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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.

Oleaginous microalgae are gaining great attention as feedstock for biofuels because of their substantial accumulation capacity for neutral lipids in the cytosolic compartment called the lipid droplet (LD). Understanding the regulatory mechanism of neutral lipid accumulation and degradation, which is mediated by LD-associated proteins, is an important issue in improving lipid productivity. However, LD-associated proteins vary among species and are waiting to be characterized in many microalgae. Stramenopile-type LD protein (StLDP) was previously identified as a primary LD protein in the marine diatom Phaeodactylum tricornutum. We produced a knockout mutant of StLDP by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing. Also, we tried to complement this mutant by expressing recognition site–modified StLDP (RSM-StLDP), which is designed to avoid an attack by Cas9 nuclease expressing in the mutant. The RSM-StLDP:enhanced green fluorescent protein was localized to both LDs and the outer chloroplast-endoplasmic reticulum. The decrease in the LD number per cell, increase in LD size and no alteration of neutral lipid content in the mutant under nitrogen deficiency clearly indicate that StLDP acts as an LD scaffold protein. The number of LDs per cell increased in the complemented strain compared to wild-type (WT) cells. The LD morphology in the mutant is probably over-rescued in the complemented strain by the strong function of the nitrate reductase promoter, which is also supported by high neutral lipid content in the complemented strain. The growth of stldp mutant showed a long lag phase relative to WT cells, suggesting that the low surface-to-volume ratio of fused LD decreased the efficiency of LD hydrolysis during the initial growth phase.

Histidine kinases are common sensory proteins used to detect environmental changes in bacteria. They respond to specific stimuli via a signal-input domain and alter gene expression through a cognate response regulator. The modulation/control of transcriptional regulation in cyanobacteria is important to reinforce the production of useful target compounds via photosynthesis without altering the growth profiles. For instance, heavy metal ions (Ni2+ and Cu2+), chemical inducers (IPTG), and a volatile compound (toluene) have been previously applied to regulate gene expression in cyanobacteria. However, most systems/regulators are only able to regulate gene expression once because it is impossible to eliminate them from the medium. To construct a reversible regulation system, a chimeric sensor, VanN_SphS, was developed by fusing the signal input domain of a quorum-sensing (QS) sensor, VanN, from Vibrio anguillarum, responding N-3-hydroxyhexanoyl-L-homoserine lactone (OHC6-HSL), with the kinase domain of SphS, a phosphate-deficiency sensor from the cyanobacterium Synechocystis sp. PCC 6803. After expression of the chimeric sensor in Synechocystis cells, responses to the various N-acyl-homoserine-lactones (AHLs) were evaluated by measuring the alkaline phosphatase (AP) activity, which is regulated by SphS. VanN_SphS responded only to OHC6-HSL and repressed AP activity. Then, the coexpression of the AHLs-degradation enzyme, Aii20J, a lactonase from Tenacibaculum sp. 20J, resumed the activity. This is the first report on the use of AHL-mediated transcriptional regulation in Synechocystis, which could be used in the future for the controlled production of useful compounds in the cyanobacterium.

Natural astaxanthin is known to be produced by green microalgae, a potent producer of the most powerful antioxidant. To increase the productivity of astaxanthin in microalgae, random mutagenesis has been extensively used to improve the yield of valuable substances. In the presented work, a newly isolated Coelastrum sp. was randomly mutagenized by exposure to ethyl methane sulfonate and further screened using two approaches; an approach for high growth mutant and an approach for high astaxanthin producing mutant with a high-throughput screening method using glufosinate. Among these, mutant G1-C1 that was selected using glufosinate showed the highest of total carotenoids (45.48±1.5 mg/L) and astaxanthin (28.32±2.5 mg/L) production, which was almost 2-fold higher than that of wild type. This study indicates that random mutagenesis via chemical mutation strategy and screening using glufosinate successfully expedited astaxanthin production in a mutated strain of a Coelastrum sp.

The availability of inorganic carbon (C

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.

Thraustochytrids have attracted attention due to the high contents of useful lipids and growth rate. Genus Schizochytrium is commonly used for docosahexaenoic acid (DHA) production, while a strain, which produces a high amount of squalene, has been reported in the genus Aurantiochytrium. These organisms are heterotrophic, and Schizochytrium degrades the extracellular macromolecules, e.g., proteins and polysaccharides, as the nutrients. However, the extracellular lytic enzymes are not well-studied yet. Here, we investigated the induction of extracellular proteases of Schizochytrium aggregatum ATCC 28209. A casein-hydrolytic activity was induced in the nitrogen-limited conditions, and that was also detected by zymography after fractionation by non-heat denatured SDS-PAGE. The proteinous band corresponding to the protease activity was analyzed by MALDI-TOF mass spectrometry after digestion with trypsin. The molecular mass data of the protein fragments were compared to the protein database of S. aggregatum ATCC 28209 in the Joint Genome Institute, and we found that the molecular masses of the six peptides were matched with the prediction from the sequence of a protein, ID 63992, which was annotated as a peptidase S8 family protein. Interestingly, we found that a paralogous protein, ID 99856, was encoded by a gene flanking at the downstream of the gene for ID 63992, and the expression of both genes was similarly induced under the nitrogen-limited conditions. These findings may provide us a key to disclose the induction mechanisms of the extracellular lytic enzymes and the function of the proteolytic enzyme for the nutrition acquisition in thraustochytrids.

A newly isolated green microalga, Coelastrum sp. has the capability to produce and accumulate astaxanthin under various stress conditions. At present, a mutant G1-C1 of Coelastrum sp. obtained through chemical mutagenesis using ethyl methane sulfonate displayed an improvement in astaxanthin accumulation, which was 2-fold higher than that of the wild-type. However, lack of genomic information limits the understanding of the molecular mechanism that leads to a high level of astaxanthin in the mutant G1-C1. In this study, transcriptome sequencing was performed to compare the transcriptome of astaxanthin hyper-producing mutant G1-C1 and wild-type of Coelastrum sp. with respect to astaxanthin biosynthesis. This is to clarify why the mutant produced higher astaxanthin yield compared to the wild-type strain. Based on the transcriptomic analysis, the differentially expressed genes involved in astaxanthin biosynthesis were significantly upregulated in the mutant G1-C1 of Coelastrum sp. Genes coding phytoene synthase, phytoene desaturase, ζ-carotene desaturase, and lycopene β-cyclase involved in β-carotene biosynthesis in the mutant cells were upregulated by 10-, 9.2-, 8.4-, and 8.7-fold, respectively. Genes coding beta-carotene ketolase and beta-carotene 3-hydroxylase involved in converting β-carotene into astaxanthin were upregulated by 7.8- and 8.0-fold, respectively. In contrast, the lycopene ε-cyclase gene was downregulated by 9.7-fold in mutant G1-C1. Together, these results contribute to higher astaxanthin accumulation in mutant G1-C1. Overall, the data in this study provided molecular insight for a better understanding of the differences in astaxanthin biosynthesis between the wild-type and mutant G1-C1 strains.

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.

Chionaster nivalis is frequently detected in thawing snowpacks and glaciers. However, the taxonomic position of this species above the genus level remains unclear. We herein conducted molecular analyses of C. nivalis using the ribosomal RNA operon sequences obtained from more than 200 cells of this species isolated from a field-collected material. Our molecular phylogenetic analyses revealed that C. nivalis is a sister to Bartheletia paradoxa, which is an orphan basal lineage of Agaricomycotina. We also showed that C. nivalis sequences were contained in several previously examined meta-amplicon sequence datasets from snowpacks and glaciers in the Northern Hemisphere and Antarctica.

Highlights • A newly isolated Coelastrum sp. improved the yield of astaxanthin by chemical mutagenesis. • Mutagenesis using chemical mutagen of EMS attempted to increase the microalgae biomass and carotenoid production in Coelastrum sp. • High-throughput screening method using glufosinate successfully expedited astaxanthin production in a mutated strain of a Coelastrum sp. • The selected mutant using glufosinate exhibited an increase of astaxanthin content with ∼2-fold higher compared to the wild type., Natural astaxanthin is known to be produced by green microalgae, a potent producer of the most powerful antioxidant. To increase the productivity of astaxanthin in microalgae, random mutagenesis has been extensively used to improve the yield of valuable substances. In the presented work, a newly isolated Coelastrum sp. was randomly mutagenized by exposure to ethyl methane sulfonate and further screened using two approaches; an approach for high growth mutant and an approach for high astaxanthin producing mutant with a high-throughput screening method using glufosinate. Among these, mutant G1-C1 that was selected using glufosinate showed the highest of total carotenoids (45.48±1.5 mg/L) and astaxanthin (28.32±2.5 mg/L) production, which was almost 2-fold higher than that of wild type. This study indicates that random mutagenesis via chemical mutation strategy and screening using glufosinate successfully expedited astaxanthin production in a mutated strain of a Coelastrum sp.

Biomineralization by calcifying microalgae is a precisely controlled intracellular calcification process that produces delicate calcite scales (or coccoliths) in the coccolithophore Emiliania huxleyi (Haptophycea). Despite its importance in biogeochemical cycles and the marine environment globally, the underlying molecular mechanism of intracellular coccolith formation, which requires calcium, bicarbonate, and coccolith-polysaccharides, remains unclear. In E. huxleyi CCMP 371, we demonstrated that reducing the calcium concentration from 10 (ambient seawater) to 0.1 mM strongly restricted coccolith production, which was then recovered by adding 10 mM calcium, irrespective of inorganic phosphate conditions, indicating that coccolith production could be finely controlled by the calcium supply. Using this strain, we investigated the expression of differentially expressed genes (DEGs) to observe the cellular events induced by changes in calcium concentrations. Intriguingly, DEG analysis revealed that the phosphatidylinositol-specific phospholipase C (PI-PLC) gene was upregulated and coccolith production by cells was blocked by the PI-PLC inhibitor U73122 under conditions closely associated with calcium-induced calcification. These findings imply that PI-PLC plays an important role in the biomineralization process of the coccolithophore E. huxleyi.

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.

Aurantiochytrium sp. is a heterotrophic microorganism that accumulates high amounts of fatty acids (FAs), including odd-chain FAs and an essential FA, docosahexaenoic acid (DHA). Since complex nutrient sources such as tryptone and yeast extract are conventionally utilized in the growth medium for Aurantiochytrium, it is difficult to investigate which individual vitamins affect growth and FA production. Thus, in the present study, we simplified the nitrogen source and defined the vitamins required for Aurantiochytrium sp. cultivation. Glutamate was found to be the most effective simple nitrogen source in cultivation media and thiamine (vitamin B1) was identified as a unique essential vitamin for this organism. Media supplemented with thiamine, as the only member of the vitamin B complex added, allowed for propagation of four strains of Aurantiochytrium. The ratio of odd-chain FAs significantly decreased in the medium containing glucose, glutamate, and thiamine when compared with the conventional medium containing glucose, tryptone, and yeast extract. This is likely caused by a decreased availability of branched-chain amino acids. Under thiamine starvation, FA content per dry cell weight gradually decreased throughout cultivation, and the specific activity of pyruvate dehydrogenase (PDH), a thiamine-dependent enzyme, was severely repressed. These results indicate that when thiamine is absent, the cells compensate some amount of acetyl-CoA through β-oxidation of FAs in vivo, instead of from the reaction by pyruvate, as occurs under normal growth conditions. Providing experimental evidence that the activity of vitamin-dependent enzymes affects biosynthesis of FAs is critical to a better understanding of the effect of vitamin supplementation in Aurantiochytrium sp. media. In the present study, we demonstrated the close relationship between PDH activity, FA content and the importance of thiamine supplementation in the culture medium in Aurantiochytrium sp.

Lipid droplets are ubiquitous cellular compartments that store neutral lipids and specific proteins localize on their surface. These proteins work as a scaffold in maintaining the lipid droplet structure or as regulators of lipogenesis or lipolysis. Previously, the most abundant lipid droplet protein, namely stramenopile-type lipid droplet protein (StLDP), was identified in the marine diatom Phaeodactylum tricornutum; however, its function remains unclear because StLDP does not reveal homology with known lipid droplet proteins and lacks a predictable domain. In this study, P. tricornutum was transformed to express a homologous StLDP gene under an fcpA promoter in order to determine its function. StLDP expression was strongly enhanced in the mutant (H8), especially in nitrogen-sufficient conditions; however, it was attenuated in nitrogen-deficient conditions. Despite the strong expression, no significant difference was observed in the lipid composition between the wild type (WT) and H8 under nitrogen-sufficient conditions. After cultivation in nitrogen-free medium for 6 days, neutral lipid content significantly increased in H8 than in WT. After 2 days of cultivation in nitrogen-free medium, 97.0% of single cells in WT formed one or two lipid droplets, whereas in H8, this proportion decreased to 78.8%, and the proportion of cells forming three or four lipid droplets increased. Thus, the function of StLDP was speculated to sequester triacylglycerol on the initial lipid droplet formation.

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.

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.

Transport systems, which perform a pivotal function in regulating the import and export of various substances via cellular membranes, are important machinery in all living organisms. The ATP-binding cassette (ABC) transporter, one of such transporters, has a crucial role in the high-affinity uptake of various nutrients in bacteria. In the cyanobacterium Synechocystis sp. PCC 6803, a model cyanobacterium, its response to phosphate (Pi)-depleted conditions and the consequential regulation of the Pi transport system have been well-studied. However, its responses and the Pi uptake under Pi-repleted conditions are not comprehended yet. This study observed the peculiar stimulation of the Pi uptake following severe chlorosis in the Synechocystis cells grown under Pi-repleted conditions. The Synechocystis cells grown under high Pi conditions (approximately 4 mM) showed the rapid Pi uptake after 3 d of cultivation, and significantly severe degradation of chlorophyll was observed. Remarkably, the Synechocystis cells showed more than 16-fold higher Pi uptake at that time. Furthermore, we revealed that sulfate (S) deficiency-induced Pi uptake and that magnesium deficiency caused chlorosis and released the intracellularly accumulated Pi. Moreover, reverse genetics revealed that the rapid Pi uptake occurred via an ABC transporter induced by S deficiency, namely, a putative sulfate transporter, SbpA-CysTWA. Our findings provide a new perspective on the Pi uptake under high Pi conditions in cyanobacteria and bacteria.

Microalgae have been used to produce useful compounds. In many cases in which target compounds are produced from CO2 via photosynthesis, there is a trade-off between the proliferation of cells and the production of the target compounds. This is due to carbon partitioning between the synthesis of cellular components and the target compounds, and/or the toxicity of the target products. Therefore, there is a requirement to develop a gene switch that can modulate the metabolism from the growth phase of cells to the production phase of the target molecules. In this study, we attempted to develop an artificial sensory protein that can respond to insoluble gaseous compounds and regulate gene expression in the model cyanobacterium Synechocystis sp. PCC 6803. We constructed a chimeric histidine kinase by fusing the signal input domain of TodS, a toluene sensor from Pseudomonas putida, and the kinase domain of SphS, a phosphate-deficient sensor from Synechocystis. Because TodS contains two kinase domains, we substituted either of the kinase domains of TodS with the kinase domain of SphS to obtain TodSS_SphS and TodSL_SphS. We introduced a gene for the chimeric sensor to Synechocystis cells and evaluated their response to toluene by measuring alkaline phosphatase (AP) activity and phoA mRNA expression. Although the cells harboring TodSL_SphS did not exhibit AP activity, both in the presence or absence of toluene, the cells harboring TodSS_SphS accumulated the transcript of the phoA gene after 5-h exposure to toluene, and only exhibited AP activity after exposure to toluene.

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.

Thraustochytrids, osmoheterotrophic protists which are decomposers in the marine ecosystem, are emerging as promising sources of valuable lipids such as docosahexaenoic acid and squalene. To decompose organic macromolecules in their environment, they are thought to secrete degradation enzymes. However, at present, the secretome analysis of thraustochytrids is still insufficiently studied. In this study, we attempted to identify extracellular proteins secreted by Aurantiochytrium sp. 18W-13a, a thraustochytrid strain that has high squalene content. Supernatants of cultures grown under standard growth media were concentrated and the obtained proteins were fractionated and identified using mass spectrometry. Two proteins, SP1 and SP2, were identified and found to be constitutively expressed. SP2 contains von Willebrand factor A and PAN/APPLE domains, which are involved in protein adhesion. Extracellular proteins from this strain may have roles related to protein or ligand interactions and may be necessary for the cell’s survival.

Eukaryotic Δ6-desaturases are microsomal enzymes which balance the synthesis of ω-3 and ω-6 C18-polyunsaturated-fatty-acids (PUFA) accordingly to their specificity. In several microalgae, includingO. tauri, plastidic C18-PUFA are specifically regulated by environmental cues suggesting an autonomous control of Δ6-desaturation of plastidic PUFA. Sequence retrieval fromO. tauridesaturases, highlighted two putative Δ6/Δ8-desaturases sequences clustering, with other microalgal homologs, apart from other characterized Δ-6 desaturases. Their overexpression in heterologous hosts, includingN. benthamianaandSynechocystis, unveiled their Δ6-desaturation activity and plastid localization.O. taurilines overexpressing these Δ6-desaturases no longer adjusted their plastidic C18-PUFA amount under phosphate starvation but didn’t show any obvious physiological alterations. Detailed lipid analyses from the various overexpressing hosts, unravelled that the substrate features involved in the Δ6-desaturase specificity importantly involved the lipid head-group and likely the non-substrate acyl-chain, in addition to the overall preference for the ω-class of the substrate acyl-chain. The most active desaturase displayed a broad range substrate specificity for plastidic lipids and a preference for ω-3 substrates, while the other was selective for ω-6 substrates, phosphatidylglycerol and 16:4-galactolipid species specific to the native host. The distribution of plastidial Δ6-desaturase products in eukaryotic hosts suggested the occurrence of C18-PUFA export from the plastid.One sentence summaryOsteococcus tauri plastidic lipid C18-PUFA remodelling involves two plastid-located cytochrome-b5 fused Δ6-desaturases with distinct preferences for both head-group and acyl-chain.

International audience; Eukaryotic Δ6-desaturases are microsomal enzymes that balance the synthesis of ω-3 and ω-6 C18-polyunsaturated fatty acids (C18-PUFAs) according to their specificity. In several microalgae, including Ostreococcus tauri, plastidic C18-PUFAs are strictly regulated by environmental cues suggesting an autonomous control of Δ6-desaturation of plastidic PUFAs. Here, we identified two putative front-end Δ6/Δ8-desaturases from Otauri that, together with putative homologs, cluster apart from other characterized Δ6-desaturases. Both were plastid-located and unambiguously displayed a Δ6-desaturation activity when overexpressed in the heterologous hosts Nicotiana benthamiana and Synechocystis sp. PCC6803, as in the native host. Detailed lipid analyses of overexpressing lines unveiled distinctive ω-class specificities, and most interestingly pointed to the importance of the lipid head-group and the nonsubstrate acyl-chain for the desaturase efficiency. One desaturase displayed a broad specificity for plastidic lipids and a preference for ω-3 substrates, while the other was more selective for ω-6 substrates and for lipid classes including phosphatidylglycerol as well as the peculiar 16:4-galactolipid species occurring in the native host. Overexpression of both Δ6-desaturases in Otauri prevented the regulation of C18-PUFA under phosphate deprivation and triggered glycerolipid fatty-acid remodeling, without causing any obvious alteration in growth or photosynthesis. Tracking fatty-acid modifications in eukaryotic hosts further suggested the export of plastidic lipids to extraplastidic compartments.

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.

Coupling of hydrothermal treatment methods with microalgae cultivation within a municipal wastewater treatment process can allow internal recycle of chemicals to improve overall waste treatment efficiency and provide biofuel as by-product. In this work, municipal sludge was treated with high temperature water in two steps to selectively recover nutrients and cellulose-rich fractions to avoid Maillard reactions. In the first step, nitrogen and phosphorous compounds were recovered in the liquid product and cellulose was recovered in the solid residue. In the second step, the cellulose-rich solid residue (ca. 60 wt%), was treated with sulfuric acid to obtain glucose. Cultivation of a mixotrophic microalgae (Euglena gracilis) and a heterotrophic microalgae (Aurantiochytrium) with the liquid product and the isolated glucose product gave positive growth indicating that the proposed two-step hydrothermal liquefaction process allows production of oils and recycle of nutrients such as sugars, nitrogen and phosphorous derived from municipal sludge. Hydrothermal treatment is an effective solid reduction technique and is a feasible method for recycling nutrients for microalgae cultivation.

Microalgae have received much attention as a next-generation source of biomass energy. However, most of the fatty acids (FAs) from microalgae are multiply unsaturated; thus, the biofuels derived from them are fluid, but vulnerable to oxidation. In this study, we attempted to synthesize cyclopropane FAs in the cyanobacterium Synechocystis sp. PCC 6803 by expressing the cfa gene for cyclopropane FA synthase from Escherichia coli with the aim of producing FAs that are fluid and stable in response to oxidization. We successfully synthesized cyclopropane FAs in Synechocystis with a yield of ~30% of total FAs. Growth of the transformants was altered, particularly at low temperatures, but photosynthesis and respiration were not significantly affected. C16:1(∆9) synthesis in the desA(-)/desD(-) strain by expression of the desC2 gene for sn-2 specific ∆9 desaturase positively affected growth at low temperatures via promotion of various cellular processes, with the exceptions of photosynthesis and respiration. Estimation of the apparent activities of desaturases suggested that some acyl-lipid desaturases might recognize the lipid side chain.

Strains of the coccolithophore Emiliania huxleyi (Haptophyta) collected from the subarctic North Pacific and Arctic oceans in 2010 were established as clone cultures and have been maintained in the laboratory at 15 °C and 32 ‰ salinity. To study the physiological responses of coccolith formation to changes in temperature and salinity, growth experiments and morphometric investigations were performed on two strains, namely MR57N isolated from the northern Bering Sea and MR70N at the Chukchi Sea. This is the first report of a detailed morphometric and morphological investigation of Arctic Ocean coccolithophore strains. The specific growth rates at the logarithmic growth phases in both strains markedly increased as temperature was elevated from 5 to 20 °C, although coccolith productivity (estimated as the percentage of calcified cells) was similar at 10–20 % at all temperatures. On the other hand, the specific growth rate of MR70N was affected less by changes in salinity in the range 26–35 ‰, but the proportion of calcified cells decreased at high and low salinities. According to scanning electron microscopy (SEM) observations, coccolith morphotypes can be categorized into Type B/C on the basis of their biometrical parameters. The central area elements of coccoliths varied from thin lath type to well-calcified lath type when temperature was increased or salinity was decreased, and coccolith size decreased simultaneously. Coccolithophore cell size also decreased with increasing temperature, although the variation in cell size was slightly greater at the lower salinity level. This indicates that subarctic and arctic coccolithophore strains can survive in a wide range of seawater temperatures and at lower salinities with change in their morphology. Because all coccolith biometric parameters followed the scaling law, the decrease in coccolith size was caused simply by the reduced calcification. Taken together, our results suggest that calcification productivity may be used to predict future oceanic environmental conditions in the polar regions.

Microalgal lipids and fatty acids are important components for achieving biofuel because of their potential high productivity. Although fatty acids that have a hydroxy group adjacent to the end of the acyl chain might be an important chemical feedstock, most algae do not accumulate it. To produce (ω−1)-hydroxy fatty acids from 3-hydroxybutylyl-CoA, an intermediate for polyhydroxybutylate biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803, we expressed a gene for the promiscuous 3-ketoacyl-ACP synthase III from Alicyclobacillus acidocalderius (aaKASIII) by the cpc560 promoter. To supply 3-hydroxybutyryl-CoAs for aaKASIII, the phaC gene for polyhydroxybutylate polymerase was deleted, and the phaAB genes for 3-hydroxybutyryl-CoA synthesis from Cupriavidus necator were overexpressed. The genetically modified strain synthesized 15-hydroxyhexadecanoic acid, 17-hydroxyoctadecanoic acid, and 17-hydroxyoctadec-9-enoic acid, and accumulated approximately 2.1 mol% of (ω−1)-hydroxy fatty acids in total fatty acids under illumination with 70 μmol photons m−2 s−1, although its growth was severely retarded. Under weak light (35 μmol photons m−2 s−1) conditions, the strain grew as well as the wild-type and showed lower hydroxy fatty acids (0.04 mol%) accumulation than that at higher illumination levels. The photosynthetic activity of this strain was lower than that of wild-type cells, suggesting that high light conditions enhanced hydroxy fatty acids production and inhibited photosynthesis. (ω−1)-Hydroxy fatty acids were not predominantly observed in the galactolipids from thin-layer chromatography, which are the major lipid classes in cyanobacteria. To the best of our knowledge, this is the first report on photoautotrophic production of fatty acids possessing a functional group near the end of the acyl chain in cyanobacteria.

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.

Various kinds of organisms, including microalgae, accumulate neutral lipids in distinct intracellular compartments called lipid droplets. Generally, lipid droplets are generated from the endoplasmic reticulum, and particular proteins localize on their surface. Some of these proteins function as structural proteins to prevent fusion between the lipid droplets, and the others could have an enzymatic role or might be involved in intracellular membrane trafficking. However, information about lipid droplet proteins in microalgae is scarce as compared with that in animals and land plants. We focused on the oil-producing, marine, pennate diatom Phaeodactylum tricornutum that forms lipid droplets during nitrogen deprivation and we investigated the proteins located on the lipid droplets. After 6 d of cultivation in a nitrate-deficient medium, the mature lipid droplets were isolated by sucrose density gradient centrifugation. Proteomic analyses revealed five proteins, with Stramenopile-type lipid droplet protein (StLDP) being the most abundant protein in the lipid droplet fraction. Although the primary sequence of StLDP did not have homology to any known lipid droplet proteins, StLDP had a central hydrophobic domain. This structural feature is also detected in oleosin of land plants and in lipid droplet surface protein (LDSP) of Nannochloropsis species. As a proline knot motif of oleosin, conservative proline residues existed in the hydrophobic domain. StLDP was up-regulated during nitrate deprivation, and fluctuations of StLDP expression levels corresponded to the size of the lipid droplets.

Cyanobacterium is an attractive host for the production of various chemicals and alternative fuels using solar energy and carbon dioxide. In previous study, we succeeded to produce isopropanol using engineered Synechococcus elongatus PCC 7942 under dark and anaerobic conditions (0.43 mM, 26.5 mg/l). In the present study, we report the further optimization of this isopropanol producing condition. We then optimized growth conditions for production of isopropanol by the engineered cyanobacteria, including the use of cells in early stationary phase and buffering of the production medium to neutral pH. We observed that shifting of cultures from dark and anaerobic to light and aerobic conditions during the production phase dramatically increased isopropanol production by conversion to isopropanol from acetate, byproduct under dark and anaerobic condition. Under the optimized production conditions, the titer of isopropanol was elevated 6-fold, to 2.42 mM (146 mg/l).