Expression of Genes for a Flavin Adenine Dinucleotide-Binding Oxidoreductase and a Methyltransferase from Mycobacterium chlorophenolicum Is Necessary for Biosynthesis of 10-Methyl Stearic Acid from Oleic Acid in Escherichia coli

In living organisms, modified fatty acids are crucial for the functions of the cellular membranes and storage lipids where the fatty acids are esterified. Some bacteria produce a typical methyl-branched fatty acid, i.e., 10-methyl stearic acid (19:0Me10). The biosynthetic pathway of 19:0Me10 in vivo has not been demonstrated clearly yet. It had been speculated that 19:0Me10 is synthesized from oleic acid (18:1Δ9) by S-adenosyl-L-methionine-dependent methyltransfer and NADPH-dependent reduction via a methylenated intermediate, 10-methyelene octadecanoic acid. Although the recombinant methyltransferases UmaA and UfaA1 from Mycobacterium tuberculosis H37Rv synthesize 19:0Me10 from 18:1Δ9 and NADPH in vitro, these methyltransferases do not possess any domains functioning in the redox reaction. These findings may contradict the two-step biosynthetic pathway. We focused on novel S-adenosyl-L-methionine-dependent methyltransferases from Mycobacterium chlorophenolicum that are involved in 19:0Me10 synthesis and selected two candidate proteins, WP_048471942 and WP_048472121, by a comparative genomic analysis. However, the heterologous expression of these candidate genes in Escherichia coli cells did not produce 19:0Me10. We found that one of the candidate genes, WP_048472121, was collocated with another gene, WP_048472120, that encodes a protein containing a domain associated with flavin adenine dinucleotide-binding oxidoreductase activity. The co-expression of these proteins (hereafter called BfaA and BfaB, respectively) led to the biosynthesis of 19:0Me10 in E. coli cells via the methylenated intermediate

Functional analysis of hisitidine kinases by expression of chimeric sensors

科学研究費助成事業 研究成果報告書：基盤研究(C)2011-2013 課題番号：2357004

Cold-induced metabolic conversion of haptophyte di- to tri-unsaturated C37 alkenones used as palaeothermometer molecules

The cosmopolitan marine haptophyte alga Emiliania huxleyi accumulates very long-chain (C37-C40) alkyl ketones with two to four trans-type carbon-carbon double bonds (alkenones). These compounds are used as biomarkers of haptophytes and as palaeothermometers for estimating sea-surface temperatures in biogeochemistry. However, the biosynthetic pathway of alkenones in algal cells remains enigmatic, although it is well known that the C37 tri-unsaturated alkenone (K37:3) becomes dominant at low temperatures, either by desaturation of K37:2 or by a separate pathway involving the elongation of tri-unsaturated alkenone precursors. Here, we present experimental evidence regarding K37:3 synthesis. Using the well-known cosmopolitan alkenone producer E. huxleyi, we labelled K37:2 with 13C by incubating cells with 13C-bicarbonate in the light at 25 °C under conditions of little if any K37:3 production. After stabilisation of the 13C-K37:2 level by depleting 13C-bicarbonate from the medium, the temperature was suddenly reduced to 15 °C. The 13C-K37:2 level rapidly decreased, and the 13C-K37:3 level increased, whereas the total 13C-K37 level—namely [K37:2 + K37:3]—remained constant. These 13C-pulse-chase-like experimental results indicate that 13C-K37:2 is converted directly to 13C-K37:3 by a desaturation reaction that is promoted by a cold signal. This clear-cut experimental evidence is indicative of the existence of a cold-signal-triggered desaturation reaction in alkenone biosynthesis

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

Characterization of cyanobacterial cells synthesizing 10-methyl stearic acid

Recently, microalgae have attracted attention as sources of biomass energy. However, fatty acids from the microalgae are mainly unsaturated and show low stability in oxygenated environments, due to oxidation of the double bonds. The branched-chain fatty acid, 10-methyl stearic acid, is synthesized from oleic acid in certain bacteria; the fatty acid is saturated, but melting point is low. Thus, it is stable in the presence of oxygen and is highly fluid. We previously demonstrated that BfaA and BfaB in Mycobacterium chlorophenolicum are involved in the synthesis of 10-methyl stearic acid from oleic acid. In this study, as a consequence of the introduction of bfaA and bfaB into the cyanobacterium, Synechocystis sp. PCC 6803, we succeeded in producing 10-methyl stearic acid, with yields up to 4.1% of the total fatty acid content. The synthesis of 10-methyl stearic acid in Synechocystis cells did not show a significant effect on photosynthetic activity, but the growth of the cells was retarded at 34 °C. We observed that the synthesis of 10-methylene stearic acid, a precursor of 10-methyl stearic acid, had an inhibitory effect on the growth of the transformants, which was mitigated under microoxic conditions. Eventually, the amount of 10-methyl stearic acid present in the sulfoquinovosyl diacylglycerol and phosphatidylglycerol of the transformants was remarkably higher than that in the monogalactosyldiacylglycerol and digalactosyldiacylglycerol. Overall, we successfully synthesized 10-methyl stearic acid in the phototroph, Synechocystis, demonstrating that it is possible to synthesize unique modified fatty acids via photosynthesis that are not naturally produced in photosynthetic organisms

A novel cell lysis system induced by phosphate deficiency in the cyanobacterium Synechocystis sp. PCC 6803

In the cultivation of microalgae for the production of useful compounds, cell disruption to extract the products of interest is a bottleneck process. To establish a cost-effective method to recover these cellular compounds, we developed a method to induce cell lysis via phosphate deficiency in the cyanobacterium Synechocystis sp. PCC 6803. In this system, the promoter from the phoA gene for alkaline phosphatase expressed bacteriophage genes encoding the lytic enzymes holin and endolysin, thus the cell lysis is induced under phosphate-deficient condition. We observed that 90% of the cells, introduced this bacteriophage genes, were lysed after 24 h of incubation under phosphate-deficient conditions. We also developed a method to induce cell lysis in highly concentrated cells for the efficient recovery of valuable cellular products and observed over 90% cell lysis after 16 h of incubation under these conditions. This inducible lysis system may contribute to decreased cell disruption costs in the algal biotechnology industry

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

Enhanced astaxanthin production by oxidative stress using methyl viologen as a reactive oxygen species (ROS) reagent in green microalgae Coelastrum sp.

Microalgae are known to be a potential resource of high-value metabolites that can be used in the growing field of biotechnology. These metabolites constitute valuable compounds with a wide range of applications that strongly enhance a bio-based economy. Among these metabolites, astaxanthin is considered the most important secondary metabolite, having superior antioxidant properties. For commercial feasibility, microalgae with enhanced astaxanthin production need to be developed. In this study, the tropical green microalgae strain, Coelastrum sp., isolated from the environment in Malaysia, was incubated with methyl viologen, a reactive oxygen species (ROS) reagent that generates superoxide anion radicals (O2-) as an enhancer to improve the accumulation of astaxanthin. The effect of different concentrations of methyl viologen on astaxanthin accumulation was investigated. The results suggested that the supplementation of methyl viologen at low concentration (0.001 mM) was successfully used as a ROS reagent in facilitating and thereby increasing the production of astaxanthin in Coelastrum sp. at a rate 1.3 times higher than in the control

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

Temperature Acclimation of the Picoalga Ostreococcus tauri Triggers Early Fatty-Acid Variations and Involves a Plastidial ?3-Desaturase

Alteration of fatty-acid unsaturation is a universal response to temperature changes. Marine microalgae display the largest diversity of polyunsaturated fatty-acid (PUFA) whose content notably varies according to temperature. The physiological relevance and the molecular mechanisms underlying these changes are however, still poorly understood. The ancestral green picoalga Ostreococcus tauri displays original lipidic features that combines PUFAs from two distinctive microalgal lineages (Chlorophyceae, Chromista kingdom). In this study, optimized conditions were implemented to unveil early fatty-acid and desaturase transcriptional variations upon chilling and warming. We further functionally characterized the O. tauri ω3-desaturase which is closely related to ω3-desaturases from Chromista species. Our results show that the overall omega-3 to omega-6 ratio is swiftly and reversibly regulated by temperature variations. The proportion of the peculiar 18:5 fatty-acid and temperature are highly and inversely correlated pinpointing the importance of 18:5 temperature-dependent variations across kingdoms. Chilling rapidly and sustainably up-regulated most desaturase genes. Desaturases involved in the regulation of the C18-PUFA pool as well as the Δ5-desaturase appear to be major transcriptional targets. The only ω3-desaturase candidate, related to ω3-desaturases from Chromista species, is localized at chloroplasts in Nicotiana benthamiana and efficiently performs ω3-desaturation of C18-PUFAs in Synechocystis sp. PCC6803. Overexpression in the native host further unveils a broad impact on plastidial and non-plastidial glycerolipids illustrated by the alteration of omega-3/omega-6 ratio in C16-PUFA and VLC-PUFA pools. Global glycerolipid features of the overexpressor recall those of chilling acclimated cells.Développement d'une infrastructure française distribuée pour la métabolomique dédiée à l'innovatio