Isolation and characterization of a Delta5-desaturase from Oblongichytrium sp.

We isolated a cDNA clone with homology to known desaturase genes from Oblongichytrium sp., recently classified as a new genus of thraustochytrids (Labyrinthulomycetes), and found that it encoded Delta5-desaturase by its heterologous expression in yeast. The enzyme had higher activity toward 20:4n-3 than 20:3n-6, indicating that this Delta5-desaturase can be used in the production of n-3 polyunsaturated fatty acids in transgenic organisms.

A new labyrinthulid isolate that produces only docosahexaenoic acid.

We show here that a new labyrinthulid strain, L72, isolated from a fallen leaf in the Seto Inland Sea of Japan, produced only docohexaenoic acid (DHA) among all the long-chain polyunsaturated fatty acids (LCPUFAs). The main fatty acid composition was 16:0 (28.9%), 18:0 (7.2%), 18:1 (5.7%), 18:2 (10.4%), and DHA (45.9%) without any other LCPUFA. The lipid content of the strain was 27.4%. The cells had many lipid bodies, which were densely located in all of the cells. On phylogenetic analysis using the 18S rDNA sequence, the strain was located in the labyrinthulids group, forming a monophyletic group with Labyrinthula sp. (strain s) and Labyrinthuila sp. (strain L59). We further tested the culture optimization of strain L72 to evaluate the ability of the strain to produce DHA. The optimum salt concentration and the temperature of the strain were 100% of artificial seawater and 20 degrees C. Strain L72 could grow well on soybean oil (SBO) or soybean lecithin (SBL) as the carbon source. When 20 g/l of SBL was added to the medium, DHA production reached the maximum amount at 0.67 g/l for 14 d. The two important facts, that the strain can use SBL as the main nutrient and contains only DHA among the LCPUFAs, will be of great advantage for industry.

High yield of long-chain polyunsaturated fatty acids by labyrinthulids on soybean lecithin-dispersed agar medium.

The aim of the present research was to provide an effective long-chain polyunsaturated fatty acid (LCPUFA) production by labyrinthulids using soybean lecithin (SBL). Use of SBL-dispersed agar medium resulted in higher LCPUFA production than soybean oil. Among the components of SBL, phosphatidylinositol (PI) and triacylglycerol (TG) were revealed to be essential factors for high growth of labyrinthulids. PI was effective for surface growth, and TG was effective for three-dimensional growth. The presence of some elements like carotenoids in SBL and the smaller droplet size of dispersed SBL were also attributed to be factors for the higher LCPUFA productivity on SBL medium. LCPUFA productivity and the volume of the oval form of the cells increased with increasing SBL concentration up to 40 g/l. Under optimum conditions, LCPUFA production of the L25 strain, isolated from Ogasawara Island in Japan, reached 2.91 g/l after 21 days.

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

Seven 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.

Biosynthesis of triacylglycerol molecular species in an oleaginous fungus, Mortierella ramanniana var. angulispora.

The incorporation of radiolabeled lipid precursors into triacylglycerol (TG) molecular species in Mortierella ramanniana var. angulispora, an oleaginous fungus, was studied to determine the biosynthetic pathways for TG molecular species. Radiolabeled TG molecular species were separated and quantified by reverse-phase high performance liquid chromatography with a radioisotope detector. The major TG molecular species labeled by [1-(14)C]oleic acid at 30 degrees C were OOP, OOO, and OPP (TG molecular species designations represent three constituent acyl groups. G, gamma-linolenic acid; L, linoleic acid; O, oleic acid; S, stearic acid; P, palmitic acid), which were abundant TG molecular species in this fungus. The incorporation of [1-(14)C]oleic acid at 15 degrees C into these molecular species was the same, while that into most other species was decreased, suggesting that biosynthesis of major molecular species such as OOP, OOO, and OPP differs from that of other TG molecular species. [1-(14)C]Linoleic acid incorporation indicated that the major labeled molecular species were LOP and LOO, which may be due to acylation of oleoyl, palmitoyl-glycerol, or dioleoyl-glycerol by exogenous linoleic acid. This is basically the same mechanism as for OOP and OOO biosynthesis from exogenous oleic acid. [(14)C(U)]Glycerol incorporation suggested that TG molecular species containing palmitic acid such as OPP were more readily synthesized through the de novo pathway. Further experiments involving inhibitors such as sodium azide and cerulenin suggested that OOO biosynthesis included a mechanism differing from that in the cases of OOP and OPP. Trifluoperazine, which inhibits the conversion from phosphatidic acid to TG, decreased [1-(14)C]oleic acid incorporation into all molecular species, suggesting that the incorporation into all molecular species included the de novo TG biosynthetic pathway via phosphatidic acid. These results revealed that the biosynthetic pathways for TG molecular species can be classified into several groups, which exhibit different sensitivities to low temperature and inhibitors of lipid metabolism. This implies that the composition of TG molecular species is regulated through different biosynthetic pathways responsible for specific TG molecular species, providing a new insight into the biosynthesis of TG molecular species.

Microbial transformations of two lupane-type triterpenes and anti-tumor-promoting effects of the transformation products.

Microbial transformation of betulin (1), a lupane-type triterpene obtained from the bark extract of white birch (Betula platyphylla Sukatshev var. japonica), and its chemical oxidation product, betulonic acid (2), by the fungus Chaetomium longirostre yielded 4,28-dihydroxy-3,4-seco-lup-20(29)-en-3-oic acid (3) and 4-hydroxy-3,4-seco-lup-20(29)-ene-3,28-dioic acid (4) from 1, and 4,7beta,17-trihydroxy-3,4-seco-28-norlup-20(29)-en-3-oic acid (5) and 7 beta,15 alpha-dihydoxy-3-oxolup-20(29)-en-28-oic acid (6) from 2. The four metabolites, 3-6, along with 1 and 2, were evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells as a primary screening test for inhibitors of tumor promotion. All of the triterpenes tested showed potent inhibitory effects, with the four metabolites 3-6 exhibiting the more potent effects.