resistance-associated aacrophage proteins; OD: Optical density; RPKM: Reads Per Kilobase of Exon Per Million Reads Mapped; VIT1: Vacuolar iron transporter 1 families; ZIPs: Zrt-, Irt-like proteins.

Enhancement of Squalene Production by Constitutive Expression of the 3-Hydroxy-3-Methylglutaryl-CoA Reductase in Aurantiochytrium sp. 18W-13a.

Squalene has a wide range of applications in the industry sectors of dietary supplements, cosmetics, immunization, and pharmaceuticals. Yet, suitable organisms as the source of squalene are limited. It is reported that the thraustochytrid Aurantiochytrium sp. strain 18W-13a can accumulate high content of squalene. However, squalene production in this organism is fluctuated under various conditions and is not yet optimized for commercialization. In this organism, the mevalonate pathway supplies isopentenyl pyrophosphate, the initial substrate for squalene production. In this pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is the rate-limiting enzyme. We found that the HMGR activity had a strong positive correlation with the squalene contents in the strain. We constitutively expressed the HMGR in this organism and found that the transformant showed increased and stable production of squalene as well as carotenoids and biomass. These results clearly indicated that the HMGR expression is the bottleneck of squalene synthesis in Aurantiochytrium sp.

Nutrient recycle from defatted microalgae (Aurantiochytrium) with hydrothermal treatment for microalgae cultivation.

Defatted heterotrophic microalgae (Aurantiochytrium limacinum SR21) was treated with high temperature water (175-350°C, 10-90min) to obtain nitrogen and phosphorous nutrients as a water soluble fraction (WS). Yields of nitrogen and phosphorous recovered in WS varied from 38 to 100% and from 57 to 99%, respectively. Maximum yields of nitrogen containing compounds in WS were proteins (43%), amino acids (12%) and ammonia (60%) at treatment temperatures of 175, 250 and 350°C, respectively. Maximum yield of phosphorous in WS was 99% at a treatment temperature of 250°C. Cultivation experiments of microalgae (A. limacinum SR21) using WS obtained at 200 and 250°C showed positive growth. Water soluble fractions from hydrothermal treatment of defatted microalgae are effective nitrogen and phosphorous nutrient sources for microalgae cultivation.

Modes of hydrocarbon oil biosynthesis revealed by comparative gene expression analysis for race A and race B strains of Botryococcus braunii.

To clarify the oil biosynthetic routes of the oil-producing green alga Botryococcus braunii, here the race-specific gene expression patterns were examined using representative strains of race A and race B producing fatty acid- and triterpene-derived hydrocarbon oils, respectively. The strain-specific gene expression patterns in the BOT-88-2 strain (race A) and the BOT-22 strain (race B) were revealed by transcriptome comparison and real-time PCR quantification. For race A, it was inferred from the gene expression patterns that the fatty acid elongation in the acyl-carrier-protein (acp)-bound form followed by further elongation in the coenzyme A (CoA)-bound form is the major route of oil biosynthesis. The fatty acids may be desaturated in both acp- and CoA-bound forms and once metabolized into glycerolipids prior to further elongation. For race B, relatively direct entry of photosynthetic products from the reductive pentose phosphate cycle into the mevalonate-independent triterpene biosynthesis was implicated.

Wavelength specificity of growth, photosynthesis, and hydrocarbon production in the oil-producing green alga Botryococcus braunii.

The effect of monochromatic light on growth, photosynthesis, and hydrocarbon production was tested in Botryococcus braunii Bot-144 (race B), which produces triterpenoid hydrocarbons. The growth was higher in order of red, blue, and green light. The color of red light-grown cells became more orange-yellow and their shape dominantly changed to grape-like with long branches. Photosynthetic carbon fixation activity was higher in order of blue, red, and green light-grown cells, but photosystem activities showed no difference. In the pulse-chase experiments with (14)CO(2), no major difference was observed in the production of lipids, hydrocarbons, polysaccharides, or proteins among the three kinds of cells, although hydrocarbon production was slightly lower in green light-grown cells. These results indicate that blue and red light were more effective for growth, photosynthetic CO(2) fixation, and hydrocarbon production than green light, and that red light is the most efficient light source when calculated based on photoenergy supplied.

Functional expression of a humanized gene for an omega-3 fatty acid desaturase from scarlet flax in transfected bovine adipocytes and bovine embryos cloned from the cells.

Long-chain n-3 fatty acids can lower the risk of lifestyle-related diseases, therefore, we introduced a plant fatty acid desaturation3 (FAD3) gene into mammalian cells. The FAD3 cDNA was isolated from the immature seeds of scarlet flax and optimized to human high-frequency codon usage for enhancement of its expression levels in mammalian cells (hFAD3). We introduced the gene into bovine muscle satellite cells, which can be differentiated into multilocular adipocytes in vitro. After hFAD3 transfection, the cells were differentiated into adipocytes and their fatty acid composition was analyzed by gas chromatography. The level of alpha-linolenic acid (18:3n-3) in transfected adipocytes increased about ten-fold compared with non-transfected adipocytes. In addition, the levels of docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in transfected adipocytes were significantly higher than those in non-transfected adipocytes. Moreover, we produced bovine cloned embryos from the hFAD3 cells by somatic cell nuclear transfer. Blastocyst rates of hFAD3 clones were the same as the control clones using the non-transfected cells (21% vs 27%, P > 0.05). hFAD3 transcripts were detected in all of the blastocysts. These results demonstrate the functional expression of a plant hFAD3 in mammalian adipocytes, and normal development of cloned embryos carrying the hFAD3 gene.

Identification and characterization of a selenoprotein, thioredoxin reductase, in a unicellular marine haptophyte alga, Emiliania huxleyi.

We found six selenoproteins (EhSEP1-6) in the coccolithophorid Emiliania huxleyi (Haptophyceae) using the (75)Se radiotracer technique. Previously, the most abundant selenoprotein, EhSEP2, was identified as a novel selenoprotein, a protein disulfide isomerase-like protein (Obata, T., and Shiraiwa, Y. (2005) J. Biol. Chem. 280, 18462-18468). The present study focused on the second abundant selenoprotein, EhSEP1, in the same cells and analyzed its molecular properties and regulation of gene expression by selenium. The cDNA sequence of EhSEP1 consists of 1950 base pairs encoding a putative product of 495 amino acids with a calculated molecular mass of 52.2 kDa. The nucleotide and amino acid sequences of EhSEP1 showed strong similarities to those of the enzyme thioredoxin reductase (TR) 1 in the public databases. The EhSEP1 protein contains redox-active cysteine residues in the putative FAD binding domain of the pyridine nucleotide-disulfide oxidoreductase class-1 domain, a dimerization domain, and a C-terminal Gly-Cys-Sec (selenocysteine)-Gly sequence that is known to function as an additional redox center. In the 3'-untranslated region of EhSEP1 cDNA, we found a selenocysteine insertion sequence (SECIS) that is similar to the SECIS found previously in animals. The expression of EhSEP1 showed almost the same pattern under both selenium-sufficient and selenium-deficient conditions. Conversely, TR activity gradually increased 4-fold within ca. 70 h when cells were transferred to the medium containing 10 nm selenite. These data show that selenium is essential for the induction of TR activity at the translational level but not at the transcriptional level in this alga.

Functional expression of a Delta12 fatty acid desaturase gene from spinach in transgenic pigs.

Linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) are polyunsaturated fatty acids that are essential for mammalian nutrition, because mammals lack the desaturases required for synthesis of Delta12 (n-6) and n-3 fatty acids. Many plants can synthesize these fatty acids and, therefore, to examine the effects of a plant desaturase in mammals, we generated transgenic pigs that carried the fatty acid desaturation 2 gene for a Delta12 fatty acid desaturase from spinach. Levels of linoleic acid (18:2n-6) in adipocytes that had differentiated in vitro from cells derived from the transgenic pigs were approximately 10 times higher than those from wild-type pigs. In addition, the white adipose tissue of transgenic pigs contained approximately 20% more linoleic acid (18:2n-6) than that of wild-type pigs. These results demonstrate the functional expression of a plant gene for a fatty acid desaturase in mammals, opening up the possibility of modifying the fatty acid composition of products from domestic animals by transgenic technology, using plant genes for fatty acid desaturases.