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微細藻類を利用したバイオ燃料は第3世代バイオ燃料と呼ばれ、生産性が高いことや食料生産と競合しないことから、次世代エネルギーの候補として注目されている。産業利用を図るには、既存のオイル産生藻類を改良することでオイルをより蓄積し、より速く増殖する株を創出する必要がある。我々はバイオ燃料候補として、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た。本発表では、暗条件下でアルケノン分解率の低かった変異株を用い、アルケノンとそれ以外のオイル(トリグリセリド：TAG)や貯蔵多糖について、暗条件下での減少パターンを解析した結果について報告する。, QST高崎サイエンスフェスタ2021

This study gives the first picture of whole RNA-Sequencing analysis of a PCB-degrading microbe, Rhodococcus jostii RHA1. Genes that were highly expressed in biphenyl-grown cells, compared with pyruvate-grown cells, were chosen based on the Reads Per Kilobase Million (RPKM) value and were summarized based on the criteria of RPKM ≥100 and fold change ≥2.0. Consequently, 266 total genes were identified as genes expressed particularly for the degradation of biphenyl. After comparison with previous microarray data that identified highly-expressed genes, based on a fold change ≥2.0 and p-value ≤0.05, 62 highly-expressed genes from biphenyl-grown cells were determined from both analytical platforms. As these 62 genes involve known PCB degradation genes, such as bph, etb, and ebd, the genes identified in this study can be considered as essential genes for PCB/biphenyl degradation. In the 62 genes, eleven genes encoding hypothetical proteins were highly expressed in the biphenyl-grown cells. Meanwhile, we identified several highly-expressed unannotated DNA regions on the opposite strand. In order to verify the encoded proteins, two regions were cloned into an expression vector. A protein was successfully obtained from one region at approximately 25 kDa from the unannotated strand. Thus, the genome sequence with transcriptomic analysis gives new insight, considering re-annotation of the genome of R. jostii RHA1, and provides a clearer picture of PCB/biphenyl degradation in this strain.

Biofuel production by microalgae, is attracting attention as a candidate for next-generation energy, because it does not compete with food production. We have been trying heavy ion beam irradiation to create useful mutant strains that are more suitable for oil production. Here, we focused on cerulenin, which is known as inhibitor of fatty acid synthesis, to get fatty acid and alkenone synthesis enhanced mutants. We picked up mutants as following 3 groups, 1) top 10% mutants with high lipid fluorescence intensity after 4-week cultivation, 2) top 10% mutants with large increase in lipid fluorescence intensity between culture after 1 week and 4 weeks, 3) top 10% mutants with high chlorophyll fluorescence intensity and high optical density after 3-week cultivation.

Alkenones are long-chain methyl/ethyl ketones (mainly in length of C37-C39) with two to four trans-unsaturated bonds produced by several kinds of marine haptophytes such as Emiliania huxleyi (coccolithophore). The physiological functions and metabolic profile of alkenones are not well known yet. In this study, we focused on elucidating how alkenones contribute to energy storage and cellular carbon partitioning in relation to other cellular components. For the purpose, we analyzed the changes in carbon allocation among various cell components like lipids, alkenones, proteins, and polysaccharides between cells exposed to N-sufficient (+N) and N-limited conditions (−N) in E. huxleyi CCMP 2090. Finally, the alkenones were found to function as main storage lipids and their accumulation was clearly increased by −N, whereas triacylglycerols (TAGs) were barely detected under any N conditions. The mobilization of carbons into alkenones was stimulated by −N from 15% under +N to 27% under −N. However, photosynthetic C allocation into other components was suppressed by −N, showing that percent C allocation into fatty acids, proteins, and polysaccharides was decreased from 9, 46, and 6.8% under +N to 7, 25, and 4.5% under −N, respectively. In addition, fatty acids such as 16:0, 18:0, 18:1, and 18:2 became dominant under −N while 18:5 became dominant under +N conditions, with no significant change in 22:6. This study revealed that alkenones function as primary carbon storage pools especially under −N condition in E. huxleyi CCMP 2090 and that N supply triggers a dynamic change in carbon metabolism by modifying membrane lipid composition and regulating carbon allocation preferences.

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。

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., U37 K and U37 K′) are temperature-dependent in culture experiments, and that alkenoate indices (e.g., U37 A, U38 A, 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.

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.

The hydrocarbons in cultures of marine haptophytes Emiliania huxleyi NIES837 and Gephyrocapsa oceanica NIES1315 were analyzed, and nonacosadienes and hentriacontadienes were detected as the major compounds in both strains. C29 and C31 monoenes and di-, tri- and tetra-unsaturated C33 alkenes were also detected as minor compounds but not C37 and C38 alkenes. The positions of the double bonds in the C29 and C31 alkenes were determined by mass spectrometry of their dimethyl disulfide (DMDS) adducts. Among the four C29 alkenes identified, the most abundant isomer was 2,20-nonacosadiene, and the other three compounds were 1,20-nonacosadiene, 3,20-nonacosadiene and 9-nonacosene, respectively. Hitherto, 2,20-nonacosadiene and 3,20-nonacosadiene were unknown to be natural products. The double bond at the n-9 (ω9) position in these C29 alkenes is hypothesized to be derived from precursors of unsaturated fatty acids possessing an n-9 double bond, such as (9Z)-9-octadecenoic acid. Nonacosadienes have the potential for being used as distinct haptophyte biomarkers.

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。

The compositions of long chain alkenes, alkenones and alkenoates in a cultured strain of the haptophyte Chrysotila lamellosa CCMP1307, isolated from a salt marsh, were investigated. The biomarker patterns were distinctive and showed a high proportion of tetraunsaturated alkenones and alkenoates, with a pronounced proportion of C 40 alkenones and a lack of C 38 methyl and C 39 ethyl alkenones. Linear regression of the alkenone unsaturation degree ( U 37 K ) with growth temperature ( T ) was obtained over the range of possible CCMP1307 growth temperature values (4–20 °C): U 37 K = 0.045 × T (°C) − 1.016, ( n = 13, r 2 0.96), while the U 37 K ′ values were weakly correlated with T : U 37 K ′ = 0.0035 T (°C) + 0.0511 ( n = 18, r 2 0.70). The U 37 K calibration exhibited a low y-intercept in comparison with that of a Chinese inland lake strain reported previously. The data show significant intraspecific variation in U 37 K for C. lamellosa between strains from different geographic origins. C. lamellosa CCMP1307 reproduced typical U 37 K values observed in C 37:4 rich lakes, especially at lower temperature ( 29:2 , C 31:1 , C 31:2 and C 31:3 alkenes, and found that the unsaturation degree of C 31 alkenes ( U 31 en ), calculated with C 31:1 and C 31:2 alkenes, might also be a useful index of growth temperature for the haptophyte C. lamellosa .

The marine coccolithophore Emiliania huxleyi (Haptophyta) requires selenium as an essential element for growth, and the active species absorbed is selenite, not selenate. This study characterized the selenite uptake mechanism using ⁷⁵Se as a tracer. Kinetic analysis of selenite uptake showed the involvement of both active and passive transport processes. The active transport was suppressed by 0.5 mM vanadate, a membrane-permeable inhibitor of H⁺-ATPase, at pH 8.3. When the pH was lowered from 8.3 to 5.3, the selenite uptake activity greatly increased, even in the presence of vanadate, suggesting that the H⁺ concentration gradient may be a motive force for selenite transport. [⁷⁵Se]Selenite uptake at selenite-limiting concentrations was hardly affected by selenate, sulfate and sulfite, even at 100 μM. In contrast, 3 μM orthophosphate increased the K(m) 5-fold. These data showed that HSeO₃⁻, a dominant selenite species at acidic pH, is the active species for transport through the plasma membrane and transport is driven by ΔpH energized by H⁺-ATPase. Kinetic analysis showed that the selenite uptake activity was competitively inhibited by orthophosphate. Furthermore, the active selenite transport mechanism was shown to be induced de novo under Se-deficient conditions and induction was suppressed by the addition of either sufficient selenite or cycloheximide, an inhibitor of de novo protein synthesis. These results indicate that E. huxleyi cells developed an active selenite uptake mechanism to overcome the disadvantages of Se limitation in ecosystems, maintaining selenium metabolism and selenoproteins for high viability.

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.

The diversity of selenoproteins raises the question of why so many life forms require selenium. Selenoproteins are found in bacteria, archaea, and many eukaryotes. In photosynthetic microorganisms, the essential requirement for selenium has been reported in 33 species belonging to six phyla, although its biochemical significance is still unclear. According to genome databases, 20 species are defined as selenoprotein-producing organisms, including five photosynthetic organisms. In a marine coccolithophorid, Emiliania huxleyi (Haptophyta), we recently found unique characteristics of selenium utilization and novel selenoproteins using 75 Se-tracer experiments. In E. huxleyi , selenite, not selenate, is the main substrate used and its uptake is driven by an ATP-dependent high-affinity, active transport system. Selenite is immediately metabolized to low-molecular mass compounds and partly converted to at least six selenoproteins, named EhSEP1–6. The most (EhSEP2) and second-most abundant selenoproteins (EhSEP1) are disulfide isomerase (PDI) homologous protein and thioredoxin reductase (TR) 1, respectively. Involvement of selenium in PDI is unique in this organism, while TR1 is also found in other organisms. In this review, we summarize physiological, biochemical, and molecular aspects of selenium utilization by microalgae and discuss their strategy of selenium utilization.

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′-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.

Several haptophyte algae produce unique neutral long-chain (C-37-C-40) ketones (alkenones) with two to four trans-carbon-carbon double (C=C) bonds and a keto-group. These molecules are of great biological interest and may have substantial commercial relevance as biofuel sources. Unfortunately, their detection and quantification, involving extraction from cells with organic solvents and fractionation of lipids through columns, are rather cumbersome. Hence, we developed a method for the rapid and reliable quantification of alkenones in intact cells using Fourier transform infrared spectroscopy (FTIR). The method is based on the absorption band at 962.5 cm(-1), which corresponds to a trans-C-C bond that is unequivocally attributable to alkenones. This peak was exclusively observed in alkenone-producing haptophytes such as Emiliania huxleyi NIES-837, Emiliania huxleyi CCMP 2090, Tisochrysis lutea (former Isochrysis galbana T-iso), Tisochrysis lutea CCMP 463, and Chrysotila lamellosa CCMP 1307, as well as in appropriate standards. We compared our FTIR method with a typical GC analysis method. The alkenone quantity determined by these two methods showed similar values. Nevertheless, the FTIR method developed in this study does not require complex extraction procedures and is therefore a much easier and rapid quantification method. This FTIR method can also be used for the screening of strains and the optimization of culture conditions for alkenone production. (C) 2016 Elsevier B.V. All rights reserved.

Selenium (Se) is an essential trace element for many organisms in order to be required for the synthesis of selenoproteins although it is very toxic at high concentration. More than 40 selenoprotein families have been identified in diverse organisms, including bacteria, archaea and eukaryotes. In photosynthetic microorganisms such as microalgae, many selenoproteins have also been identified by using bioinformatics approaches. Essentiality of Se requirement was experimentally proved in several algae, such as haptophyte algae, that possess selenoproteins. On the other hand, all of them that possess selenoproteins do not always show Se essential requirement in certain microalgae, such as a green algae. Se presents as four inorganic forms of selenide (−2), selenium (0), selenite (+2) and selenite (+4) and organic selenium-containing compounds. This Chapter focuses on the function of Se, membrane transport system, intracellular accumulation, metabolism to non-toxic organic compounds, and then the synthesis of selenoproteins involving a selenocysteine in which Se is replaced with sulfur. Especially, this Chapter introduces a unique property of E. huxleyi (coccolithophore, haptophyte) that possess two pathways for Se-compound production, namely both animal-like property to synthesize selenoproteins and land plant-like property to synthesize non-toxic organic compounds pathways. Algae can be considered to have evolved their properties for obtaining high viability by adjusting their Se-metabolism.

Metagenome research has brought much attention for the identification of important and novel genes of industrial and pharmaceutical value. Here, using a metagenome library constructed from bacteria associated with the marine sponge, Styllisa massa, a high-throughput screening technique using radioisotope was implemented to screen for cadmium (Cd) binding or accumulation genes. From a total of 3301 randomly selected clones, a clone 247-11C was identified as harboring an open reading frame (ORF) showing Cd accumulation characteristics. The ORF, termed as ORF5, was further analyzed by protein functional studies to reveal the presence of a protein, Cdae-1. Cdae-1, composed of a signal peptide and domain harboring an E(G/A)KCG pentapeptide motif, enhanced Cd accumulation when expressed in Escherichia coli. Although showing no direct binding to Cd in vitro, the presence of important amino acid residues related to Cd detoxification suggests that Cdae-1 may possess a different mechanism from known Cd binding proteins such as metallothioneins (MTs) and phytochelatins (PCs). In summary, using the advantage of bacterial metagenomes, our findings in this work suggest the first report on the identification of a unique protein involved in Cd accumulation from bacteria associated with a marine sponge.

Lipid body (LB) is recognized as the cellular carbon and energy storage organelle in many organisms. LBs have been observed in the marine haptophyte alga Tisochrysis lutea that produces special lipids such as long-chain (C37 -C40) ketones (alkenones) with 2-4 trans-type double bonds. In this study, we succeeded in developing a modified method to isolate LB from T. lutea. Purity of isolated LBs was confirmed by the absence of chlorophyll auto-fluorescence and no contamination of the most abundant cellular protein ribulose-1,5-bisphosphate carboxylase/oxygenase. As alkenones predominated in the LB by GC-MS analysis, the LB can be more appropriately named as "alkenone body (AB)." Extracted AB-containing proteins were analyzed by the combination of 1DE (SDS-PAGE) and MS/MS for confident protein identification and annotated using BLAST tools at National Center for Biotechnology Information. Totally 514 proteins were identified at the maximum. The homology search identified three major proteins, V-ATPase, a hypothetical protein EMIHUDRAFT_465517 found in other alkenone-producing haptophytes, and a lipid raft-associated SPFH domain-containing protein. Our data suggest that AB of T. lutera is surrounded by a lipid membrane originating from either the ER or the ER-derived four layer-envelopes chloroplast and function as the storage site of alkenones and alkenes.

A glucose-tolerant strain of the cyanobacterium Synechocystis sp. PCC 6803, generally referred to as wild type, produces a hemolysin-like protein (HLP) located on the cell surface. To analyze the function of HLP, we constructed a mutant in which the hlp gene was disrupted. The growth rate of the mutant was reduced when the cells were stressed by treatment with CuSO(4), CdCl(2), ZnCl(2), ampicillin, kanamycin, or sorbitol in liquid medium, suggesting that HLP may increase cellular resistance to the inhibitory effects of these compounds. Uptake assays with (109)Cd(2+) using the silicone-oil layer centrifugation technique revealed that both wild type and mutant cells were labeled with (109)Cd(2+) within 1 min. Although the total radioactivity was much higher in the wild-type cells, (109)Cd(2+) incorporation was clearly much higher in the mutant cells after adsorbed (109)Cd(2+) was removed from the cell surface by washing with EDTA. These findings suggest that HLP functions as a barrier against the adsorption of toxic compounds.

We investigated the uptake and bioconcentration of the essential element selenium by a coccolithophorid, Emiliania huxleyi, using [75Se]selenite. The time course of 75Se uptake showed a biphasic pattern, namely a primary phase and a subsequent secondary phase. The primary and secondary phases are due to a rapid selenite uptake process that attained a stationary level within 2 min and a slow Se-accumulation process that continued at a constant rate for 4 h or longer, respectively. Kinetic analysis revealed that the selenite uptake process consists of two components, one saturable and one linearly related to substrate concentration. The Km of the saturable component was 29.8 nM selenite; the uptake activity of this component was suppressed by inhibitors of ATP biogenesis, suggesting that selenite uptake is driven by a high-affinity, active transport system. During a 6-h incubation of cells with [75Se]selenite, 70% of the intracellular 75Se was incorporated into low-molecular-mass compounds (LMCs), and 17% was incorporated into proteins, but [75Se]selenite was barely detectable. A pulse-chase experiment demonstrated that the 75Se that had accumulated in LMCs was transferred into proteins. When the syntheses of amino acids and proteins were each separately inhibited, 75Se incorporation into LMCs and proteins was decreased. These results suggest that E. huxleyi rapidly absorbs selenite, filling a small intracellular pool. Then, Se-containing LMCs are immediately synthesized from the selenite, creating a pool of LMCs that are then metabolized to selenoproteins.