Sesamin is a potent and specific inhibitor of Δ5 desaturase in polyunsaturated fatty acid biosynthesis

Incubation with sesame oil increases the mycelial dihomo-γ-linolenic acid content of an arachidonic acid-producing fungus,Mortierella alpina, but decreases its arachidonic acid content [Shimizu, S., K. Akimoto, H. Kawashima, Y. Shinmen and H. Yamada (1989)J. Am. Oil Chem. Soc. 66, 237–241]. The factor causing these effects was isolated and identified to be (+)-sesamin. The results obtained in experiments with both a cell-free extract of the fungus and with rat liver microsomes demonstrated that (+)-sesamin specifically inhibits Δ5 desaturase at low concentrations, but does not inhibit Δ6, Δ9 and Δ12 desaturases. Kinetic analysis showed that (+)-sesamin is a noncompetitive inhibitor (K_i for rat liver Δ5 desaturase, 155 μM). (+)-Sesamolin, (+)-sesaminol and (+)-episesamin, also inhibited only Δ5 desaturases of the fungus and liver. These results demonstrate that (+)-sesamin and related lignan compounds present in sesame seeds or its oil are specific inhibitors of Δ5 desaturase in polyunsaturated fatty acid biosynthesis in both microorganisms and animals. On leave from Suntory Ltd.     

Production of dihomo-γ-linolenic acid byMortierella alpina 1S-4

The mycelial dihomo-γ-linolenic acid content of an arachidonic acid-producing fungus,Mortierella alpina 1S-4, was found to increase, with an accompanying marked decrease in its arachidonic acid content, on cultivation with sesame oil. The resultant mycelia were found to be a rich source of dihomo-γ-linolenic acid. This unique phenomenon was suggested to be due to specific repression of the conversion of dihomo-γ-linolenic acid to arachidonic acid by the oil. After fractionation of the oil with acetone into oil and non-oil fractions, it was found that the effective factor(s) was present in the non-oil fraction. In a study on optimization of the culture conditions for the production of dihomo-γ-linolenic acid byM. alpina 1S-4, a medium containing glucose, yeast extract and the non-oil fraction was found to be suitable for the production. Under the optimal conditions in a 50-1 fermentor, the fungus produced 107 mg of dihomo-γ-linolenic acid/g dry mycelia (2.17 g/l of culture broth). This value accounted for 23.1% of the total fatty acids in the lipids extracted from the mycelia. The mycelia were also rich in arachidonic acid (53.5 mg/g dry mycelia, 11.2%). Other major fatty acids in the lipids were palmitic acid (24.1%), stearic acid (7.0), oleic acid (20.1), linoleic acid (6.6) and γ-linolenic acid (4.1). On leave from Suntory Ltd.     

Regiospecific analysis by ethanolysis of oil with immobilized <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase

A mixture of oil/ethanol (1∶3, w/w) was shaken at 30°C with 4% immobilized Candida antarctica lipase by weight of the reaction mixture. The reaction regiospecifically converted FA at the 1- and 3-positions to FA ethyl esters, and the lipase acted on C₁₄−C₂₄ FA to a similar degree. The content of 2-MAG reached a maximum after 4 h; the content was 28–29 mol% based on the total amount of FA in the reaction mixture at 59–69% ethanolysis. Only 2-MAG were present in the reaction mixture during the first 4 h, and 1(3)-MAG were detected after 7 h. After removal of ethanol from the 4-h reaction mixture by evaporation, 2-MAG were fractionated by silica gel column chromatography. The contents of FA in the 2-MAG obtained by ethanolysis of several oils coincided well with FA compositions at the 2-position, which was analyzed by Grignard degradation. It was shown that ethanolysis of oil with C. antarctica lipase can be applied to analysis of FA composition at the 2-position in TAG.     

Production of 5,8,11-cis-eicosatrienoic acid by a Δ12-desaturase-defective mutant ofMortierella alpina 1S-4

A mutant defective in Δ12-desaturase of an arachidonic-acid producing fungus,Mortierella alpina 1S-4, was shown to be a novel potent producer of Mead acid (5,8,11-cis-eicosatrienoic acid, 20:3ω9). The fungus produced several fatty acids of the n-9 family,i.e., 6,9-cis-octadecadienoic acid (18:2ω9), 8,11-cis-eicosadienoic acid (20:2ω9) and 20:3ω9. Significantly high levels of these fatty acids were produced during growth at low temperatures (12–20°C). On submerged cultivation at 20°C for 10 days in a 5-L fermenter containing 2% glucose plus 1% yeast extract (pH 6.0), the production of 20:3ω9 reachedca. 0.8 g/L (56 mg/g dry mycelia), accounting for 15% (by wt) of the total mycelial fatty acids. The other major fatty acids were palmitic acid (6%), stearic acid (11%), oleic acid (45%), 18:2ω9 (12%) and 20:2ω9 (3%). Studies on the distribution of fatty acids among lipid classes showed that, irrespective of the growth temperature employed (12–28°C),ca. 70% (by mol) of 20:3ω9 was present in the triglyceride and the remainder in the phospholipid fraction, especially in phosphatidylcholine (PC). When the fungus was grown at 12°C, the proportion of 20:3ω9 in the PC fraction wasca. 55%. On leave from Suntory Ltd.     

Production of eicosapentaenoic acid bySaprolegnia sp. 28YTF-1

Saprolegnia sp. 28YTF-1, isolated from a freshwater sample, is a potent producer of 5,8,11,14,17-cis-eicosapentaenoic acid (EPA). The fungus used various kinds of carbon sources, such as starch, dextrin, sucrose, glucose, and olive oil for growth, and olive oil was the best carbon source for EPA production. The EPA content reached 17 mg/g dry mycelium (0.25 mg/L) when the fungus was grown in a medium that contained 2.5% olive oil and 0.5% yeast extract, at pH 6.0 and 28°C for 6 d with shaking. Accompanying production of arachidonic acid (AA; 3.2 mg/g dry mycelia, EPA/AA = 5.1) and other ω6 polyunsaturated fatty acids was low. Both EPA content and EPA/AA ratio increased in parallel by lowering growth temperature. Triglyceride was the major mycelial lipid (ca. 84%), but EPA comprised only 2.2% of the total fatty acids of this lipid. About 40% of the EPA produced was found in polar lipids, such as phosphatidylethanolamine (EPA content, 28.2%), phosphatidylcholine (13.6%), and phosphatidylserine (21.2%).     

Production of 8,11,14,17-cis-eicosatetraenoic acid by Δ5 desaturase-defective mutants of an arachidonic acid-producing fungus, Mortierella alpina

Δ5 Desaturase-defective mutants of an arachidonic acid-producing fungus, Mortierella alpina 1S-4, accumulate large amounts of 8,11,14,17-cis-eicosatetraenoic acid (20:4ω3) when grown with linseed oil. One of the mutants, the S14 strain, produced 1.65 mg of 20:4ω3 per mL of culture medium (corresponding to 66.0 mg/g dry mycelia and 11.6% of total cellular fatty acids) when grown in a medium containing 1% glucose, 1% yeast extract, and 4% linseed oil methyl ester at 28°C for 2 d, and then at 16°C for 7 d. In a bench-scale fermentation in a 5-L jar fermenter, 20:4ω3 production reached 1.60 g/L of culture medium on the eighth day (corresponding to 77.3 mg/g dry mycelia and 26.0% of total cellular fatty acids). The cellular lipids of the S14 strain comprised 75.8% triacylglycerol (TG), 6.7% diacylglycerol, and 13.3% phospholipids (PL). The percentage of 20:4ω3 was higher in PL than in TG, and highest in phosphatidylcholine (32.6%).     

Enhancement of arachidonic acid production by Mortierella alpina 1S-4

The effect of mineral addition on arachidonic acid (AA) production by Mortierella alpina 1S-4 was evaluated. At first, the addition of minerals such as sodium, potassium, calcium, and magnesium was examined in flask cultures, and then the addition of phosphorus with the optimal amounts of the minerals was investigated in a 10-L jar-fermenter. As a result, 1.5% soy flour medium with the addition of 0.3% KH₂PO₄, 0.1% Na₂SO₄, 0.05% CaCl₂·2H₂O, and 0.05% MgCl₂·6H₂O was found to enhance the AA yield 1.7-fold over that without mineral addition. When 1% yeast extract with the above mineral mixture was used, the AA yield was enhanced 1.35-fold over that without minerals. We also verified that an increase in the polar lipid content occurred in the case of only KH₂PO₄ addition, and that the above-mentioned increase in the AA yield was due to the minerals themselves, not a pH buffer effect.     

Effects of oleic,arachidonic and 5,8,11,14-nonadecatetraenoic acids on lipid secretion and ketogenesis in perfused rat liver

The effects of perfused oleic (18∶1n−9), arachidonic (20∶4n−6) and 5,8,11,14-nonadecatetraenoic (19∶4n−5) acids on triglyceride and cholesterol secretion and ketone body production were studied in isolated rat liver. As compared to oleic and 19∶4n−5 acids, both ketone body production and triglyceride secretion were significantly lowered when arachidonic acid was perfused. The concentration of triglyceride in the post-perfused liver was lower upon perfusion with arachidonic acid than upon perfusion with oleic acid or 19∶4n−5 acid. Cholesterol secretion in the liver perfused with arachidonic acid or 19∶4n−5 acid was significantly higher than with oleic acid. The concentration of cholesterol in the post-perfused liver was slightly but significantly higher with 19∶4n−5 acid than with the other fatty acids. The results suggest that 19∶4n−5 acid when compared with arachidonic acid affects lipid metabolism in liver differently.