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ω3 Polyunsaturated fatty acids are currently obtained mainly from fisheries; thus, sustainable alternative sources such as oleaginous microorganisms are required. Here, we describe the isolation, characterization, and application of 3 novel ω3 desaturases with ω3 polyunsaturated fatty acid–producing activity at ordinary temperatures (28 °C). First, we selected Pythium sulcatum and Plectospira myriandra after screening for oomycetes with high eicosapentaenoic acid/arachidonic acid ratios and isolated the genes psulω3 and pmd17 , respectively, which encode ω3 desaturases. Subsequent characterization showed that PSULω3 exhibited ω3 desaturase activity on both C18 and C20 ω6 polyunsaturated fatty acids while PMD17 exhibited ω3 desaturase activity exclusively on C20 ω6 polyunsaturated fatty acids. Expression of psulω3 and pmd17 in the arachidonic acid–producer Mortierella alpina resulted in transformants that produced eicosapentaenoic acid/total fatty acid values of 38% and 40%, respectively, at ordinary temperatures. These ω3 desaturases should facilitate the construction of sustainable ω3 polyunsaturated fatty acid sources. [ABSTRACT FROM AUTHOR]

Oleaginous fungus Mortierella alpina 1S-4 accumulates arachidonic acid (20:4ω6) as a major polyunsaturated fatty acid. However, 20:4ω6 is not essential for the growth of M. alpina 1S-4, because various mutants that produce no 20:4ω6 were isolated. M. alpina JT-180, a Δ12 desaturation-defective mutant that was derived from M. alpina 1S-4 by treatment with a chemical mutagen, accumulates Mead acid (20:3ω9) instead of 20:4ω6. p -Anisidine was found to be a Δ6 desaturation inhibitor in this study. The concentration of 0.1 mg/ml of p -anisidine had an inhibitory effect on the growth of M. alpina JT-180. The addition of p -anisidine to the medium caused M. alpina JT-180 to produce only monoenoic acids such as oleic acid (18:1ω9) and eicosenoic acid (20:1ω9) as unsaturated fatty acids. The effects of exogenous fatty acids were investigated when M. alpina JT-180 was cultivated in medium containing p -anisidine. The addition of linoleic acid (18:2ω6) and 6 Z ,9 Z -octadecadienoic acid (18:2ω9) restored the growth of M. alpina JT-180 cultivated on the medium containing p -anisidine, but palmitic acid (16:0), 18:1ω9, and vaccenic acid (18:1ω7) had no effect. For the growth of oleaginous fungus M. alpina , 18-carbon length fatty acids with more than two double bonds are considered to be essential. [ABSTRACT FROM AUTHOR]

To develop an efficient gene-targeting system in Mortierella alpina 1S-4, we identified the ku80 gene encoding the Ku80 protein, which is involved in the nonhomologous end-joining pathway in genomic double-strand break (DSB) repair, and constructed ku80 gene-disrupted strains via single-crossover homologous recombination. The Δku80 strain from M. alpina 1S-4 showed no negative effects on vegetative growth, formation of spores, and fatty acid productivity, and exhibited high sensitivity to methyl methanesulfonate, which causes DSBs. Dihomo-γ-linolenic acid (DGLA)-producing strains were constructed by disruption of the Δ5- desaturase gene, encoding a key enzyme of bioconversion of DGLA to ARA, using the Δku80 strain as a host strain. The significant improvement of gene-targeting efficiency was not observed by disruption of the ku80 gene, but the construction of DGLA-producing strain by disruption of the Δ5- desaturase gene was succeeded using the Δku80 strain as a host strain. This report describes the first study on the identification and disruption of the ku80 gene in zygomycetes and construction of a DGLA-producing transformant using a gene-targeting system in M. alpina 1S-4. [ABSTRACT FROM AUTHOR]

Strain D2 was isolated from a natural sample as a crude glycerol-assimilating microorganism. The ITS-5.8S rDNA sequence of strain D2 was most similar to that of Fusarium solani deposited in the NCBI database. Strain D2 accumulated 10-hydroxy- cis -12-octadecenoic acid (HYA), 10-hydroxyoctadecanoic acid (HYB), and 10-oxooctadecanoic acid (KetoB) in a medium containing crude glycerol, as a carbon source, and yeast extract, named CG medium. The growth and HYB production of strain D2 depended on the crude glycerol concentration in the medium. Strain D2 produced 2.20 g/L (40% of total fatty acids) of HYB on cultivation in CG medium containing 8% crude glycerol. When strain D2 was cultivated in CG medium containing 6% crude glycerol, the yield of HYB on cultivation under 4 days-shaking and 3 days-static conditions reached 1.19 g/L, which was 2.2 times higher than that under 7 days-shaking conditions and accounted for 53% of total fatty acids. The fungus was found not only to efficiently produce fatty acids utilizing crude glycerol, but also to be the first filamentous fungus to produce hydroxy and oxo fatty acids such as HYB, HYA, and KetoB. • Strain D2, which produces hydroxy fatty acids by using crude glycerol, was isolated. • Strain D2 was found to be closely related to Fusarium solani. • 10-Hydroxy- cis -12-octadecenoic acid and 10-hydroxyoctadecanoic acid were detected. • The yield of hydroxy fatty acid increased under microaerobic conditions. [ABSTRACT FROM AUTHOR]

Mead acid (MA; 20:3ω9) is one of the ω9 series of polyunsaturated fatty acids (PUFAs). MA is used to inhibit the inflammation of joints and is applied to the medicinal or health food field. We aimed to construct MA-producing strains with disruption of the Δ12-desaturase gene (Δ1 2ds) via an efficient gene-targeting system using the lig4 -disrupted strain of Mortierella alpina 1S-4 as the host. The transformants showed a unique fatty acid composition that only comprised ω9-PUFAs and saturated fatty acids, while ω6-and ω3-PUFAs were not detected, and the total composition of ω9-PUFAs, including oleic acid (18:1ω9), 18:2ω9, 20:1ω9, 20:2ω9, and MA, was up to 68.4% of the total fatty acids. The MA production in the Δ1 2ds -disruptant reached 0.10 g/L (8.5%), which exceeded 0.050 g/L (4.6%) in the conventional Δ1 2ds -defective mutant JT-180. [ABSTRACT FROM AUTHOR]