Papers - HONDA Daisuke
-
PUFA synthase-independent DHA synthesis pathway in Parietichytrium sp. and its modification to produce EPA and n-3DPA Reviewed International journal
Yohei Ishibashi, Hatsumi Goda, Rie Hamaguchi, Keishi Sakaguchi, Takayoshi Sekiguchi, Yuko Ishiwata, Yuji Okita, Seiya Mochinaga, Shingo Ikeuchi, Takahiro Mizobuchi, Yoshitake Takao, Kazuki Mori, Kosuke Tashiro, Nozomu Okino, Daiske Honda, Masahiro Hayashi, Makoto Ito
Communications Biology 4 ( 1 ) 1378 2021.12
Joint Work
Publisher：Springer Science and Business Media LLC
Abstract
The demand for n-3 long-chain polyunsaturated fatty acids (n-3LC-PUFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), will exceed their supply in the near future, and a sustainable source of n-3LC-PUFAs is needed. Thraustochytrids are marine protists characterized by anaerobic biosynthesis of DHA via polyunsaturated fatty acid synthase (PUFA-S). Analysis of a homemade draft genome database suggested that Parietichytrium sp. lacks PUFA-S but possesses all fatty acid elongase (ELO) and desaturase (DES) genes required for DHA synthesis. The reverse genetic approach and a tracing experiment using stable isotope-labeled fatty acids revealed that the ELO/DES pathway is the only DHA synthesis pathway in Parietichytrium sp. Disruption of the C20 fatty acid ELO (C20ELO) and ∆4 fatty acid DES (∆4DES) genes with expression of ω3 fatty acid DES in this thraustochytrid allowed the production of EPA and n-3docosapentaenoic acid (n-3DPA), respectively, at the highest level among known microbial sources using fed-batch culture.DOI： 10.1038/s42003-021-02857-w
Other Link： https://www.nature.com/articles/s42003-021-02857-w
-
Draft Genome Sequence of Sicyoidochytrium minutum DNA Virus Strain 001 Reviewed International journal
Yumi Murakoshi, Takayuki Shimeki, Daiske Honda, Yoshitake Takao
Microbiology Resource Announcements 10 ( 23 ) e00418-21 2021.6
Joint Work
Publisher：American Society for Microbiology
Sicyoidochytrium minutum DNA virus strain 001 (SmDNAV 001) is a double-stranded DNA (dsDNA) virus that infects the marine fungoid protist Sicyoidochytrium minutum . We report the draft genome sequence of SmDNAV 001. The 236,345-bp genome contained 358 coding sequences (CDSs) and three tRNA-coding sequences.
DOI： 10.1128/mra.00418-21
-
Taming chlorophylls by early eukaryotes underpinned algal interactions and the diversification of the eukaryotes on the oxygenated Earth. Reviewed International coauthorship International journal
Kashiyama, Y., Yokoyama, A., Shiratori, T., Hess, S., Not, F., Bachy, C., Gutierrez-Rodriguez, A., Kawahara, J., Suzaki, T., Nakazawa, M., Ishikawa, T., Maruyama, M., Wang, M., Chen, M., Gong, Y., Seto, K., Kagami, M., Hamamoto, Y., Honda, D., Umetani, T., Shihongi, A., Kayama, M., Matsuda, M., Taira, J., Yabuki, A., Tsuchiya, M., Hirakawa, Y., Kawaguchi, A., Nomura, M., Nakamura, A., Namba, N., Matsumoto, M., Tanaka, T., Yoshino, T., Higuchi, R., Yamamoto, A., Maruyama, T., Yamaguchi, A., Uzuka, A., Miyagishima, S., Tanifuji, G., Kawachi, M., Kinoshita, Y., Tamiaki, H.
The ISME Journal 13 1899 - 1910 2019.2
Joint Work
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.
DOI： 10.1038/s41396-019-0377-0
Other Link： https://www.nature.com/articles/s41396-019-0377-0
-
Nutritional intake of Aplanochytrium (Labyrinthulea, Stramenopiles) from living diatoms revealed by culture experiments suggesting the new prey–predator interactions in the grazing food web of the marine ecosystem. Reviewed International journal
Hamamoto, Y., Honda, D.
PLoS ONE 14 ( 1 ) e0208941 2019.1
Joint Work
Authorship：Last author, Corresponding author
Labyrinthuleans (Labyrinthulea, Stramenopiles) are recognized as decomposers in marine ecosystems but their nutrient sources are not fully understood. We conducted two-mem- bered culture experiments with labyrinthuleans and diatoms to discover where labyrinthu- leans obtain their nutrients from. The results showed that Aplanochytrium strains obtained nutrients by consuming living diatoms. Aplanochytrium cells did not release digestive enzymes into the medium, but adhered to diatom cells via the tip of their characteristic ecto- plasmic net system to obtain nutrients from them. The chloroplast and cell contents of the diatoms shrank and were absorbed, and then the number of Aplanochytrium cells rapidly increased as multiple aplanospores were released. To estimate the effect of labyrinthulean organisms including Aplanochytrium on marine ecosystem, we explored the dataset gener- ated by the Tara Oceans Project from a wide range of oceanic regions. The average propor- tion of all labyrinthulean sequences to diatom sequences at each station was about 10%, and labyrinthulids, oblongichytrids, and aplanochytrids were the major constituent genera, accounting for more than 80% of labyrinthuleans. Therefore, these groups are suggested to greatly affect the marine ecosystem. There were positive correlations between aplanochy- trids and phototrophs, green algae, and diatoms. At many stations, relatively large propor- tions of aplanochytrid sequences were detected in the size fraction larger than their cell size. This implied that Aplanochytrium cells increased their particle size by adhering to each other and forming aggregates with diatoms that are captured by larger zooplankton in the environment, thereby bypassing the food web pathway via aplanochytrids to higher preda- tors. The intake of nutrients from diatoms by aplanochytrids represents a newly recognized pathway in the grazing food chain in the marine ecosystem.
DOI： 10.1371/journal.pone.0208941
Other Link： https://doi.org/10.1371/journal.pone.0208941
-
Erratum to “Nutritional Intake by Ectoplasmic Nets of Schizochytrium aggregatum (Labyrinthulomycetes, Stramenopiles" (Protist (2018) 169(5) (727–743), (S1434461018300622) (10.1016/j.protis.2018.06.002)) Reviewed International journal
Iwata I, Honda D.
Protist 169 ( 6 ) 978 - 979 2018.12
Joint Work
Authorship：Last author, Corresponding author
DOI： 10.1016/j.protis.2018.11.005
Other Link： http://orcid.org/0000-0002-3436-4561
-
Correction: Novel lysophospholipid acyltransferase plat1 of aurantiochytrium limacinum f26-b responsible for generation of palmitate-docosahexaenoate-phosphatidylcholine and phosphatidylethanolamine (PLoS ONE (2014) 9: 8 (e102377) DOI: 10.1371/journal.pon Reviewed International journal
Abe E, Ikeda K, Nutahara E, Hayashi M, Yamashita A, Taguchi R, Doi K, Honda D, Okino N, Ito M.
PLoS ONE 13 ( 8 ) e0203016 - e0203016 2018.8
-
Elemental composition and ultrafine structure of the skeleton in shellbearing protists—A case study of phaeodarians and radiolarians Reviewed International journal
Nakamura, Y., Iwata, I., Hori, R. S., Uchiyama, N., Tuji, A., Fujita, M. J., Honda, D., Ohfuji, H.
Journal of Structural Biology 204 45 - 51 2018.7
Joint Work
Cross-sections were prepared by ultramicrotome (UM) and focused ion beam (FIB) system in order to examine the skeletal structure of ecologically and geologically important shell-bearing protists: phaeodarians and radi- olarians. The elemental composition of the skeleton was clarified by the energy dispersive X-ray spectroscopy, suggesting that the skeletons of both groups are mainly made of amorphous silica (SiO2·nH2O) with other minor elements (Na, Mg, Al, Cl, K, Ca and Fe) and that these two groups have similar elemental composition, compared with other siliceous organisms (diatoms and sponges). However, the structural difference among the two groups was confirmed: phaeodarian skeletons are porous, unlike radiolarians with solid skeletons. It was also revealed that the phaeodarian skeleton contains concentric layered structure with spaces, presumably related to the ontogenetic skeleton formation. The distinction in the skeletal ultrafine structure (porous/solid and non-dense/ dense) would reflect the ecological difference among the two groups and could be an effective criterion to determine whether microfossils belong to Radiolaria or Phaeodaria. The UM and FIB combined method pre- sented in this study could be a useful approach to examine the chemical and structural characteristics of un- culturable and/or rare microorganisms.
-
Nutritional intake by ectoplasmic nets of Schizochytrium aggregatum (Labyrinthulomycetes, Stramenopiles). Reviewed International journal
Iwata, I., Honda, D.
Protist 169 727 - 743 2018.6
Joint Work
Authorship：Last author, Corresponding author
Thraustochytrid cells attach to their food via ectoplasmic nets (ENs). Here, we analyzed the cause and effect relationship between the various forms and functions of ENs of Schizochytrium aggregatum. The ENs spread out over a large area forming a fine network to efficiently search for the experimental food source. After recognizing the experimental food source, the ENs that attached to the food source became thicker, and net elements developed. The thick ENs on the surface at the attachment site were enveloped in dense materials (fibrous materials), which were visualized as fibrous layers under a transmission electron microscope. Experiments using fluorescein diacetate and the fluorescent glu- cose analog 2-NBDG showed that the production rate of hydrolytic enzymes and the absorption rate of glucose by ENs of S. aggregatum increased in the presence of an experimental food source. Our results reveal that ENs change their shape and function according to the presence/absence of a food source.
-
Possible aplanochytrid (Labyrinthulea) prey detected using 18S metagenetic diet analysis in the key copepod species Calanus sinicus in the coastal waters of the subtropical western North Pacific Reviewed International journal
Hirai, J., Hamamoto, Y., Honda, D., Hidaka, K.
Plankton and Benthos Research 13 ( 2 ) 75 - 82 2018.5
Joint Work
Metagenetic diet analyses of the 18S V9 region were conducted in 40 adult female Calanus sinicus dur- ing winter in Tosa Bay (Japan). The majority of prey items were small crustaceans (of Copepoda and Cirripedia) and diatoms, taxa that are dominant in the environment and have been previously reported as important prey items of Cala- nus. The abundance of sequences attributable to Dinophyta and Chlorophyta was significantly lower in C. sinicus gut contents than in environmental plankton communities, suggesting that C. sinicus avoids prey from these groups. Hy- drozoans were also observed, and aplanochytrids (Labyrinthulea) were detected for the first time as a major prey of C. sinicus. Additionally, high proportions of unclassified eukaryote material were observed, suggesting undetected preda- tor–prey relationships in key copepod species in marine ecosystems. The dietary importance of aplanochytrids, hetero- trophic protists that accumulate unsaturated fatty acids such as docosahexaenoic acid, has been overlooked in previous research. Calanus sinicus is a key copepod species in the subtropical coastal regions of the western North Pacific, and a major food source for the larvae of commercially important fish; therefore, further investigation into novel prey items such as aplanochytrids is recommended to understand the complex food web structures in marine ecosystems.
DOI： 10.3800/pbr.13.75
-
Proposal of Monorhizochytrium globosum gen. nov., comb. nov.(Stramenopiles, Labyrinthulomycetes) for former Thraustochytrium globosum based on morphological features and phylogenetic relationships Reviewed International journal
Doi, K., Honda, D.
Phycological Research 65 ( 3 ) 188 - 201 2017.7
Joint Work
Authorship：Last author, Corresponding author
Thraustochytrium is the type genus of the family Thraustochy- triaceae in the class Labyrinthulomycetes. This genus is char- acterized by zoospore formation, namely, shape of the cell wall of sporangia and presence or absence of a proliferous body. However, there are several issues associated with the taxonomy of this genus, and these include polyphyletic taxa and overlapping of taxonomic features among species. In par- ticular, the first and second species, T. proliferum and T. glo- bosum, were described based on observations of the morphological features of natural samples in the absence of culture conditions. Before addressing the taxonomic issues associated with this genus, it is important to consider the taxo- nomic features of each species, i.e., the life history under cul- ture conditions and the phylogenetic position. The objective of the present study was to isolate T. globosum, the second described species in the genus Thraustochytrium, from the type locality. We successfully isolated strain NBRC 112723, which exhibited characteristic features of T. globosum. Under culture conditions, strain NBRC 112723 exhibited taxonomic features observed in other thraustochytrid species. Our molec- ular phylogeny indicated that this strain isolated from the type locality was located in an unidentified thraustochytrid group; moreover, some strains located in this group exhibited charac- teristic features of strain NBRC 112723. We clearly distin- guished T. globosum based on the taxonomic criteria used to classify the T. proliferum type species. Therefore, we propose the establishment of a new genus, Monorhizochytrium, for the species T. globosum in the family Thraustochytriaceae.
DOI： 10.1111/pre.12175
-
Bothrosome formation in Schizochytrium aggregatum (Labyrinthulomycetes, Stramenopiles) during zoospore settlement Reviewed International journal
Izumi Iwata, Kei Kimura, Yuji Tomaru, Taizo Motomura, Kanae Koike, Kazuhiko Koike, Daiske Honda
Protist 168 ( 2 ) 206 - 219 2017.4
Joint Work
Authorship：Last author, Corresponding author
Labyrinthulomycetes are characterized by the presence of ectoplasmic nets originating from an organelle known as the bothrosome, whose evolutionary origin is unclear. To address this issue, we investigated the developmental process from a zoospore to a vegetative cell in Schizochytrium aggregatum. After disappearance of the flagellum during zoospore settlement, the bothrosome emerged at the anterior-ventral pole of the cells. A new Golgi body also appeared at this stage, and the bothrosome was positioned close to both the new and the old Golgi bodies. This observation suggested that the Golgi body is related to the formation of the bothrosome. Actin appeared as a spot in the same location as the newly appeared bothrosome, as determined by immunofluorescence labeling. An immunoelectron microscopic analysis revealed that actin was present in the ectoplasmic nets and in the cytoplasm around the bothrosome, indicating that the electron-dense materials of the bothrosome are not the polar center of F-actin. This suggests that actin filaments pull the endoplasmic reticulum to the bothrosome and induce the membrane to become evaginated within ectoplasmic nets.
-
Ecological dynamics of two distinct viruses infecting marine eukaryotic decomposer thraustochytrids (Labyrinthulomycetes, Stramenopiles) Reviewed International journal
Yoshitake Takao, Yuji Tomaru, Keizo Nagasaki, Daiske Honda
PLOS ONE 10 ( 7 ) e0133395 2015.7
Joint Work
Authorship：Last author, Corresponding author
Thraustochytrids are cosmopolitan osmotrophic or heterotrophic microorganisms that are considered as important decomposers in coastal ecosystems. However, because of a lack of estimation method for each genus or systematic group of them, relatively little is known about their ecology in situ. Previously, we reported two distinct types of virus infecting thraustochytrids (AuRNAV: reported as SssRNAV, and SmDNAV) suggesting they have wide distributions in the host-virus systems of coastal environments. Here we conducted a field survey from 2004 through 2005 to show the fluctuation pattern of thraustochytrids and their viruses in Hiroshima Bay, Japan. During the field survey, we monitored the dynamics of the two types of thraustochytrid-infecting virus: small viruses causing lysis of Aurantio- chytrium sp. NIBH N1-27 (identified as AuRNAV) and the large viruses of Sicyoidochytrium minutum NBRC 102975 (similar to SmDNAV in physiology and morphology). Fluctuation patterns of the two distinct types of virus were different from each other. This may reflect the difference in the preference of organic substrates; i.e., it may be likely the host of AuRNAV (Aurantiochytrium sp.) increases utilizing algal dead bodies or feeble cells as the virus shows a large increase in abundance following raphidophyte blooms; whereas, the trophic nutrient supply for S. minutum may primarily depend on other constantly-supplied organic compounds because it did not show any significant change in abundance throughout the survey. Further study concerning the population composition of thraustochytrids and their viruses may demonstrate the microbial ecology (especially concerning the detrital food web) of marine environments.
DOI： 10.1371/journal.pone.0133395
Other Link： http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133395
-
Seasonal dynamics of culturable thraustochytrids (Labyrinthulomycetes, Stramenopiles) in estuarine and coastal waters Reviewed International journal
Ueda, M., Nomura, Y., Doi, K., Nakajima, M., Honda, D.
Aquatic Microbial Ecology 74 187 - 204 2015.3
Joint Work
Authorship：Last author, Corresponding author
Regular monitoring of estuarine and coastal areas at fixed points was carried out to investigate changes in biomass and species composition of thraustochytrids, which are colorless heterotrophs in the class Labyrinthulomycetes, Stramenopiles. Cell number counts using the most probable number (MPN) method with pine pollen baiting showed that 1 or 2 conspicuous peaks in abundance (termed ‘thraustochytrid spikes’) occurred between spring and late summer in most sampling years. Changes in thraustochytrid biomass had no correlation with phytoplankton abundance, although spikes seemed to occur after reductions in salinity and associated rapid rises of water temperature. The average cell density at the estuary site, excluding values during spike periods, was 4670 cells l–1. Although thraustochytrid biomass was only 1.59% that of bacterial biomass, the fixed energy (as biomass) transferred directly from thraustochytrids to zooplankton was estimated to be 15.9% of that transferred from bacterioplankton via phagotrophic protists. This is because, per the theory of energy efficiency, energy transfer between trophic levels only creates 10% of the net production in the next trophic level relative to the first. The phylogenetic identification of established strains revealed an unexpectedly high diversity of thraustochytrids, including 10 unidentified lineages. A similar seasonal succession of phylogenetic groups was observed in each year of sampling. The differences in thraustochytrids isolated at each monitoring site and date suggest that habitat segregation may occur as a result of differences in environmental factors such as water temperature, salinity, and nutrient sources.
DOI： 10.3354/ame01736
-
Novel lysophospholipid acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b responsible for generation of palmitate-docosahexaenoate-phosphatidylcholine and phosphatidylethanolamine. Reviewed International journal
Abe, E., Ikeda, K., Nutahara, E., Hayashi, M., Yamashita A., Taguchi, R., Doi K., Honda D., Okino N., Ito, M.
PLoS ONE 9 ( 8 ) e102377 2014.8
Joint Work
N-3 polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA, 22:6n-3), have been reported to play roles in preventing cardiovascular diseases. The major source of DHA is fish oils but a recent increase in the global demand of DHA and decrease in fish stocks require a substitute. Thraustochytrids, unicellular marine protists belonging to the Chromista kingdom, can synthesize large amounts of DHA, and, thus, are expected to be an alternative to fish oils. DHA is found in the acyl chain(s) of phospholipids as well as triacylglycerols in thraustochytrids; however, how thraustochytrids incorporate DHA into phospholipids remains unknown. We report here a novel lysophospholipid acyltransferase (PLAT1), which is responsible for the generation of DHA-containing phosphatidylcholine and phosphatidylethanolamine in thraustochytrids. The PLAT1 gene, which was isolated from the genomic DNA of Aurantiochytrium limacinum F26-b, was expressed in Saccharomyces cerevisiae, and the FLAG-tagged recombinant enzyme was characterized after purification with anti-FLAG affinity gel. PLAT1 shows wide specificity for donor substrates as well as acceptor substrates in vitro, i.e, the enzyme can adopt lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidylinositol as acceptor substrates, and 15:0/16:0-CoA and DHA-CoA as donor substrates. In contrast to the in vitro experiment, only lysophosphatidylcholine acyltransferase and lysophosphatidylethanolamine acyltransferase activities were decreased in plat1-knockout mutants, resulting in a decrease of 16:0-DHA-phosphatidylcholine (PC) [PC(38∶6)] and 16:0-DHA-phosphatidylethanolamine (PE) [PE(38∶6)], which are two major DHA-containing phospholipids in A. limacinum F26-b. However, the amounts of other phospholipid species including DHA-DHA-PC [PC(44∶12)] and DHA-DHA-PE [PE(44∶12)] were almost the same in plat-knockout mutants and the wild-type. These results indicate that PLAT1 is the enzyme responsible for the generation of 16:0-DHA-PC and 16:0-DHA-PE in the thraustochytrid.
DOI： 10.1371/journal.pone.0102377
Other Link： http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102377
-
Bacillus ligniniphilus sp. nov., an alkaliphilic and halotolerant bacterium isolated from sediments of the South China Sea. Reviewed International coauthorship International journal
Zhu, D., Tanabe, S., Xie, C., Honda, D., Sun, J., Ai, L.
International Journal of Systematic and Evolutionary Microbiology 64 1712 - 1717 2014.5
Joint Work
An alkaliphilic and halotolerant Gram-stain-positive bacterium, which was isolated from sediment samples from the South China Sea, was subjected to a taxonomic study. The isolate, strain L1T, grew well at a wide range of temperatures and pH values, 10.0–45.0 °C and pH 6–11, with optima at 30 °C and pH 9.0, respectively. The growth of strain L1T occurred at total salt concentrations of 0–10 % (w/v) with an optimum at 2 % (w/v). Phylogenetic analysis based on 16S rRNA sequence comparison indicated that the isolate represented a member of the genus Bacillus . The strains most closely related to strain L1T were Bacillus nanhaiisediminis JCM 16507T, Bacillus halodurans DSM 497T and Bacillus pseudofirmus DSM 8715T, with 16S rRNA similarities of 96.5 %, 95.9 % and 95.7 %, respectively. DNA–DNA hybridization of strain L1T with the type strains of the most closely related species, B. nanhaiisediminis JCM 16507T, B. halodurans DSM 497T and B. pseudofirmus DSM 8715T, showed reassociation values of about 21.7 %, 14.3 % and 13.9 %, respectively. The DNA G+C content of strain L1 T was 40.76 mol%. The predominant isoprenoid quinone was menaquinone 7 (MK-7). The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant cellular fatty acids of strain L1T were iso-C14 : 0 and anteiso-C15 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Based on the phenotypic and phylogenetic characteristics, it is proposed that strain L1T ( = JCM 18543T = DSM 26145T) should be classified as the type strain of Bacillus ligniniphilus sp. nov.
-
TLC screening of thraustochytrid strains for squalene production. Reviewed International coauthorship International journal
Nakazawa, A., Kokubun, Y., Matsuura, H., Yonezawa, N., Kose, R., Yoshida, M., Tanabe, Y., Kusuda, E., Thang, D. V., Ueda, M., Honda, D., Mahakhant, A., Kaya, K., Watanabe, M. M.
Journal of Applied Phycology 26 ( 1 ) 29 - 41 2014.2
Joint Work
Screenings of thraustochytrids (Labyrinthulomycetes) have been conducted for 176 strains isolated from various sites in the Asian region to investigate what type of species and strains accumulate high levels of squalene. Thin layer chromatography (TLC) screening for squalene production revealed that 38 strains were rated as “+” (high), 29 as “±” (medium), and 109 as “−” (low). Further, high performance liquid chromatography analysis strongly supported the TLC screening results. Besides the 18W-13a strain of Aurantiochytrium sp., which was previously recognized as a squalene-rich strain, several strains produced squalene at approximately 1 g L−1 of culture volume. Squalene production was strongly related to locality, colony color, and phylogenetic clade. Most strains with “+” squalene spots were isolated from Okinawa, a subtropical region of Japan, while the strains with “±” and “−” squalene spots were isolated from wide geographical regions from tropical to subarctic. Approximately half the strains with orange colonies on GTY medium plates produced a high amount of squalene, whereas the other strains with different colors showed less or no squalene spots on TLC. All the squalene-rich strains were assigned to the Aurantiochytrium clade. Overall, our results suggest that (1) the thraustochytrids show tendentious locality in terms of squalene production, (2) a relationship exists between the metabolic synthesis of carotenoid pigments and squalene production, and (3) the Aurantiochytrium clade may have evolved to accumulate squalene.
-
Effect of trace elements on growth of marine eukaryotes, tharaustochytrids Reviewed International journal
Nagano N, Taoka Y, Honda D, Hayashi M.
Journal of Bioscience and Bioengineering 116 ( 3 ) 337 - 339 2013.9
Joint Work
We determined the effect of trace elements on the growth of thraustochytrids. The growth of the strains cultured with the trace elements was much higher than that of the strains cultured without any trace element. Iron and zinc were particularly important to obtaining the optimum growth of thraustochytrids.
-
Analysis of delta-12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304 Reviewed International journal
Matsuda T, Sakaguchi K, Hamaguchi R, Kobayashi T, Abe E, Hama Y, Hayashi M, Honda D, Okita Y, Sugimoto S, Okino N, Ito M
Journal of Lipid Research 53 1210 - 1222 2012.2
Joint Work
Thraustochytrids are known to synthesize PUFAs such as docosahexaenoic acid (DHA). Accumulating evidence suggests the presence of two synthetic pathways of PUFAs in thraustochytrids: the polyketide synthase-like (PUFA synthase) and desaturase/elongase (standard) pathways. It remains unclear whether the latter pathway functions in thraustochytrids. In this study, we report that the standard pathway produces PUFA in Thraustochytrium aureum ATCC 34304. We isolated a gene encoding a putative Δ12-fatty acid desaturase (TauΔ12des) from T. aureum. Yeasts transformed with the tauΔ12des converted endogenous oleic acid (OA) into linoleic acid (LA). The disruption of the tauΔ12des in T. aureum by homologous recombination resulted in the accumulation of OA and a decrease in the levels of LA and its downstream PUFAs. However, the DHA content was increased slightly in tauΔ12des-disruption mutants, suggesting that DHA is primarily produced in T. aureum via the PUFA synthase pathway. The transformation of the tauΔ12des-disruption mutants with a tauΔ12des expression cassette restored the wild-type fatty acid profiles. These data clearly indicate that TauΔ12des functions as Δ12-fatty acid desaturase in the standard pathway of T. aureum and demonstrate that this thraustochytrid produces PUFAs via both the PUFA synthase and the standard pathways.
DOI： 10.1194/jlr.M024935
-
Versatile Transformation System That Is Applicable to both Multiple Transgene Expression and Gene Targeting for Thraustochytrids Reviewed International journal
Sakaguchi K, Matsuda T, Kobayashi T, Ohara J, Hamaguchi R, Abe E, Nagano N, Hayashi M, Ueda M, Honda D, Okita Y, Taoka Y, Sugimoto S, Okino N, Ito M
Applied and Environmental Microbiology 78 ( 9 ) 3193 - 3202 2012.2
Joint Work
A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neo r), driven with an ubiquitin or an EF-1α promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neo r marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neo r mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C20:3n-6) and eicosatetraenoic acid (C20:4n-3), substrates for Δ5 desaturase, and a decrease of arachidonic acid (C20:4n-6) and eicosapentaenoic acid (C20:5n-3), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.
DOI： 10.1128/AEM.07129-11
-
On the bio-rearrangement into fully saturated fatty acidscontaining triglyceride in Aurantiochytrium sp. Reviewed International journal
Matsuura H, Nakazawa A, Ueda M, Honda D, Watanabe MM, Kaya K
Procedia Environmental Sciences 15 66 - 72 2012
Joint Work
A strain of Aurantiochytrium sp. was grown in media with various concentrations of glucose to monitor triglyceride production as a potential source of oil for biodiesel. The fatty acid composition of triglyceride in the strain was unique, because the fatty acids consisted of only 6 molecular species, and the major species were myristic, pentadecanoic, palmitic, heptadecanoic, docosapentaenoic, and docosahexaenoic acids. When cells were cultured in glucose-rich (over 9%) medium for 4 days, the triglyceride yields were 0.5-1.0 g/L. After culture for 4 days, the fatty acid composition of triglyceride was nearly identical in all cells grown in media containing various concentrations of glucose. However, when cells were grown in medium containing 12% glucose for 12 days, unique triglyceride containing only saturated fatty acids accumulated. This bio-rearrangement into fully-saturated fatty acids-containing triglyceride may be utilized for the preparation of biodiesel oil.