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 75Se 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\u27-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

Overexpression of Tisochrysis lutea Akd1 identifies a key cold-induced alkenone desaturase enzyme

Alkenones are unusual long-chain neutral lipids that were first identified in oceanic sediments. Currently they are regarded as reliable palaeothermometers, since their unsaturation status changes depending on temperature. These molecules are synthesised by specific haptophyte algae and are stored in the lipid body as the main energy storage molecules. However, the molecular mechanisms that regulate the alkenone biosynthetic pathway, especially the low temperature-dependent desaturation reaction, have not been elucidated. Here, using an alkenone-producing haptophyte alga, Tisochrysis lutea, we show that the alkenone desaturation reaction is catalysed by a newly identified desaturase. We first isolated two candidate desaturase genes and found that one of these genes was drastically upregulated in response to cold stress. Gas chromatographic analysis revealed that the overexpression of this gene, named as Akd1 finally, increased the conversion of di-unsaturated C37-alkenone to tri-unsaturated molecule by alkenone desaturation, even at a high temperature when endogenous desaturation is efficiently suppressed. We anticipate that the Akd1 gene will be of great help for elucidating more detailed mechanisms of temperature response of alkenone desaturation, and identification of active species contributing alkenone production in metagenomic and/or metatranscriptomic studies in the field of oceanic biogeochemistry

Novel alkenone-producing strains of genus Isochrysis (Haptophyta) isolated from Canadian saline lakes show temperature sensitivity of alkenones and alkenoates

Alkenone-producing species have been recently found in diverse lacustrine environments, albeit with taxonomic information derived indirectly from environmental genomic techniques. In this study, we isolated alkenone-producing algal species from Canadian saline lakes and established unialgal cultures of individual strains to identify their taxonomical and molecular biological characteristics. Water and sediments collected from the lakes were first enriched in artificial seawater medium over a range of salinities (5–40 g/L) to cultivate taxa in vitro. Unialgal cultures of seven haptophyte strains were isolated and categorized in the Isochrysis clade using SSU and LSU rRNA gene analysis. The alkenone distributions within isolated strains were determined to be novel compared with other previously reported alkenone-producing haptophytes. While all strains produced the typical C37 and C38 range of isomers, one strain isolated from Canadian salt lakes also produced novel C41 and C42 alkenones that are temperature sensitive. In addition, we showed that all alkenone unsaturation indices (e.g., UK37 and UK'37) are temperature-dependent in culture experiments, and that alkenoate indices (e.g., UA37, UA38, RIA38 and A37/A38) provide alternative options for temperature calibration based on these new lacustrine algal strains. Importantly, these indices show temperature dependence in culture experiments at temperatures below 10 °C, where traditional alkenone proxies were not as sensitive. We hypothesize that this suite of calibrations may be used for reconstructions of past water temperature in a broad range of lakes in the Canadian prairies

Novel alkenone-producing strains of genus Isochrysis (Haptophyta) isolated from Canadian saline lakes show temperature sensitivity of alkenones and alkenoates

Alkenone-producing species have been recently found in diverse lacustrine environments, albeit with taxonomic information derived indirectly from environmental genomic techniques. In this study, we isolated alkenone-producing algal species from Canadian saline lakes and established unialgal cultures of individual strains to identify their taxonomical and molecular biological characteristics. Water and sediments collected from the lakes were first enriched in artificial seawater medium over a range of salinities (5–40 g/L) to cultivate taxa in vitro. Unialgal cultures of seven haptophyte strains were isolated and categorized in the Isochrysis clade using SSU and LSU rRNA gene analysis. The alkenone distributions within isolated strains were determined to be novel compared with other previously reported alkenone-producing haptophytes. While all strains produced the typical C37 and C38 range of isomers, one strain isolated from Canadian salt lakes also produced novel C41 and C42 alkenones that are temperature sensitive. In addition, we showed that all alkenone unsaturation indices (e.g., UK37 and UK'37) are temperature-dependent in culture experiments, and that alkenoate indices (e.g., UA37, UA38, RIA38 and A37/A38) provide alternative options for temperature calibration based on these new lacustrine algal strains. Importantly, these indices show temperature dependence in culture experiments at temperatures below 10 °C, where traditional alkenone proxies were not as sensitive. We hypothesize that this suite of calibrations may be used for reconstructions of past water temperature in a broad range of lakes in the Canadian prairies

Selenium Utilization Strategy by Microalgae

molecule

Mutagenesis of the Oil-producing Algae by Heavy Ion Irradiation

藻類を利用したバイオ燃料は次世代エネルギーとして注目されており、我々は、その候補として、これまで5種のハプト藻でしか合成することが知られていない超長鎖脂質アルケノンに着目している。これまで、アルケノンを産生するハプト藻の1種であるTisochrysis lutea（T-Iso株）にイオンビーム照射を行い、高オイル産生変異株の選抜を行ってきた。アルケノンは暗条件ではエネルギー源として使われるため分解が進むことが知られている。これまでに単離したアルケノン高生産株を明条件で2週間培養後、暗条件で1週間培養を行い、ナイルレッド染色により脂肪酸量の推移を調査した。その結果、アルケノンの分解が遅いと思われる9系統を選抜した。さらに、実際のアルケノンの含量を測定する試験を行い、暗条件処理前の88%以上のアルケノンを含んでいた5系統を選抜した

Mutagenesis of the oil-producing algae by heavy ion beam irradiation

藻類を利用したバイオ燃料は次世代エネルギーとして注目されており、我々は、その候補として、これまで5種のハプト藻でしか合成することが知られていない超長鎖脂質アルケノンに着目している。しかし、アルケノンの生合成についてはほとんど分かっていないため、アルケノンを産生するハプト藻の1種であるTisochrysis lutea（T-Iso株）に重イオンビーム照射を行い、高オイル産生変異株の創出を行ってきた。ここでは、オイル高産生の１つの要因と考えられるアルケノンの分解が抑制されている変異株の探索と解析を行った。アルケノンは暗条件ではエネルギー源として使われるため分解が進むことが知られている。これまでに単離したアルケノン高生産株を明条件で2週間培養後、暗条件で1週間培養を行い、ナイルレッド染色により脂肪酸量の推移を調査した。その結果、アルケノンの分解が遅いと思われる9系統を選抜した

Mutagenesis of the Oil-producing Algae by Ion Beam Irradiation

For biofuel production using microalgae, it is necessary to mutate the algae to improve the productivity of biomass and oil contents. In this study, we focused on very-long-alkyl ketones so-called alkenone that are thought to be good candidates for biofuels. Previously, we reported dose-dependency of the colony formation after 0-320 Gy of carbon ion irradiation. We screened mutants that showed high alkenone productivity from the irradiated cells. In this study, we irradiated carbon ion beams to the high alkenone-producing mutants to investigate the effects of the second-round mutagenesis on the mutant. The cells cultured for 14 d in a 96-well plate were stained with Nile Red and the amount of lipids was evaluated by a microplate reader. The results suggested that it is possible to further improve the productivity of high alkenone-producing strain by the second-round mutagenesis. In the precious study, 30 Gy was suggested to be a suitable dose to enhance oil productivity, however, it seemed better to use a lower dose for the second-round irradiation