Saeree Jareonkitmongkol

Fatty acid desaturation-defective mutants of an arachidonic-acid-producing fungus, Mortierella alpina 1S-4

SUMMARY: Three mutants, which were defective in the desaturation of fatty acids, were isolated from an arachidonic-acidproducing fungus, Mortierella alpina 1S-4, after treating wild-type spores with N-methyl-N′-nitro-N-nitrosoguanidine. They were designated Mut44, Mut48 and Mut49. Mut44 was a mutant with low Δ5-desaturase activity. It accumulated a high level of dihomo-λ-linolenic acid (DGLA) (28.6%, w/w) but a low level of arachidonic acid (Ara) (10.6%), compared with the wild type, which had levels of 6.3 and 47.0%, respectively. Mut48 was unable to desaturate oleic acid (18:1) to linoleic acid (18:2), i.e. Δ12-desaturation, and therefore a large amount of 18:1 (49.5%) accumulated and no fatty acid of the ω-6 family was detected. In addition, several fatty acids of the ω-9 family, such as 5,8,11-cis-eicosatrienoic acid, were found. In Mut49, 18:2 (46%) accumulated markedly, but only small amounts of DGLA and Ara were detected. Thus, Mut49 was considered to be defective in Δ6-desaturation. These mutants showed a somewhat longer lag phase than the wild type on cultivation at both 28 and 12 °C.

Production of an eicosapentaenoic acid-containing oil by a Δ12 desaturase-defective mutant ofMortierella alpina 1S-4

A Δ12 desaturase-defective mutant of an arachidonic acid (AA)-producing fungus,Mortierella alpina 1S-4, converted α-linolenic acid (18:3ω3) to 5(Z),8(Z),11(Z),14(Z),17(Z)-eicosapentaenoic acid (EPA). On submerged cultivation at 20°C for 10 d in a 5-L fermentor containing medium comprising 1% glucose, 1% yeast extract and 3% (vol/vol) linseed oil, EPA production amounted toca. 1 g/L culture broth (64 mg/g dry mycelium), which accounted forca. 20% of the total mycelial fatty acids. AA content was 26 mg/g dry mycelium (0.4 g/L), accounting for 7.8% of the total mycelial fatty acids. The other major mycelial fatty acids were palmitic acid (4.5%), oleic acid (20.4%), linoleic acid (10.0%), 18:3ω3 (20.3%) and lignoceric acid (4.3%). Most of the EPA produced (ca. 90 mol%) was in triglyceride form.

Production of a novel ω1-eicosapentaenoic acid by Mortierella alpina 1S-4 grown on 1-hexadecene

An arachidonic-producing fungus, Mortierella alpina 1S-4, was found to accumulate ω-unsaturated fatty acids of C-20 chain length together with ω1-hexadecenoic acid, ω1-octadecenoic acid and so on, when grown on 1-alkenes, i.e., 1-hexadecene and 1-octadecene. The results of mass spectroscopy and proton NMR showed that a C20 polyunsaturated fatty acid (PUFA) is a novel cis-5,8,11,14,19-eicosapentaenoic acid (20:5ω1). This PUFA was obtained at a yield of 0.13 mg/ml culture broth (2.8% of the fungal total fatty acid content) on cultivation of the fungus in a medium containing 4% (v/v) 1-hexadecene and 1% yeast extract at 28°C for 1 week. Investigation of the distribution of fatty acids showed that about 90% (by mol.) of the PUFA was present in the triglycerides and 10% was in the phospholipid fraction. About 70% of that found in the phospholipids was phosphatidylcholine (PC) and the value accounted for ca. 10% of the total fatty acid content. The formation of these ω-unsaturated fatty acids was presumed to occur through the arachidonic acid biosynthetic pathway (n-6 route).

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%.

Occurrence of two nonmethylene-interrupted Δ5 polyunsaturated fatty acids in a Δ6-desaturase-defective mutant of the fungus Mortierella alpina 1S-4

Abstract Two non-methylene-interrupted fatty acids, 5(Z),11(Z),14(Z)-eicosatrienoic acid (20:3Δ5) and 5(Z),11(Z),14(Z),17(Z)-eicosa tetraenoic acid (20:4Δ5), were found in a Δ6-desaturase-defective mutant of Mortierella alpina 1S-4. 20:4Δ5 was detected only when growth was at a temperature lower than 24°C or in a culture medium supplemented with either α-linolenic acid or 11(Z),14(Z),17(Z)-eicosatrienoic acid. 20:3Δ5 was found when growth was at 12–28°C. The amount of 20:3Δ5 was greatest (27 mg g−1 dry mycelia) when growth was at 20°C, and it accounted for about 7 wt% of the total fatty acids of the mycelia. The optimum temperature for the accumulation of 20:4Δ5 was 12°C; there was 6.4 mg 20:4Δ5/g dry mycelia. Most of these two fatty acids (more than 75 mol%) was in the triacylglycerol fraction.

Inhibitory effect of curcumin on fatty acid desaturation inMortierella alpina 1S-4 and rat liver microsomes

An extract of rhizomes ofCurcuma longta L. (turmeric) inhibited the desaturation of dihomo-γ-linolenic acid (DGLA) in the arachidonic acid (AA) producing fungusMortierella alpina 1S-4. The factor responsible for this phenomenon was isolated and identified as curcumin (diferuloyl methane). Mycelial DGLA levels increased about two-fold (22.3 mg/g dry weight) with a concomitant decrease in AA levels when the fungus was cultivated with curcumin. The 50% inhibitory concentration against Δ5 desaturase was 27.2 μM. Curcumin also inhibited rat liver microsomal Δ5 and Δ6 desaturases.

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.

Isolation and characterization of a Δ--desaturation-defective mutant of an arachidonic acid-producing fungus, Mortierella alpina 1S-4

A mutant, considered to be defective in Δ9-desaturation, of an arachidonic acid-producing fungus, Mortierella alpina 1S-4, was isolated after treating the wild-type spores with N-methyl-N′-nitro-N-nitrosoguanidine. The mycelial FA of this mutant included 38% stearic acid, the level being only 5% for the wild type. Upon growth at 24–28°C, its mycelial lipids included a high level (up to 50 mol% of total mycelial lipids) of FFA, of which about 90 mol% was stearic acid. However, the level of FFA was markedly decreased with a concomitant increase in the IAG level when the mutant was grown at 20°C or lower, or when it was grown in a culture medium supplemented with PUFA.

Inhibitory effects of lignan compounds on the formation of arachidonic acid in a Δ5-desaturase-defective mutant of Mortierella alpina 1S-4

Abstract The level of arachidonic acid (AA) in a Δ5-desaturase-defective mutant of an AA-producing fungus, Mortierella alpina 1S-4, could be decreased by growing the fungus in the presence of a mixture of Δ5-desaturase-specific inhibitors, sesamin and episesamin. Under submerged batch culture conditions using a culture medium supplemented with 0.075 g· l −1 sesamin mixture, the production of dihomo-γ-linoleic acid was 2.1 g· l −1 culture broth (16.1% of total fatty acids) and the arachidonic acid production was 0.2 g· l −1 (1.3%).