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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]

The filamentous fungus Mortierella alpina 1S-4 is capable of accumulating a large amount of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs). Indeed, triacylglycerol production by M. alpina 1S-4 can reach 20 g/L of culture broth, and the critical cellular signaling and structural PUFA arachidonic acid (ARA) comprises 30%–70% of the total fatty acid. The demonstrated health benefits of functional PUFAs have in turn encouraged the search for rich sources of these compounds, including fungal strains showing enhanced production of specific PUFAs. Screening for mutants and targeted gene manipulation of M. alpina 1S-4 have elucidated the functions of various enzymes involved in PUFA biosynthesis and established lines with improved PUFA productivity. In some cases, these strains have been used for indistrial-scale production of PUFAs, including ARA. In this review, we described practical ARA production through mutant breeding, functional analyses of genes encoding enzymes involved in PUFA biosynthesis, and recent advances in the production of specific PUFAs through molecular breeding of M. alpina 1S-4. [ABSTRACT FROM AUTHOR]

The aim of this work was to study the molecular breeding of oleaginous filamentous Mortierella alpina for high production of linoleic (LA) or oleic acid (OA). Heterologous expression of the Δ12-desaturase (DS) gene derived from Coprinopsis cinerea in the Δ6DS activity-defective mutant of M. alpina increased the LA production rate as to total fatty acid to 5 times that in the wild strain. By suppressing the endogenous Δ6I gene expression by RNAi in the Δ12DS activity-defective mutant of M. alpina , the OA accumulation rate as to total fatty acid reached 68.0%. The production of LA and OA in these transformants reached 1.44 and 2.76 g/L, respectively, on the 5th day. The Δ6I transcriptional levels of the RNAi-treated strains were suppressed to 1/10th that in the parent strain. The amount of Δ6II RNA in the Δ6I RNAi-treated strain increased to 8 times that in the wild strain. [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]

We constructed dihomo-γ-linolenic acid (DGLA)-producing strains with disruption of the Δ5-desaturase ( Δ5ds ) gene, which encodes a key enzyme catalyzing the bioconversion of DGLA to arachidonic acid (ARA), by efficient gene-targeting, using Δlig4 strain of Mortierella alpina 1S-4 as the host. In previous study, we had already identified and disrupted the lig4 gene encoding DNA ligase 4, which involves in non-homologous end joining, in M. alpina 1S-4, and the Δlig4 strain had showed efficient gene-targeting. In this study, the uracil auxotroph of Δlig4 strain was constructed, and then transformed for disruption of Δ5ds . The isolation of nine Δ5ds -disruptants out of 18 isolates indicated that the disruption efficiency was 50%. The ratio of DGLA among the total fatty acids of the Δ5ds -disruptants reached 40.1%; however, no ARA was detected. To our knowledge, this is the first study to report the construction of DGLA-producing transformants by using the efficient gene-targeting system in M. alpina 1S-4. [ABSTRACT FROM AUTHOR]

The oleaginous fungus Mortierella alpina is known to accumulate eicosapentaenoic acid (EPA) only when cultivated at a low temperature (below 15°C). Here, we investigated EPA production at an ordinary temperature (28°C) by expressing the Saprolegnia diclina D17 desaturase gene (sdd17m) in M. alpina ST1358, an ω3-desaturation activity-defective mutant derived from M. alpina 1S-4. Expression of the exogenous gene was confirmed by EPA accumulation in transformants at both 28 and 12°C. The EPA content in total lipids produced by transformants was over 20% at 28°C. Bench-scale fermentation with a 5-L jar fermentor showed that EPA content reached 26.4% of total fatty acids, and EPA production reached 1.8 g/L. This is the first study to report the accumulation of EPA in M. alpina at an ordinary temperature, and provide a platform technology for the industrial production of EPA using M. alpina as a promising source for EPA. [ABSTRACT FROM AUTHOR]

A ω3-fatty acid desaturase gene (maw3) which is involved in biosynthesis of n-3 polyunsaturated fatty acids (PUFAs) was previously isolated from Mortierella alpina 1S-4. In this report, we investigated the products of MAW3 catalyzing reaction with endogenous and exogenous fatty acids in the yeast transformant. Two unusual fatty acids de novo synthesized in the yeast transformant expressing maw3 gene were identified as n-4 hexadecadienoic acid (16:29cis,12cis ) and n-1 hexadecatrienoic acid (16:39cis,12cis,15) by GC–MS and 1H NMR analyses. In addition to the desaturation activity at the ω3-position for 18- and 20-carbon PUFAs, MAW3 in the yeast transformant inserted a double bond at Δ12-position of endogenous palmitoleic acid (16:19cis ) and further at Δ15-position of the resulting 16:29cis,12cis to result in the formation of 16:39cis,12cis,15 leading to a bifunctional Δ12/Δ15-desaturase for 16-carbon fatty acids. Moreover, we evaluated the activity of MAW3 in the yeast transformant under different temperatures. The MAW3 did not have desaturation activities in M. alpina 1S-4 at 28°C but it had in the yeast transformant for various fatty acids. The MAW3 was demonstrated to be a trifunctional Δ12/Δ15/ω3-desaturase, exhibiting Δ12-desaturation for 16:19cis , Δ15-desaturation for 16- and 18-carbon fatty acids that had a preexisting cis-double bond at Δ12 position, and ω3-desaturation for 20-carbon fatty acids having that at Δ14-position. It is the first report that the fatty acid desaturase (MAW3) is shown to have Δ12- and Δ15-desaturation activities for a 16-carbon fatty acid, in addition to its major function, ω3-desaturation activity. [ABSTRACT FROM AUTHOR]

Researches related with the application of functional lipids such as polyunsaturated fatty acids (PUFAs) have been conducted in various fields with a view to health and dietary requirements. Novel rich sources other than known natural sources such as plant seeds and fish oils are required for increasing demands of PUFAs. The filamentous fungus Mortierella alpina 1S-4 produces triacylglycerols rich in arachidonic acid, i.e., ones reaching 20 g/l in concentration and containing 30–70% arachidonic acid as total fatty acids. Various mutants derived from M. alpina 1S-4 have led to the production of oils containing various PUFAs. Molecular breeding of M. alpina strains by means of manipulation of the genes involved in PUFA biosynthesis facilitates improvement of PUFA productivity and elucidation of the functions of their enzymes. This review describes practical PUFA production through mutant breeding, functional analyses of the genes of the enzymes involved in PUFA biosynthesis, and recent advances in unique PUFA production through molecular breeding. [Copyright &y& Elsevier]

Abstract: A filamentous fungus, Mortierella alpina 1S-4, was obtained, through extensive screening, as an potential producer of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs) such as arachidonic acid. With this discovery as a starting point, we conducted employing methods from metabolic engineering and molecular biology for controlling cultures and breeding mutant strains. These parental and mutant strains are now used for large-scale production of a variety of PUFAs. [Copyright &y& Elsevier]

Discusses the isolation of three Δ6 desaturase-defective mutants, designated YB214, HR95, and ST66, from Mortiella alpina 1S-4, after treating wild-type spores with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Finding that two functional Δ6 desaturases exist in M. alpina 1S-4; Identification of the mutation sites on the Δ6I gene in the Δ6 desaturase-defective mutants; Amino acid replacement; Polyunsaturated fatty acid.

Describes the cloning and sequencing of the ura3 and ura5 genes, and the isolation and characterization of uracil auxotrophs of the fungus Mortierella alpina 1S-4.

Abstract: The mutation sites on ω3 desaturase genes in two ω3 desaturase-defective mutants derived from arachidonic acid-producing Mortierella alpina 1S-4 were identified. The mutations each resulted in an amino acid replacement (W232Stop or W386Stop) which caused a lack of ω3 desaturase activity in these mutants. [Copyright &y& Elsevier]

We investigated the omega-3 eicosatetraenoic acid (ETA) production by molecular breeding of the oleaginous fungus Mortierella alpina , which can slightly accumulate ETA only when cultivated at a low temperature. The endogenous ω3-desaturase gene or the heterologous Saprolegnia diclina Δ17 ( sdd17m ) desaturase gene were overexpressed in M. alpina S14, a Δ5-desaturation activity-defective mutant derived from M. alpina 1S-4. M. alpina S14 transformants introduced with the endogenous ω3-desaturase gene showed ETA at 42.1% content in the total lipids that was 84.2-fold and 3.2-fold higher than that of the wild-type strain 1S-4 and host strain S14, respectively, when cultivated at 12°C. No accumulation of ETA was observed at 28°C. In contrast, transformants with the heterologous sdd17m gene showed 24.9% of the content of total lipids at 28°C. These results indicated that these M. alpina S14 transformants are promising strains for the production of ETA, which is hard to obtain from natural sources. [ABSTRACT FROM AUTHOR]

Abstract: We report the production of arachidonic acid (ARA) in seeds of soybean [Glycine max (L.) Merrill] by seed-specific expression of the genes encoding Δ6 desaturase, fatty acid elongase, and Δ5 desaturase from a filamentous fungus, Mortierella alpina 1S-4, as well as the down-regulation of an endogenous gene of soybean, Δ15 desaturase. Several fatty acids, γ-linolenic acid (GLA), eicosa-8, 11-dienoic acid (EDA), dihomo-γ-linolenic acid (DGLA), and ARA, which the host plant does not produce, accumulated up to 11.0 and 8.4% of the total fatty acids in the transgenic histodifferentiated somatic embryos and the transgenic mature seeds, respectively. The ability to synthesize these novel fatty acids was inherited to their progenies. These results indicate that metabolic engineering of the fatty acid biosynthetic pathway to produce desired very long-chain polyunsaturated fatty acids in oilseed crops is feasible. [Copyright &y& Elsevier]

Abstract: The Δ6-fatty-acid desaturase gene isolated from Mortierella alpina 1S-4 was introduced into Lotus japonicus and Vigna angularis by Agrobacterium-mediated transformation and constitutively expressed. Two transgenic L. japonicus lines accumulated γ-linolenic acid (GLA, C18:3, n-6), which was not found in the host, at levels of up to 11.34 and 9.66% of the total fatty acids in the leaves, respectively. These results indicate that the legume plant can synthesize GLA by expressing the fungus gene. The ability to synthesize GLA was also inherited by their progenies, which accumulated levels of up to 12.82 and 13.75% of the total fatty acids in the leaves, and 2.39 and 2.65% of those in the seeds. The GLA also accumulated at levels of up to 0.44 and 1.26% of the total fatty acids in the leaves and seeds, respectively, of a transgenic V. angularis line and at 0.69% in the leaves of its progeny. These findings reveal that the modification of the fatty-acid biosynthetic pathway by genetic manipulation in order to produce specific polyunsaturated fatty acids in grain legume crops is a promising technique. [Copyright &y& Elsevier]