Yoshifumi Shinmen

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 (Ki 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.

Production of eicosapentaenoic acid byMortierella fungi

Mycelia of arachidonic acid-producing fungi belonging to the genusMortierella were found to be rich sources of 5,8,11,14,17-cis-eicosapentaenoic acid (EPA). Production of EPA by these fungi was observed only when they were grown at low temperature (6–16 C). EPA comprised 5–20% of the total extractable mycelial fatty acids in most strains tested. No significant accumulation of EPA was observed on incubation at high temperature (20–28 C), at which the other major mycelial C-20 fatty acid, arachidonic acid, was still efficiently produced. In a study on the optimization of the culture conditions for EPA production by a selected fungiM. alpina 20–17, a medium containing glucose and yeast extract as major carbon and nitrogen sources, respectively, was found to be suitable. Periodic feeding of glucose during growth of the fungus and cultivation at high temperature (20 C) during the early growth phase followed by temperature shift to 12 C were found to be effective at increasing mycelial yield and reducing cultural period, respectively. Under the optimal culture conditions, the EPA production reached 0.49 mg/ml of culture broth (29 mg/g dry mycelia). This value accounted for 13.5% of the total fatty acids in the extracted lipids. Other major fatty acids in the lipids were palmitic acid (6.0%, by weight), stearic acid (5.3), oleic acid (6.2), linoleic acid (3.0), γ-linolenic acid (3.5) and arachidonic acid (60.0).

Fungal mycelia as a novel source of eicosapentaenoic acid: Activation of enzyme(s) involved in eicosapentaenoic acid production at low temperature

Several filamentous fungi belonging to the genus Mortierella were found to produce large amounts of 5,8,11,14,17-cis-eicosapentaenoic acid (EPA) in their mycelia only when grown at low temperature (12 degrees C), i.e., not at physiological growth temperature (20-28 degrees C). The results of experiments with cell-free extracts suggested that this unique phenomenon is due to activation of enzyme(s) involved in EPA formation at low temperature. Mortierella alpina 1S-4 produced 0.3 g/l of EPA (27 mg/g dry mycelia). This high productivity show the practical significance of these novel EPA producers.

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).

Microbial conversion of an oil containing α-linolenic acid to an oil containing eicosapentaenoic acid

Mycelia of arachidonic acid-producing fungi belonging to the genusMortierella were found to convert an oil containing α-linolenic acid to an oil containing 5,8,11,14,17-cis-eicosapentaenoic acid (EPA). This conversion was observed when they were grown in a medium containing the oil, glucose and yeast extract at 28 C. On the screening of various oils, linseed oil, in which α-linolenic acid amounts to about 60% of the total fatty acids, was found to be the most suitable for EPA production. Under the optimal culture conditions, a selected strain,Mortierella alpina 20-17, converted 5.1% of the α-linolenic acid in the added oil into EPA, the EPA production reaching 1.35 g/l of culture broth (41.5 mg/g dry mycelia). This value corresponded to 7.1% (by weight) of the total fatty acids in the extracted lipids. The lipid was also found to be rich in arachidonic acid (12.3%). Other major fatty acids in the lipid were palmitic acid (4.4%), stearic acid (3.2%), oleic acid (13.5%), linoleic acid (13.7%), α-linolenic acid (38.5%) and γ-linolenic acid (0.9%).

Conversion of linseed oil to an eicosapentaenoic acid-containing oil by Mortierella alpina 1S-4 at low temperature

SummaryThe production of 5,8,11,14,17-cis-eicosapentaenoic acid (EPA) by Mortierella fungi was hgghly stimulated when they were grown in a medium containing linseed oil at low temperature. This phenomenon was suggested to be mainly due to low temperature-dependent production of EPA from arachidonic acid formed through the n-6 route, although conversion of α-linolenic acid in the added linseed oil to EPA through the n-3 route took place at the same time. The amount of EPA accumulated reached 1.88 mg/ml of culture broth (66.6 mg/g dry mycelia) on cultivation of M. alpina 1S-4 with linseed oil at 12° C.

Luminol chemiluminescence reaction catalyzed by a microbial peroxidase

A peroxidase produced by microorganisms belonging to the genera Arthromyces and Coprinus was found to be a potent catalyst for the chemiluminescent oxidation of luminol, the luminescence produced per unit of microbial peroxidase protein being well over 100 times as strong as that produced by horseradish peroxidase. No large difference in Km value for H2O2 in the presence of luminol was found between Arthromyces ramosus peroxidase and horseradish peroxidase (7.0 and 15.5 microM, respectively), but Vmax of the Arthromyces peroxidase was 500 times greater than that of the horseradish peroxidase. It was also found that the Arthromyces peroxidase surpasses, beyond expectation, the horseradish peroxidase in the initial velocity of the chemiluminescence reaction with the stopped-flow method. The Arthromyces peroxidase was used for the glucose and cholesterol assays, which were notably more sensitive than the corresponding assays involving the horseradish peroxidase.

Production of odd chain polyunsaturated fatty acids byMortierella fungi

A soil isolate,Mortierella alpina 1S-4, was found to show high production of odd chain polyunsaturated fatty acids (PUFAs) among various arachidonic acid-producingMortierella strains tested. The fungus mainly accumulated 5,8,11,M-cis-nonadecatetraenoic acid. With 5%n-hepta-decane and 1% yeast extract as growth substrates, the amount of C19:4:4 acid accumulated reached 44.4 mg/g dry mycelia (0.68 mg/mL of culture broth). This value accounted for 11.2% of the total fatty acids in the extracted lipids from mycelia, and odd chain fatty acids comprised over 95% of the total mycelial fatty acids. The addition of sesamin, a specific inhibitor of A5 desaturation, caused an increase in C19:3 acid and an accompanying decrease in C19:4 acid. On the other hand, species ofMortierella that could not produce C-20 PUFAs accumulated C-17 acids, but no C-19 PUFAs, when grown with fatty substrates with an odd chain skeleton. The odd chain PUFAs were distributed in both neutral and polar lipids. The biosynthetic route to C19:4 acid was presumed to mimic the n-6 route to arachidonic acid as follows: C17:0 → C17:1→ C17:2→ C17:3 → C19:3 → C19:4 acids.

Production of arachidonic acid and eicosapentaenoic acids by Marchantia polymorpha in cell culture

Abstract The contents of arachidonic acid (ARA) and eicosapentaenoic acid (EPA) in several bryophytes were determined. Marchantia polymorpha was selected and assayed for the production of ARA and EPA in cell culture. It showed a high growth rate (13 gl −1 , in 3 weeks) under photomixotrophic conditions, and produced high amounts of ARA and EPA (92 and 48 mgl −1 , respectively). The polyunsaturated acids were mainly found in diacylglycerols.

Concentration of eicosapentaenoic acid and docosahexaenoic acid in an arachidonic acid-producing fungus, Mortierella alpina 1S-4, grown with fish oil

The intracellular concentration of 5,8,11,14,17-cis-eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-cis-docosahexaenoic acid (DHA) was carried out by Mortierella alpina 1S-4 in a medium containing fish oil as the main carbon source. The EPA and DHA contents reached 29.2% and 20.0% of total fatty acids, respectively, when the fungus was grown in a medium containing salmon oil (EPA and DHA contents, 14.0% and 17.3%, respectively) as the main carbon source in a 5-1 bench-scale fermentor. EPA and DHA in the added fish oil were incorporated into both the mycelial polar lipids and triglycerides.