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.

Docosahexaenoic acid production and lipid-body formation in Schizochytrium limacinum SR21.

Schizochytrium limacinum SR21, a thraustochytrid (Labyrinturomycota), is a heterotrophic marine microorganism. SR21 has attracted recent attention because of the production of docosahexaenoic acid (DHA). We obtained highly concentrated SR21 zoospores and successfully observed synchronous growth. We investigated changes of lipid content and fatty acid composition during the growth. The morphological features of the lipid bodies were also described via fluorescent and electron microscopy. The cells developed quickly after zoospore settlement. Lipid bodies developed in accordance with an increase in lipid content during the 8-h synchronous growth. The total lipid was composed mainly of triacylglycerol, sterol esters, and phosphatidylcholine. The proportion of neutral lipids (triacylglycerol and sterol esters) in the total lipid was fairly constant during growth. The fatty acid composition of neutral lipids, primary components of the lipid body, and phospholipids, primary components of the cell membranes, was nearly unchanged during the synchronous growth. However, the DHA content of the phospholipids decreased drastically after a 10-day culture. Electron micrographs prepared using a high-pressure freeze substitution technique revealed a fine structure of light- and dark-staining bands inside the lipid bodies in many stages of the cells.

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.

Codenitrification and denitrification are dual metabolic pathways through which dinitrogen evolves from nitrate in Streptomyces antibioticus.

We screened actinomycete strains for dinitrogen (N(2))-producing activity and discovered that Streptomyces antibioticus B-546 evolves N(2) and some nitrous oxide (N(2)O) from nitrate (NO(3)(-)). Most of the N(2) that evolved from the heavy isotope ([(15)N]NO(3)(-)) was (15)N(14)N, indicating that this nitrogen species consists of two atoms, one arising from NO(3)(-) and the other from different sources. This phenomenon is similar to codenitrification in fungi. The strain also evolved less, but significant, amounts of (15)N(15)N from [(15)N]NO(3)(-) in addition to (15)N(15)NO with concomitant cell growth. Prior to the production of N(2) and N(2)O, NO(3)(-) was rapidly reduced to nitrite (NO(2)(-)) accompanied by distinct cell growth, showing that the actinomycete strain is a facultative anaerobe that depends on denitrification and nitrate respiration for anoxic growth. The cell-free activities of denitrifying enzymes could be reconstituted, supporting the notion that the (15)N(15)N and (15)N(15)NO species are produced by denitrification from NO(3)(-) via NO(2)(-). We therefore demonstrated a unique system in an actinomycete that produces gaseous nitrogen (N(2) and N(2)O) through both denitrification and codenitrification. The predominance of codenitrification over denitrification along with oxygen tolerance is the key feature of nitrate metabolism in this actinomycete.