Chromosome-Scale Genome Assembly of the Marine Oleaginous Diatom Fistulifera solaris.

Microalgae including diatoms are of interest for environmentally friendly manufacturing such as production of biofuels, chemicals, and materials. The highly oil-accumulating marine diatom Fistulifera solaris has been studied as a promising host organism to be employed for these applications. Recently reported large-scale genetic engineering based on episomal vectors for diatoms could be useful to further enhance the potential of F. solaris, whereas we need to understand more the mode-of-action of diatom centromeres to rationally design the episomal vectors for stable extrachromosomal maintenance. Our previous genome analysis with pyrosequencing (short read sequencing) had generated the fragmented scaffolds which were not useful to predict centromeres on each chromosome. Here, we report the almost complete chromosomal structure of the genome of F. solaris using a long-read nanopore sequencing platform MinION. From just one single run using a MinION flow-cell, the chromosome-scale assembly with telomere-to-telomere resolution was achieved for 41 out of 44 chromosomes. Putative centromere regions were predicted from the 16 chromosomes, and we discovered putative consensus motifs in the predicted centromeres. Similar motif search had been performed in model diatoms, but no consensus motif was found. Therefore, this is the first study to successfully estimate consensus motifs in diatom centromeres. The chromosome-scale assembly also suggests the potential existence of multi-copy mini-chromosomes and tandemly repeated lipogenesis genes related to the oleaginous phenotype of F. solaris. Findings of this study are useful to understand and further engineer the oleaginous phenotype of F. solaris.

Assessment on the oil accumulation by knockdown of triacylglycerol lipase in the oleaginous diatom Fistulifera solaris.

Microalgae are promising producers of biofuel due to higher accumulation of triacylglycerol (TAG). However, further improvement of the lipid metabolism is critical for feasible application of microalgae in industrial production of biofuel. Suppression of lipid degradation pathways is a promising way to remarkably increase the lipid production in model diatoms. In this study, we established an antisense-based knockdown (KD) technique in the marine oleaginous diatom, Fistulifera solaris. This species has a capability to accumulate high content of lipids. Tgl1 KD showed positive impact on cell growth and lipid accumulation in conventional culture in f/2 medium, resulting in higher oil contents compared to wild type strain. However, these impacts of Tgl1 KD were slight when the cells were subjected to the two-stage growth system. The Tgl1 KD resulted in slight change of fatty acid composition; increasing in C14:0, C16:0 and C16:1, and decreasing in C20:5. This study indicates that, although Tgl1 played a certain role in lipid degradation in F. solaris, suppression of only a single type of TAG lipase was not significantly effective to improve the lipid production. Comprehensive understanding of the lipid catabolism in this microalga is essential to further improve the lipid production.

Engineered chlorophyll catabolism conferring predator resistance for microalgal biomass production.

Production of valuable compounds including biofuels and pharmaceutical precursors derived from microalgae has garnered significant interest. Stable production of algal biomass is essential to make the microalgal industry commercially feasible. However, one of the largest issues is severe biological contamination by predators grazing the algal biomass, resulting in the crash of outdoor cultures. In the present study, we propose a novel engineering strategy for microalgae to cope with predators. The overexpression of plant chlorophyllase (CLH) in a microalga resulted in the enhancement of resistance to the predator. This result supported our hypothesis that CLH promotes chlorophyll breakdown in the chloroplasts of the microalgae when they are digested by the predator, generating the phototoxic catabolite chlorophyllide that damages the predator. To the best of our knowledge, this is the first study to establish predator-resistant microalgae by enhancing the CLH activity.

Prostaglandin production by the microalga with heterologous expression of cyclooxygenase.

Prostaglandins (PGs) are the physiologically active compounds synthesized from C20 polyunsaturated fatty acids (PUFAs) by cyclooxygenase (COX) and a series of PG synthases, and are utilized as pharmaceuticals. Currently, commercialized PGs are mainly produced by chemical synthesis under harsh conditions. By contrast, bioproduction of PGs can be an alternative, environmental-friendly, and inexpensive process with genetic engineering of model plants, although these conventional host organisms contain a limited quantity of PG precursors. In this study, we established an efficient PG production process using the genetically engineered microalga Fistulifera solaris which is rich in C20 PUFAs. A cox gene derived from the red alga Agarophyton vermiculophyllum was introduced into F. solaris. As a result, a transformant clone with high cox expression produced PGs (i.e., PGD2 , PGE2 , PGF2α , and 15-ketoPGF2α derived from arachidonic acid, and PGD3 , PGE3 , and PGF3α derived from eicosapentaenoic acid) as revealed by liquid chromatography/mass spectrometry. The total content of PGs was 1290.4 ng/g of dry cell weight, which was higher than that produced in the transgenic plant reported previously. The results obtained in this study indicate that the C20 PUFA-rich microalga functionally expressing COX is a promising host for PG bioproduction.

Two bacterial glycosphingolipid synthases responsible for the synthesis of glucuronosylceramide and α-galactosylceramide.

Bacterial glycosphingolipids such as glucuronosylceramide and galactosylceramide have been identified as ligands for invariant natural killer T cells and play important roles in host defense. However, the glycosphingolipid synthases required for production of these ceramides have not been well-characterized. Here, we report the identification and characterization of glucuronosylceramide synthase (ceramide UDP-glucuronosyltransferase [Cer-GlcAT]) in Zymomonas mobilis, a Gram-negative bacterium whose cellular membranes contain glucuronosylceramide. On comparing the gene sequences that encode the diacylglycerol GlcAT in bacteria and plants, we found a homologous gene that is widely distributed in the order Sphingomonadales in the Z. mobilis genome. We first cloned the gene and expressed it in Escherichia coli, followed by protein purification using nickel-Sepharose affinity and gel filtration chromatography. Using the highly enriched enzyme, we observed that it has high glycosyltransferase activity with UDP-glucuronic acid and ceramide as sugar donor and acceptor substrate, respectively. Cer-GlcAT deletion resulted in a loss of glucuronosylceramide and increased the levels of ceramide phosphoglycerol, which was expressed in WT cells only at very low levels. Furthermore, we found sequences homologous to Cer-GlcAT in Sphingobium yanoikuyae and Bacteroides fragilis, which have been reported to produce glucuronosylceramide and α-galactosylceramide, respectively. We expressed the two homologs of the cer-glcat gene in E. coli and found that each gene encodes Cer-GlcAT and Cer-galactosyltransferase, respectively. These results contribute to the understanding of the roles of bacterial glycosphingolipids in host-bacteria interactions and the function of bacterial glycosphingolipids in bacterial physiology.

Taming chlorophylls by early eukaryotes underpinned algal interactions and the diversification of the eukaryotes on the oxygenated Earth.

Extant eukaryote ecology is primarily sustained by oxygenic photosynthesis, in which chlorophylls play essential roles. The exceptional photosensitivity of chlorophylls allows them to harvest solar energy for photosynthesis, but on the other hand, they also generate cytotoxic reactive oxygen species. A risk of such phototoxicity of the chlorophyll must become particularly prominent upon dynamic cellular interactions that potentially disrupt the mechanisms that are designed to quench photoexcited chlorophylls in the phototrophic cells. Extensive examination of a wide variety of phagotrophic, parasitic, and phototrophic microeukaryotes demonstrates that a catabolic process that converts chlorophylls into nonphotosensitive 132,173-cyclopheophorbide enols (CPEs) is phylogenetically ubiquitous among extant eukaryotes. The accumulation of CPEs is identified in phagotrophic algivores belonging to virtually all major eukaryotic assemblages with the exception of Archaeplastida, in which no algivorous species have been reported. In addition, accumulation of CPEs is revealed to be common among phototrophic microeukaryotes (i.e., microalgae) along with dismantling of their secondary chloroplasts. Thus, we infer that CPE-accumulating chlorophyll catabolism (CACC) primarily evolved among algivorous microeukaryotes to detoxify chlorophylls in an early stage of their evolution. Subsequently, it also underpinned photosynthetic endosymbiosis by securing close interactions with photosynthetic machinery containing abundant chlorophylls, which led to the acquisition of secondary chloroplasts. Our results strongly suggest that CACC, which allowed the consumption of oxygenic primary producers, ultimately permitted the successful radiation of the eukaryotes throughout and after the late Proterozoic global oxygenation.

Marine microalgae for production of biofuels and chemicals.

Marine microalgae are recognized as promising feedstocks for biofuels and chemicals owing to their higher growth rates than those of terrestrial crop plants. We aimed to summarize the production of biofuels and chemicals by marine microalgae and to discuss their advantages and potential from the aspect of bioprocess. The present circumstances of the microalgae industry were briefly described and large-scale industrial plants for microalgae production, where some marine microalgae are cultivated, were introduced. The advantages of marine microalgae in terms of water and land usage were also discussed. Finally, novel genome editing tools that could further exploit the potential of marine microalgae were reviewed. The present study provided comprehensive information regarding current biotechnology using marine microalgae.

Production of eicosapentaenoic acid by high cell density cultivation of the marine oleaginous diatom Fistulifera solaris.

Polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA), have attracted attention owing to their health benefits for humans, as well as their importance in aquaculture and animal husbandry. Establishing a sustainable PUFA supply based on fish oils has been difficult due to their increasing demand. Therefore, alternative sources of PUFAs are required. In this research, we examined the potential of the marine oleaginous diatom Fistulifera solaris as an alternative producer of PUFAs. Optimization of culture conditions was carried out for high cell density cultivation, and a maximal biomass productivity of 1.32±0.13g/(L·day) was achieved. By slightly adjusting the culture conditions for EPA production, the maximal EPA productivity reached 135.7±10.0mg/(L·day). To the best of our knowledge, this is the highest EPA productivity among microalgae cultured under photoautotrophic conditions. This result indicates that F. solaris is a promising candidate host for sustainable PUFA production.

UV-C irradiation accelerates neutral lipid synthesis in the marine oleaginous diatom Fistulifera solaris.

This study investigated the induction of oil synthesis in the oleaginous diatom, Fistulifera solaris, following irradiation with small doses of UV-C. A rapid induction of oil accumulation was confirmed within 6h following UV-C radiation of the diatom cells, with increases in cell oil body volumes after 24h of approximately 4- to 6-fold from the initial volume. Reactive oxygen species (ROS), which can be generated by a UV-C-mediated reaction, were detected in irradiated cells and the correlation between ROS generation and oil accumulation was confirmed. The smallest UV-C intensity required for oil induction in the cells was 10mJ/cm2. Based on the ideal biodiesel profile, the most suitable FAME composition was obtained when UV255 was used to irradiate the cells. The UV-C radiation method is therefore a solution for shortening the oil accumulation period and improving biodiesel productivity.

Outdoor Cultivation of Marine Diatoms for Year-Round Production of Biofuels.

Biofuel production using microalgae is believed to have the advantage of continuous year-round production over crop plants, which have strong seasonality. However, actual year-round production of microalgal lipids using outdoor mass cultivation has rarely been demonstrated. In our previous study, it was demonstrated that the oleaginous diatom, Fistulifera solaris, was culturable in outdoor bioreactors from spring to autumn, whereas biomass and lipid production in winter failed because F. solaris did not grow below 15 °C. Therefore, another candidate strain that is culturable in winter is required. In this study, a cold-tolerant diatom, Mayamaea sp. JPCC CTDA0820, was selected as a promising candidate for biofuel production in winter. Laboratory-scale characterization revealed that this diatom was culturable at temperatures as low as 10 °C. Subsequently, F. solaris (April-October) and Mayamaea sp. JPCC CTDA0820 (November-March) were cultured in outdoor open-pond bioreactors, wherein year-round production of diatom lipids was successfully demonstrated. The maximal values of areal productivities of biomass and lipids reached to 9.79 and 1.80 g/(m² day) for F. solaris, and 8.62 and 0.92 g/(m² day) for Mayamaea sp. JPCC CTDA0820, respectively. With the combined use of these two diatom species, stable year-round production of microalgal lipids became possible.

Isolation and characterization of bacterium producing lipid from short-chain fatty acids.

Anaerobic fermentation generates propionic acid, which inhibits microbial growth and accumulates in wastewater containing increased amounts of organic matter. We therefore isolated a propionic acid-assimilating bacterium that could produce triacylglycerol, for use in wastewater treatment. Nitratireductor sp. strain OM-1 can proliferate in medium containing propionic, acetic, butyric, and valeric acids as well as glycerol, and produces triacylglycerol when both propionic and acetic acids or glycerol are present. In composite model wastewater containing acetic acid, propionic acid and glycerol, this strain shows an even higher conversion rate, suggesting that it is suitable for wastewater treatment. Further, nitrogen depletion in medium containing an acetic-propionic acid mixture resulted in the production of the light oil 2-butenoic acid 1-methylethyl ester, but not triacylglycerol. Collectively, our data indicate that strain OM-1 has the potential to reduce accumulation of activated sludge in wastewater treatment and may contribute to the production of biodiesel.

Enhancement of glycerol metabolism in the oleaginous marine diatom Fistulifera solaris JPCC DA0580 to improve triacylglycerol productivity.

BACKGROUND: Microalgal oil is a promising alternative feedstock for biodiesel fuel (BDF). Mixotrophic cultivation with glycerol, the primary byproduct of BDF production, may be used to optimize BDF production. This strategy would reduce costs through glycerol recycling and improve lipid productivity and biomass productivity by overcoming the growth retardation caused by decreased light penetration in high-density culture. RESULTS: Overexpression of the endogenous glycerol kinase (GK) gene in an oleaginous marine diatom, Fistulifera solaris JPCC DA0580, accelerates glycerol metabolism and improves lipid and biomass productivities. Two candidates were selected from a collection of 90 G418-resistant clones, based on growth and confirmation of genome integration. GK gene expression was higher in the selected clones (GK1_7 and GK2_16) than in the wild-type culture. The GK2_16 clone achieved a 12% increase in lipid productivity. CONCLUSION: We have demonstrated the potential of metabolic engineering in oleaginous microalgae to improve lipid productivity. Metabolic engineering techniques can be used to optimize BDF production.

Oil accumulation by the oleaginous diatom Fistulifera solaris as revealed by the genome and transcriptome.

Oleaginous photosynthetic organisms such as microalgae are promising sources for biofuel production through the generation of carbon-neutral sustainable energy. However, the metabolic mechanisms driving high-rate lipid production in these oleaginous organisms remain unclear, thus impeding efforts to improve productivity through genetic modifications. We analyzed the genome and transcriptome of the oleaginous diatom Fistulifera solaris JPCC DA0580. Next-generation sequencing technology provided evidence of an allodiploid genome structure, suggesting unorthodox molecular evolutionary and genetic regulatory systems for reinforcing metabolic efficiencies. Although major metabolic pathways were shared with nonoleaginous diatoms, transcriptome analysis revealed unique expression patterns, such as concomitant upregulation of fatty acid/triacylglycerol biosynthesis and fatty acid degradation (ß-oxidation) in concert with ATP production. This peculiar pattern of gene expression may account for the simultaneous growth and oil accumulation phenotype and may inspire novel biofuel production technology based on this oleaginous microalga.

Profiling of polar lipids in marine oleaginous diatom Fistulifera solaris JPCC DA0580: prediction of the potential mechanism for eicosapentaenoic acid-incorporation into triacylglycerol.

The marine oleaginous diatom Fistulifera solaris JPCC DA0580 is a candidate for biodiesel production because of its high lipid productivity. However, the substantial eicosapentaenoic acid (EPA) content in this strain would affect the biodiesel quality. On the other hand, EPA is also known as the essential health supplement for humans. EPAs are mainly incorporated into glycerolipids in the microalgal cell instead of the presence as free fatty acids. Therefore, the understanding of the EPA biosynthesis including the incorporation of the EPA into glycerolipids especially triacylglycerol (TAG) is fundamental for regulating EPA content for different purposes. In this study, in order to identify the biosynthesis pathway for the EPA-containing TAG species, a lipidomic characterization of the EPA-enriched polar lipids was performed by using direct infusion electrospray ionization (ESI)-Q-TRAP-MS and MS/MS analyses. The determination of the fatty acid positional distribution showed that the sn-2 position of all the chloroplast lipids and part of phosphatidylcholine (PC) species was occupied by C16 fatty acids. This result suggested the critical role of the chloroplast on the lipid synthesis in F. solaris. Furthermore, the exclusive presence of C18 fatty acids in PC highly indicated the biosynthesis of EPA on PC. Finally, the PC-based acyl-editing and head group exchange processes were proposed to be essential for the incorporation of EPA into TAG and chloroplast lipids.

Seasonal variation of biomass and oil production of the oleaginous diatom Fistulifera sp. in outdoor vertical bubble column and raceway-type bioreactors.

To evaluate the feasibility of industrial biodiesel production, outdoor mass cultivation of the marine oleaginous diatom, Fistulifera sp. strain JPCC DA0580, was conducted in bench-scale photobioreactors (∼200 L, raceway- and column-types) and seasonal variation of biomass and oil content were monitored. Through three seasons (from spring to autumn), the microalgae showed steady growth and oil accumulation in both reactors in spite of fluctuating temperature and solar irradiation. When comparing the both reactors, the column-type bioreactor was better with regard to energy conversion efficiency compared to the raceway-type bioreactor. The areal oil productivity of 3.23 g/m(2)/day is comparable or even higher level as compared with the one from other oleaginous microalgae prepared in outdoor mass cultivation. Furthermore, repeated batch culture experiments resulted in success at least 5 cycles. Through the experimental period, little bacterial contamination was observed while protozoal contamination was a fatal issue. The microalgal cell was robust enough to be handled by an automated pump system in inoculation and harvesting processes, and cell adhesion to the bioreactor wall was not observed. These beneficial features could realize ease of oil production and system maintenance. These findings ensure promising innovation by means of outdoor mass cultivation with this strain toward biodiesel production.

Biosynthesis of polyunsaturated fatty acids in the oleaginous marine diatom Fistulifera sp. strain JPCC DA0580.

Studies of polyunsaturated fatty acid (PUFA) biosynthesis in microalgae are of great importance for many reasons, including the production of biofuel and variable omega 3-long chain PUFAs. The elucidation of the PUFA biosynthesis pathway is necessary for bioengineering to increase or decrease PUFA content in certain microalgae. In this study, we identified the PUFA synthesis pathway in the oleaginous marine diatom, Fistulifera sp. strain JPCC DA0580, a promising candidate for biodiesel production. The data revealed not only the presence of the desaturases and elongases involved in eicosapentaenoic acid (EPA) synthesis, but also the unexpected localization of ω3-desaturase expression in the chloroplast. This suggests that this microalga might perform the final step of EPA synthesis in the chloroplast and not in the endoplasmic reticulum (ER) like other diatoms. The detailed fatty acid profile suggests that the EPA was synthesized only through the ω6-pathway in this strain, which was also different from other diatoms. Finally, the transcriptome analysis demonstrated an overall down-regulation of desaturases and elongases over incubation time. These genetic features might explain the decrease of PUFA percentage over incubation time in this strain. The important insights into metabolite synthesis acquired here will be useful for future metabolic engineering to control PUFA content in this diatom.

Isolation and characterization of a thermotolerant ammonia-oxidizing bacterium Nitrosomonas sp. JPCCT2 from a thermal power station.

A thermotolerant ammonia-oxidizing bacterium strain JPCCT2 was isolated from activated sludge in a thermal power station. Cells of JPCCT2 are short non-motile rods or ellipsoidal. Molecular phylogenetic analysis of 16S rRNA gene sequences demonstrated that JPCCT2 belongs to the genus Nitrosomonas with the highest similarity to Nitrosomonas nitrosa Nm90 (100%), Nitrosomonas sp. Nm148 (99.7%), and Nitrosomonas communis Nm2 (97.7%). However, G+C content of JPCCT2 DNA was 49.1 mol% and clearly different from N. nitrosa Nm90, 47.9%. JPCCT2 was capable of growing at temperatures up to 48 °C, while N. nitrosa Nm90 and N. communis Nm2 could not grow at 42°C. Moreover, JPCCT2 grew similarly at concentrations of carbonate 0 and 5 gL(-1). This is the first report that Nitrosomonas bacterium is capable of growing at temperatures higher than 37°C.

Identification and functional analysis of delta-9 desaturase, a key enzyme in PUFA Synthesis, isolated from the oleaginous diatom Fistulifera.

Oleaginous microalgae are one of the promising resource of nonedible biodiesel fuel (BDF) feed stock alternatives. Now a challenge task is the decrease of the long-chain polyunsaturated fatty acids (PUFAs) content affecting on the BDF oxidative stability by using gene manipulation techniques. However, only the limited knowledge has been available concerning the fatty acid and PUFA synthesis pathways in microalgae. Especially, the function of Δ9 desaturase, which is a key enzyme in PUFA synthesis pathway, has not been determined in diatom. In this study, 4 Δ(9) desaturase genes (fD9desA, fD9desB, fD9desC and fD9desD) from the oleaginous diatom Fistulifera were newly isolated and functionally characterized. The putative Δ(9) acyl-CoA desaturases in the endoplasmic reticulum (ER) showed 3 histidine clusters that are well-conserved motifs in the typical Δ(9) desaturase. Furthermore, the function of these Δ(9) desaturases was confirmed in the Saccharomyces cerevisiae ole1 gene deletion mutant (Δole1). All the putative Δ(9) acyl-CoA desaturases showed Δ(9) desaturation activity for C16∶0 fatty acids; fD9desA and fD9desB also showed desaturation activity for C18∶0 fatty acids. This study represents the first functional analysis of Δ(9) desaturases from oleaginous microalgae and from diatoms as the first enzyme to introduce a double bond in saturated fatty acids during PUFA synthesis. The findings will provide beneficial insights into applying metabolic engineering processes to suppressing PUFA synthesis in this oleaginous microalgal strain.

A process design and productivity evaluation for oil production by indoor mass cultivation of a marine diatom, Fistulifera sp. JPCC DA0580.

The present study involved the designing of a culture process and the evaluation of productivity of oil products from a highly oleaginous marine diatom, Fistulifera sp. JPCC DA0580, which had been cultured in a commercial-scale factory. The culture facility had a capacity of 48,000 L and held 96 flat-type 500-L photobioreactors (PBRs) equipped with artificial light, which secures a stable, perennial supply of the products. A 10 days culture that had reached a cell density of 6.5 g dry weight L(-1) possessing a cellular oil content of 48% (wt/wt) was found to provide the highest oil yield. On considering a production area of 1500 m(2), annual algal mass and oil productivity is 68.7 and 33.3 t ha(-1) year(-1), respectively. This study thus provides a reproducible prediction of a theoretical maximum oil yield from a highly oleaginous microalgal strain based on industrially practical production area.

Altererythrobacter ishigakiensis sp. nov., an astaxanthin-producing bacterium isolated from a marine sediment.

A Gram-negative, non-motile, non-spore-forming, halophilic rod, designated JPCCMB0017(T), was isolated from a marine sediment of the coastal area of Okinawa, Japan. The isolate formed orange-red colonies on marine agar. Bacteriochlorophyll α was absent and sphingoglycolipid 1 and other carotenoids, including astaxanthin, adonixanthin and zeaxanthin, were present. Ubiquinone-10 (Q-10) was the main respiratory quinone and C(18:1)ω7c was the major cellular fatty acid. The G+C content of DNA was 59.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the isolate was a member of the genus Altererythrobacter in the family Erythrobacteraceae. Strain JPCCMB0017(T) exhibited 96.8% 16S rRNA gene sequence similarity with Altererythrobacter marinus H32(T). Unlike other members of the genus Altererythrobacter, strain JPCCMB0017(T) reduced nitrate. On the basis of genotypic and phenotypic data, a novel species is proposed to accommodate this isolate, with the name Altererythrobacter ishigakiensis sp. nov. The type strain is JPCCMB0017(T) (= NITE-AP48(T)= ATCC BAA-2084(T)=NBRC 107699(T)).