Production of docosahexaenoic acid by a novel isolated Aurantiochytrium sp. 6-2 using fermented defatted soybean as a nitrogen source for sustainable fish feed development.

We focused on the production of docosahexaenoic acid (DHA)-containing microbial lipids by Aurantiochytrium sp. using of defatted soybean (DS) as a nitrogen source. Defatted soybean is a plant biomass that could provide a sustainable supply at a low cost. Results showed that Aurantiochytrium sp. could not directly assimilate the DS as a nitrogen source but could grow well in a medium containing DS fermented with rice malt. When cultivated in a fermented DS (FDS) medium, Aurantiochytrium sp. showed vigorous growth with the addition of sufficient sulfate and chloride ions as inorganic nutrients without seawater salt. A novel isolated Aurantiochytrium sp. 6-2 showed 15.8 ± 3.4 g/L DHA productivity (in 54.8 ± 12.1 g/L total fatty acid production) in 1 L of the FDS medium. Therefore, DHA produced by Aurantiochytrium sp. using FDS enables a stable and sustainable DHA supply and could be an alternative source of natural DHA derived from fish oil.

Mead acid production by disruption of Δ12-desaturase gene in Mortierella alpina 1S-4.

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 (Δ12ds) 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 Δ12ds-disruptant reached 0.10 g/L (8.5%), which exceeded 0.050 g/L (4.6%) in the conventional Δ12ds-defective mutant JT-180.

Isolation and characterization of the ω3-docosapentaenoic acid-producing microorganism Aurantiochytrium sp. T7.

ω3-Docosapentaenoic acid (ω3-DPA), an ω3-polyunsaturated fatty acid (ω3-PUFA), is expected to have beneficial physiological functions to humans; however, because of its rarity in nature, it has not been fully analyzed. We isolated an ω3-DPA producing microorganism strain T7 from brackish areas in Japan. Although most oleaginous microorganisms rarely accumulate ω3-DPA (<5% of total lipid), strain T7 accumulated ω3-DPA with more than 20% of total fatty acids. The strain T7 was identified as a related species of Aurantiochytrium. In Aurantiochytrium sp. T7, ω3-DPA production reached 164 mg/L culture broth, and the ω3-DPA content reached 23.5% of the total fatty acids when cultivated in a medium containing 2% glucose as the carbon source and 1% yeast extract as the nitrogen source, with a salinity equivalent to 50% of that of seawater and a pH in the acidic range (pH < 5.5). Aurantiochytrium sp. T7 is a promising producer of high-purity ω3-DPA containing-lipid for the functional analysis of ω3-DPA whose physiological function has hardly been elucidated, and a useful strain for investigating the novel metabolic pathway of fatty acids.

Characterization of ω3 fatty acid desaturases from oomycetes and their application toward eicosapentaenoic acid production in Mortierella alpina.

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.

Metabolic engineering of oleaginous fungus Mortierella alpina for high production of oleic and linoleic acids.

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 2.76g/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.

Production of ricinoleic acid-containing monoestolide triacylglycerides in an oleaginous diatom, Chaetoceros gracilis.

Ricinoleic acid (RA), a hydroxyl fatty acid, is suitable for medical and industrial uses and is produced in high-oil-accumulating organisms such as castor bean and the ergot fungus Claviceps. We report here the efficient production of RA in a transgenic diatom Chaetoceros gracilis expressing the fatty acid hydroxylase gene (CpFAH) from Claviceps purpurea. In transgenic C. gracilis, RA content increased at low temperatures, reaching 2.2 pg/cell when cultured for 7 d at 15 °C, without affecting cell growth, and was enhanced (3.3 pg/cell) by the co-expression of a palmitic acid-specific elongase gene. Most of the accumulated RA was linked with monoestolide triacylglycerol (ME TAG), in which one RA molecule was esterified to the α position of the glycerol backbone and was further esterified at its hydroxy group with a fatty acid or second RA moiety, or 1-OH TAG, in which RA was esterified to the glycerol backbone. Overall, 80% of RA was accumulated as ME TAGs. Furthermore, exogenous RA-methyl ester suppressed the growth of wild-type diatoms in a dose-dependent manner and was rapidly converted to ME TAG. These results suggest that C. gracilis masks the hydroxyl group and accumulates RA as the less-toxic ME TAG.

Microbial production of dihomo-γ-linolenic acid by Δ5-desaturase gene-disruptants of Mortierella alpina 1S-4.

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.

Disruption of lig4 improves gene targeting efficiency in the oleaginous fungus Mortierella alpina 1S-4.

The oil-producing zygomycete Mortierella alpina 1S-4 is known to accumulate beneficial polyunsaturated fatty acids. We identified the lig4 gene that encodes for a DNA ligase 4 homolog, which functions to repair double strand breaks by non-homologous end joining. We disrupted the lig4 gene to improve the gene targeting efficiency in M. alpina. The M. alpina 1S-4 Δlig4 strains showed no defect in vegetative growth, formation of spores, and fatty acid production, but exhibited high sensitivity to methyl methansulfonate, an agent that causes DNA double-strand breaks. Importantly, gene replacement of ura5 marker by CBXB marker occurred in 67% of Δlig4 strains and the gene targeting efficiency was 21-fold greater than that observed in disruption of the lig4 gene in the M. alpina 1S-4 host strain. Further metabolic engineering of the Δlig4 strains is expected to result in strains that produce higher levels of rare and beneficial polyunsaturated fatty acids and contribute to basic research on the zygomycete.

Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4.

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.

Gene targeting in the oil-producing fungus Mortierella alpina 1S-4 and construction of a strain producing a valuable polyunsaturated fatty acid.

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.

Selection and characterization of promoters based on genomic approach for the molecular breeding of oleaginous fungus Mortierella alpina 1S-4.

To express a foreign gene effectively, a good expression system is required. In this study, we investigated various promoters as useful tools for gene manipulation in oleaginous fungus Mortierella alpina 1S-4. We selected and cloned the promoter regions of 28 genes in M. alpina 1S-4 on the basis of expression sequence tag abundance data. The activity of each promoter was evaluated using the ß-glucuronidase (GUS) reporter gene. Eight of these promoters were shown to enhance GUS expression more efficiently than a histone promoter, which is conventionally used for the gene manipulation in M. alpina. Especially, the predicted protein 3 and the predicted protein 6 promoters demonstrated approximately fivefold higher activity than the histone promoter. The activity of some promoters changed along with the cultivation phase of M. alpina 1S-4. Seven promoters with constitutive or time-dependent, high-level expression activity were selected, and deletion analysis was carried out to determine the promoter regions required to retain activity. This is the first report of comprehensive promoter analysis based on a genomic approach for M. alpina. The promoters described here will be useful tools for gene manipulation in this strain.

Characterization of galactose-dependent promoters from an oleaginous fungus Mortierella alpina 1S-4.

An inducible promoter is a useful tool for the controlled expression of a given gene. In this report, we describe galactose-dependent promoters for potential use in an oleaginous fungus Mortierella alpina. We cloned the putative promoter regions of two genes encoding galactose metabolic enzymes, GAL1 and GAL10, from the genome of M. alpina 1S-4. The ß-glucuronidase (GUS) reporter gene assay in M. alpina 1S-4 revealed that regulation of these promoters was dependent on the presence of galactose in the medium both with and without other sugars. With the GAL10 promoter, an approximately 50-fold increase of GUS activity was demonstrated by addition of galactose into the culture media at any cultivation phase. The 5' deletion analysis of the GAL10 promoter revealed that a promoter region of over 2,000 bp length was required for its high-level activity and sufficient inducible response. Significantly, this is the first report of inducible promoters of zygomycetes. The GAL10 promoter will be a valuable tool for gene manipulation in M. alpina 1S-4.