Daiske Honda

Optimization of docosahexaenoic acid production by Schizochytrium limacinum SR21

Culture conditions of Schizochytrium limacinum SR21 for the purpose of microbial docosahexaenoic acid (DHA) production were investigated. The strain SR21 showed a wide tolerance to salinity; that is, the optimum salinity was between 50% and 200% that of sea water. Monosaccharides (glucose and fructose) and glycerol supported good cell growth and DHA yield. Di- and polysaccharides, oleic acid, and linseed oil gave low DHA yields. A high content of DHA (more than 30% of total fatty acids) was obtained from culture on glucose, fructose, and glycerol, and also the strain had simple polyunsaturated fatty acid profiles. The major polyunsaturated fatty acids other than DHA were n-6 docosapentaenoic acid only, and the contents of icosapentaenoic acid and arachidonic acid were less than 1%. Using corn steep liquor as a nitrogen source, a high total fatty acid content was obtained. The total fatty acid content in the dry cell weight increased as the concentration of the nitrogen source decreased, reached more than 50%. An increase in carbon source concentration led to a high DHA yield. A maximum DHA yield of more than 4 g/l was obtained in both glucose and glycerol media at 9% and 12% respectively. S. limacinum SR21 was thought to be a promising resource for microbial DHA production yielding a good level of productivity as well as a simple polyunsaturated fatty acid profile.

Production of docosahexaenoic and docosapentaenoic acids bySchizochytrium sp. isolated from Yap Islands

A marine microbe (strain SR21) from the coral reef area of the Yap Islands was isolated by a screening test for polyunsaturated fatty acids and was found to accumulate lipid that contained 22:5n-6 docosapentaenoic acid (DPA) as well as 22:6n-3 docosahexaenoic acid (DHA). Strain SR21 was identified as genusSchizochytrium in Labyrinthulomycota, owing to its ultrastructural character and life cycle, which is composed of vegetative cell, zoosporangium, and zoospore stages. After cultural optimization, both in flask and fermenter, the highest DHA and DPA productivities of 2.0 and 0.44 g/L per day, respectively, were obtained in a medium of 60 g/L glucose and corn steep liquor/ammonium sulfate in a half salt concentration of seawater in fermenter culture at 28°C and pH 4. This productivity was almost twice that obtained with flask culture, indicating its high resistance to mechanical stirring. The lipid extracted from the cell was about 50% of the dry cell weight and was composed of 93% triacylglycerol (TG). DHA content of the lipid was 34% of total fatty acids. The TG profile was simple, and the content of the most dominant TG, 1,3-dipalmitoyl-2-DHA-TG, was 27%. TG that contained DHA and n-6-DPA amounted to 57 and 17%, respectively, of total TG molecules. Strain SR21 was revealed to be an excellent source of microbial DHA and n-6 DPA.

Molecular Phylogeny of Labyrinthulids and Thraustochytrids Based On the Sequencing of 18s Ribosomal Rna Gene

ABSTRACT Labyrinthulids and thraustochytrids are unicellular heterotrophs, formerly considered as fungi, but presently are recognized as members in the stramenopiles of the kingdom Protista sensu lato. We determined the 18S ribpsomal RNA gene sequences of 14 strains from different species of the six genera and analyzed the molecular phylogenetic relationships. the results conflict with the current classification based on morphology, at the genus and species levels. These organisms are separated, based on signature sequences and unique inserted sequences, into two major groups, which were named the labyrinthulid phylogenetic group and the thraustochytrid phylogenetic group. Although these groupings are in disagreement with many conventional taxonomic characters, they correlated better with the sugar composition of the cell wall. Thus, the currently used taxonomic criteria need serious reconsideration.

Phylogenetic analysis of psychrophilic bacteria isolated from the Japan Trench, including a description of the deep-sea species Psychrobacter pacificensis sp. nov.

Phylogenetic positions of psychrophilic bacteria isolated from the Japan Trench were determined by sequencing analysis of PCR-amplified bacterial small subunit (16S) rRNA genes. Between surface and deep-sea psychrophiles, distinct positions clearly differed within the gamma-Proteobacteria. In phylogenetic analysis using neighbour-joining, maximum-parsimony and maximum-likelihood, strains from surface seawater were inferred to be located in the Halomonas aquamarina-meridiana clade within the family Halomonadaceae. Strains from deep seawater (5000-6000 m), however, formed a novel monophyletic clade within the Moraxella-Psychrobacter branch in the family Moraxellaceae, showing separation from terrestrial and Antarctic relatives. These deep-sea strains were also discriminated from other known Psychrobacter species in phenotype, e.g. limited growth in the absence of NaCl (optimum at about 3% NaCl), positive urease activity, acid production from xylose and arabinose, and the presence of multiple fimbriae. DNA relatedness values among six deep-sea strains were > 85% in DNA-DNA hybridization experiments and > 98% in aligned 16S rDNA sequences. From this evidence, a new species, Psychrobacter pacificensis, is proposed for these deep-sea psychrophiles; the type strain of Psychrobacter pacificensis is strain NIBH P2K6T (= IFO 16270T). Occurrence of psychrobacters in cold Japan Trench deep seawater and at the Antarctic sea surface suggests that deep-sea bacterial habitation and evolution have been mediated by global deep-ocean circulation linked to the sinking of cooled seawater in polar regions.

Schizochytrium limacinum sp. nov., a new thraustochytrid from a mangrove area in the west Pacific Ocean

Schizochytrium limacinum sp. nov, a thraustochytrid, is described from sea water from a mangrove area of the Yap Islands in the west Pacific Ocean. It is closest to S. aggregatum , and differs from other Schizochytrium spp. in its limaciform amoeboid cells, the size of zoospores, released without an amoeboid stage, and the assimilation profile of carbon sources.

Grouping newly isolated docosahexaenoic acid-producing thraustochytrids based on their polyunsaturated fatty acid profiles and comparative analysis of 18S rRNA genes.

AbstractSeven strains of marine microbes producing a significant amount of docosahexaenoic acid (DHA; C22:6, n-3) were screened from seawater collected in coastal areas of Japan and Fiji. They accumulate their respective intermediate fatty acids in addition to DHA. There are 5 kinds of polyunsaturated fatty acid (PUFA) profiles which can be described as (1) DHA/docosapentaenoic acid (DPA; C22:5, n-6), (2) DHA/DPA/eicosapentaenoic acid (EPA; C20:5, n-3), (3) DHA/EPA, (4) DHA/DPA/EPA/arachidonic acid (AA; C20:4, n-6), and (5) DHA/DPA/EPA/AA/docosatetraenoic acid (C22:4, n-6). These isolates are proved to be new thraustochytrids by their specific insertion sequences in the 18S rRNA genes. The phylogenetic tree constructed by molecular analysis of 18S rRNA genes from the isolates and typical thraustochytrids shows that strains with the same PUFA profile form each monophyletic cluster. These results suggest that the C20-22 PUFA profile may be applicable as an effective characteristic for grouping thraustochytrids.

Optimization of culture conditions of the thraustochytrid Aurantiochytrium sp. strain 18W-13a for squalene production

Optimum conditions of temperature, salinity and glucose concentration were investigated for squalene production of the strain of Aurantiochytrium sp. 18 W-13a, with a high content of squalene. Squalene production by this strain was optimum at 25 °C, 25-50% seawater concentration and 2-6% glucose concentration. When this strain was grown in the optimum condition, the squalene content and production of approximately 171 mg/g dry weight and 0.9 g/L were much higher than that previously reported in thraustochytrids, plants and yeasts, respectively. Therefore, 18 W-13a could be used as an alternative source of commercial squalene.

The development, ultrastructural cytology, and molecular phylogeny of the basal oomycete Eurychasma dicksonii, infecting the filamentous phaeophyte algae Ectocarpus siliculosus and Pylaiella littoralis

The morphological development, ultrastructural cytology, and molecular phylogeny of Eurychasma dicksonii, a holocarpic oomycete endoparasite of phaeophyte algae, were investigated in laboratory cultures. Infection of the host algae by E. dicksonii is initiated by an adhesorium-like infection apparatus. First non-walled, the parasite cell developed a cell wall and numerous large vacuoles once it had almost completely filled the infected host cell (foamy stage). Large-scale cytoplasmic changes led to the differentiation of a sporangium with peripheral primary cysts. Secondary zoospores appeared to be liberated from the primary cysts in the internal space left after the peripheral spores differentiated. These zoospores contained two phases of peripheral vesicles, most likely homologous to the dorsal encystment vesicles and K-bodies observed in other oomycetes. Following zoospore liberation the walls of the empty cyst were left behind, forming the so-called net sporangium, a distinctive morphological feature of this genus. The morphological and ultrastructural features of Eurychasma were discussed in relation to similarities with other oomycetes. Both SSU rRNA and COII trees pointed to a basal position of Eurychasma among the Oomycetes. The cox2 sequences also revealed that the UGA codon encoded tryptophan, constituting the first report of stop codon reassignment in an oomycete mitochondrion.

Taxonomy, molecular phylogeny, and ultrastructural morphology of Olpidiopsis porphyrae sp. nov. (Oomycetes, straminipiles), a unicellular obligate endoparasite of Bangia and Porphyra spp. (Bangiales, Rhodophyta)

Olpidiopsis porphyrae sp. nov., a marine oomycete endoparasite that infects the commercially cultivated red alga Porphyra yezoensis, is described and its phylogenetic position based on molecular data and ultrastructural morphology is discussed. O. porphyrae infects the host Porphyra by means of encysted zoospores. Spherical-shaped holocarpic thalli develop within the cytoplasm of its algal host, which produce monoplanetic, subapically biflagellate zoospores. The characteristic features of this isolate are the ellipsoidal, unicellular thallus and simple holocarpic zoosporangial development, which show morphological similarity with the genus Olpidiopsis. Laboratory infection experiments with a wide range of green, brown, and red algae revealed that O. porphyrae infects several stages of the bangialean red algae (the genera Bangia and Porphyra). Molecular phylogenetic analyses inferred from both SSU rRNA and cox2 genes showed O. porphyrae branched before the main saprolegnian and peronosporalean lineages within the monophyletic oomycete clade, indicating its phylogenetic separation from them. A single or double K-body-like organelle, which contains tubular inclusions, is found located to one side of the zoospore nucleus and shows similarities to homologous organelles previously described in O. saprolegniae. The ultrastructural morphology of O. porphyrae with zoospore initials containing K-bodies and tubular mitochondrial cristae is characteristic of oomycetes. Group I intron-like multiple insertions were found in the SSU rRNA gene of O. porphyrae. This is the first report of SSU group I introns in the class Oomycetes.

Analysis of Δ12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304

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.