Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2015 www.elsevier.com/locate/jbiosc
Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4 Tomoyo Okuda,1 Akinori Ando,1, 2 Hiroaki Negoro,1 Hiroshi Kikukawa,1 Takaiku Sakamoto,1 Eiji Sakuradani,3 Sakayu Shimizu,1, 4 and Jun Ogawa1, 2, * Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan,1 Research Unit for the Physiological Chemistry, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan,2 Institute of Technology and Science, The University of Tokushima, 2-1 Minami-josanjima, Tokushima 770-8506, Japan,3 and Department of Bioscience and Biotechnology, Faculty of Bioenvironmental Science, Kyoto Gakuen University, 1-1 Nanjo-otani, Sogabe-cho, Kameoka, Kyoto 621-8555, Japan4 Received 10 November 2014; accepted 16 January 2015 Available online xxx
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 u3desaturase gene or the heterologous Saprolegnia diclina D17 (sdd17m) desaturase gene were overexpressed in M. alpina S14, a D5-desaturation activity-defective mutant derived from M. alpina 1S-4. M. alpina S14 transformants introduced with the endogenous u3-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. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Omega-3 polyunsaturated fatty acids; Lipid-producing fungus; Mortierella alpina; Rare fatty acid; Eicosatetraenoic acid]
Omega-3 polyunsaturated fatty acids (u3-PUFAs) are found in natural sources. Each u3-PUFA and its derivative have been reported to have some particular physiological function. For example, Eicosapentaenoic acid (EPA, 20:5u3) and docosahexaenoic acid (DHA, 20:6u3) now typically derived from a natural marine source and supplied sufficiently, have been known as precursors of the eicosanoids of signaling molecules including prostaglandins, thromboxanes, and leukotrienes (1). Recently, EPA and DHA have attracted much attention for the beneficial effects on human health in reducing the risk for sudden death caused by cardiac arrhythmias and all-cause mortality in patients with known coronary heart disease (2e4). In addition, there are some reports suggestive of effects on prevention of rheumatoid arthritis and asthma (5e7). Alpha-linolenic acid (ALA, 18:3u3), which is contained in natural oils from higher plants such as linseed and soya, has been also reported to contribute to prevention several diseases, such as coronary heart disease and depressive illness (8,9). Furthermore, u3PUFA derivatives were also important as anti-inflammatory lipid mediators and candidates of anti-influenza agent (10,11). Therefore, the demand for u3-PUFAs is rapidly increasing in the pharmaceutical, medical and nutritional fields.
* Corresponding author at: Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan. Tel.: þ81 75 753 6115; fax: þ81 75 753 6113. E-mail addresses: [email protected] (H. Kikukawa), [email protected] (T. Sakamoto), [email protected] (J. Ogawa).
EPA, DHA, and ALA, which can be prepared from natural sources, have been well studied because of their sufficient natural supply. On the other hand, there are few reports on the other u3-PUFAs, such as stearidonic acid (SDA, 18:4u3), docosapentaenoic acid (DPA, 22:5u3), and eicosatetraenoic acid (ETA, 20:4u3), because their supply has been limited to date. These minor u3-PUFAs have been expected to possess beneficial function as precursors of bioactive substances as well as EPA and so on, therefore sufficient supply of such rare PUFAs has been required for elucidation of their physiological function. Recently, SDA has been reported to be accumulated in Echium plantagineum (12), and the development of its purification method is now under way. In addition, the SDAaccumulating soybean derived by the molecular breeding has been commoditized already (13). DPA also has been reported to be present in several natural oils produced by earless seal (14), Cyanea capillata, and Mola mola (15). Thus, sources of SDA and DPA are being developed, therefore research on their physiological function will be advancing in the near future. On the other hand, ETA is hard to find in nature. In addition, there are only few reports of ETA production at a low level by overexpression of D6 desaturase gene in E. plantagineum (16), by mutation in Mortierella alpina (17), and molecular breeding in Arabidopsis thaliana (18). Although ETA has been expected to show beneficial effects on human health, the detailed bioactivity of ETA has remained almost unknown because of lack of its sources. Therefore establishment of a stable supply of ETA is needed for subsequent studies of physiological function of ETA
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.01.014
Please cite this article in press as: Okuda, T., et al., Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.014
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followed by expansion of its applications in medical and pharmaceutical fields. Mortierella alpina 1S-4, an oleaginous fungus, is known as an industrial strain that produces arachidonic acid (ARA, 20:4u6) commercially (19). To date, we have reported considerable accumulation of EPA by overexpressing endogenous u3-desaturase gene in M. alpina 1S-4 as a novel alternative source of u3-PUFAs (20). We have also succeeded in the industrial production of various kinds of PUFAs by using mutants derived from M. alpina 1S4 through chemical mutagenesis (21,22). M. alpina S14 is a D5desaturation activity-defective mutant derived from M. alpina 1S4, after treating the parental spores with a chemical mutagen (23) (Fig. 1). The strain S14 produces only a trace (about 1%) amount of ARA, and the ratio of dihomo-g-linolenic acid (DGLA, 18:4u6) to total fatty acids is markedly high, accounting for as much as 43% (24) and has been applied to the industrial production of DGLA (25). ETA can be biosynthesized by u3- (e.g., similar to D17-) desaturation from DGLA; therefore, we hypothesized that this strain would be a good host strain for ETA production by expressing u3- or D17desaturase gene. In this study, we aimed to evaluate ETA production using expression of the endogenous u3-desaturase gene and the heterologous D17-desaturase gene in M. alpina S14. MATERIALS AND METHODS Strain, media, and growth conditions M. alpina S14, a D5-desaturation activity-defective mutant derived from M. alpina 1S-4 (24), was maintained on Potato Dextrose Agar medium (Difco, MI, USA), and the uracil auxotrophs (ura5 strain) derived from M. alpina S14 on the PDA medium containing uracil (0.05 mg/ mL). GY medium consisting of 2% (w/v) glucose and 1% yeast extract was used for fatty acid composition analysis and for extracting genomic DNA. Czapek-Dox minimal medium consisting of 3% sucrose, 0.2% NaNO3, 0.1% KH2PO4, 0.05% KCl, 0.05% MgSO4$7H2O, and 0.001% FeSO4$7H2O, pH 6.0 was used for the sporulation of the fungi. SC medium was used as a uracil-free synthetic medium, which contained 5 g of Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate (Difco), 1.7 g of (NH4)2SO4, 20 g of glucose, 20 mg of adenine, 30 mg of tyrosine, 1 mg of methionine, 2 mg of arginine, 2 mg of histidine, 4 mg of lysine, 4 mg of tryptophan, 5 mg of threonine, 6 mg of isoleucine, 6 mg of leucine, and 6 mg of phenylalanine per liter. Solid media contained 1.5% agar. Escherichia coli DH5a cells were used for DNA manipulation and cultivated at 37 C with vigorous shaking at 300 rpm. Isolation of uracil auxotrophs of M. alpina S14 Isolation of uracil auxotrophs was performed as described previously (26). Mutant S14 was incubated on Czapek-Dox agar medium at 28 C for 1 month, and allowed to sporulate at 12 C for 1 month. Spores of S14 were harvested from the surface of Czapek-Dox (2.6 108 spores/225 cm2); 2.6 107 spores were spread on a GY agar medium containing 1.0 mg/mL of 5-fluoroorotic acid (5-FOA) and 0.05 mg/mL of uracil. By means of 5-FOA positive selection, uracil auxotrophs that acquired 5-FOA resistance could be isolated. Fatty acid analysis All strains were inoculated in GY medium and then the culture was carried out at 12 C or 28 C with reciprocal shaking (120 strokes/min) for
a desired period. The mycelia were harvested by suction filtration and dried at 120 C. The dried cells were weighed and transmethylated with 10% methanolic HCl and dichloromethane at 55 C for 2 h, containing 0.2 mg of n-tricosanoic acid as an internal standard. The resultant fatty acid methylesters were extracted with nhexane, concentrated and then analyzed using gas chromatography. Isolation of the ura5 genomic gene of uracil auxotrophs of S14 The ura5 genomic gene was amplified using forward primer ura5upF (50 TTTCTGATGTGTCTCCCACC-30 ) and reverse primer ura5downR (50 -TTCCAACAGAACCTTCCCTCG-30 ) with uracil auxotrophic S14 genomic DNA as the template. A 700-bp PCR product was cloned into the pUC118 vector using Reagent Set for Mighty Cloning Kit (Takara, Shiga, Japan). DNA sequencing Nucleotide sequences were identified using the dideoxy chain termination method by means of a CEQ Dye Terminator Cycle Sequencing kit (Beckman Coulter Inc., CA, USA) and an automated sequencer DNA Analysis System CEQ 8000 (Beckman Coulter Inc.). The sequence data were analyzed in the GenetyxWindows software, ver. 11.0.3 (Software Development, Japan). Construction of a transformation vector for M. alpina S14 ura5L strain Transformation vectors pSDura5u3 and pSDura5u32 were constructed by the modification of pSDura5 z (27,28). The u3-desaturase gene was amplified using a forward primer, w3F2PciI (50 -GGGAATATTAAGCTTACATGTCCCC-30 ) and a reverse primer, w3R2BamHI (50 -GCCGGATCCAAATTGTTAATGCTTG-30 ) at 56 C with M. alpina 1S-4 cDNA as a template. The 2 primers contained a PciI and a BamHI site, respectively (underlined). About 1.3-kb of PCR product was ligated to the pT7 Blue T-Vector (Novagen, Darmstadt, Germany), resulting in construction of a plasmid named pT7u3. We checked its sequence. The u3-desaturase gene was digested with PciI and BamHI, followed by ligation into pBlueshtp treated with NcoI and BamHI to construct pBluesu3 (29). The u3-desaturase expression unit including a promoter and a terminator was cut out by EcoRI from pBluesu3 and ligated into pSDura5 digested with the same enzyme to generate pSDura5u3 and pSDura5u32 (Fig. 2). The latter plasmid possessed two u3-desaturase expression units. Saprolegnia diclina D17 desaturase gene (sdd17m) was synthesized with optimized codon usage to reflect the codon bias of M. alpina 1S-4 (obtained from the Kazusa database; http://www.kazusa.or.jp/codon/), with additional SpeI and BamHI restriction enzyme sites at the 50 and 30 ends, respectively. The sdd17m expression cassette, with the SSA2 promoter (30) and SdhB terminator (19), was generated by fusion PCR with XbaI and NheI restriction sites at the 50 and 30 ends of the cassette, respectively. This cassette was then digested with XbaI and NheI and ligated into pBIG35ZhSSA2psdd17m, which had been digested with same restriction enzymes (Fig. 2). Transformation of M. alpina by microprojectile bombardment A spore suspension from the M. alpina S14 ura5 strain was freshly prepared from cultures growing on Czapek-Dox agar medium supplemented with 0.05 mg/mL uracil; the suspension was filtered through Miracloth (Calbiochem) and spread on a uracilfree SC medium. A PDS-1000/He Particle Delivery System (Bio-Rad Laboratories Inc., CA, USA) was used for the transformation. Tungsten particles (0.4 mm in diameter) coated with pSDura5, pSDura5u3 and pSDura5u32 were prepared according to the manufacturer’s manual. Bombardment was performed in the plate placed on the device under a helium pressure of 1100 psi (7580 kPa). After the bombardment, the plate was incubated at 28 C (3e6 days) (28). Transformation of M. alpina using the ATMT method The spore suspension from the M. alpina S14 ura5 strain was prepared as mentioned at the description of microprojectile bombardment method. Transformation of the M. alpina S14 ura5 strain with pBIG35ZhSSA2psdd17m was performed using the Agrobacterium tumefaciens-mediated transformation (ATMT) method described previously (20) with slight modification. Initially, A. tumefaciens C58C1 was transformed with each vector via electroporation as
COOH 16:0
Δ5 desaturase defetive COOH 18:0
COOH OA, 18:1ω9
18:2ω9
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GLA, 18:3ω6
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ω3 desaturase COOH ALA, 18:3ω3
SDA, 18:4ω3
COOH
ETA, 20:4ω3
COOH
COOH EPA, 20:5ω3
FIG. 1. A biosynthetic pathway of polyunsaturated fatty acids (PUFA) in the mutant strain S14 derived from Mortierella alpina 1S-4. OA, oleic acid; LA, linoleic acid; GLA, g-linolenic acid; DGLA, dihomo-g-linolenic acid; ARA, arachidonic acid; ALA, a-linolenic acid; SDA, stearidonic acid; ETA, u3 eicosatetraenoic acid; EPA, u3 eicosapentaenoic acid.
Please cite this article in press as: Okuda, T., et al., Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.014
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FIG. 2. Vector constructs used for expression of (A) u3-desaturase and (B) sdd17m in M. alpina S14. u3, Mortierella alpina u3-desaturase; hisH 4.1p, M. alpina histone H4.1 promoter; trpC t, Aspergillus nidulans trpC transcription terminator; rDNA, M. alpina 1S-4 18S rDNA fragment; bla, ampicillin resistance gene; ura5, orotate phosphoribosyl transferase gene of M. alpina; SSA2 p, M. alpina SSA2 promoter; SdhB t, M. alpina SdhB transcription terminator; Sdd17m, codon-optimized D17 fatty acid desaturase gene from S. diclina; NPTIII, neomycin phosphotransferase III gene; TrfA, TrfA locus, which produces 2 proteins that promote replication of the plasmid; ColEI ori, ColEI origin of replication; oriV, pRK2 origin of replication; RB, right border; LB, left border.
described previously (31) and its transformants were isolated on LB-Mg agar plates supplemented with kanamycin (20 mg/mL), ampicillin (50 mg/mL) and rifampicin (50 mg/mL). A. tumefaciens transformants were cultivated in 100 mL of MM supplemented with kanamycin (20 mg/mL) and ampicillin (50 mg/mL) at 28 C for 48 h with shaking (120 rpm). Bacterial cells were harvested by centrifugation at 8000 g, washed once with fresh IM, and then diluted to an optical density of 660 nm (OD660) of 0.1e0.2 in 10 mL of fresh IM. After pre-incubation for 12e16 h at 28 C with shaking (300 rpm) to an OD660 of 1.5e2.0, 100 mL of the bacterial cell suspension was mixed with an equal volume of a spore suspension (108 spores/mL) from the M. alpina 1S-4 ura5 strain, and then spread on membranes (Whatman 50 Hardened Circles 70 mm, Whatman International Ltd., UK) which were put on cocultivation media (IM in 1.5% agar) and incubated at 23 C for 5 days. After cocultivation, the membranes were transferred to uracil-free SC agar plates that contained 0.03% Nile blue A (SigmaeAldrich Japan) to discriminate between fungal colonies and the white color of the membrane. After 2 days of incubation at 28 C, hyphae from visible fungal colonies were transferred to fresh uracil-free SC agar plates; this process was repeated 3 times to obtain candidates. Integration of the vector into the chromosome of the host strain was verified by PCR, as described previously (28).
RESULTS Isolation and characterization of uracil auxotrophs of M. alpina S14 M. alpina S14, a D5-desaturase defective mutant derived from M. alpina 1S-4, was used as the host strain for heterologous gene expression. The strain S14 accumulates a higher amount of DGLA, a precursor for ETA, than does the wildtype strain 1S-4. To develop a transformation system using M. alpina S14, we obtained 4 uracil auxotrophic S14 mutants (S14-1, 2, 3, and 6) that grow on the SC medium with uracil, but not on uracil-free SC medium (Fig. 3A). We evaluated these S14 uracil auxotrophs as a host strain for molecular breeding based on growth and fatty acid production and composition. As a result, we found that all 4 uracil auxotrophs showed vigorous growth and as much fatty acid production and composition as the wild-type strain S14 (data not shown). To assess whether the homologous ura5 gene was suitable as a selective marker for uracil auxotrophic mutants, the ura5 gene in the genome of these mutants was sequenced (26). As shown in Fig. 3B, in case of a mutant S14-2, the mutation of base substitution was observed in the ura5 gene: the substitution of G for A and G for T were observed at the þ211 and þ212 nucleotide positions, leading to amino acid replacement, G 71 I. A base-pair change was detected in that of S14-3: the substitution of A to G at þ1 caused the deficiency of start codon. S14-1 and -6 were found to have no mutations point in their ura5 gene. Consequently, the strain S14-2 was used as a host strain for transformation in subsequent studies because of the evident multiple mutational points in its ura5 gene.
Transformation of the M. alpina S14 uracil auxotroph with pSDura5u332 and fatty acid analysis We performed transformation of the M. alpina S14 uracil auxotroph with pSDura5u32 using the microprojectile bombardment method and selected one stable transformant (u3#1). Subsequently, we evaluated ETA production of the 1S-4 wild-type strain, S14 host strain, and the transformant u3#1 during cultivation at 12 C (Fig. 4). The amount of ETA in u3#1 remarkably increased with the elapse of cultivation time compared to wild-type 1S-4 and host S14. The ETA contents of u3#1 reached 42.1% in the total fatty acids, while those of wild-type 1S-4 and S14 were just 0.5% and 13.1%: ETA content of u3#1 was up to 84.2-fold and 3.2-fold higher compared to wild-type 1S-4 and S14, respectively. ALA and SDA
(A) wild
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G 211 A G 212 T
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FIG. 3. Characterization of uracil auxotrophic strains of M. alpina S14. (A) Growth of M. alpina 1S-4, S14 and uracil auxotrophic S14 on SC medium with or without uracil. (B) A mutation site of the ura5 gene in M. alpina uracil auxotrophic S14.
Please cite this article in press as: Okuda, T., et al., Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.014
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FIG. 4. Time course of growth, fatty acid production, and composition of the M. alpina 1S-4 wild-type strain, S14 host strain, and its transformant u3#1 overexpressing u3desaturase gene. All strains were cultivated in 10 mL of the GY liquid medium at 12 C for 5, 7, 9, and 11 days. The data are shown as mean SD from 3 individual experiments. For all other abbreviations, see the legend of Fig. 1.
contents of u3#1 were up to 3.7% and 14.2% of total fatty acid on Day 5. In contrast, no accumulation of u3-PUFAs including ETA was observed in u3#1 when cultivated at 28 C (data not shown). Transformation of the M. alpina S14 uracil auxotroph with pBIG35Zhsdd17m and fatty acid analysis In order to produce ETA in M. alpina S14 at a normal temperature, we used the S. diclina fatty acid D17-desaturase gene (sdd17m). S. diclina is an oleaginous microorganism producing u3-PUFAs at a normal temperature, and Sdd17 is able to catalyze a desaturation at u3 position of DGLA resulting in ETA biosynthesis (32) as well as endogenous u3 desaturase in M. alpina. The expression vector pBIG35ZhSSA2psdd17m (Fig. 2), carrying the codon-optimized sdd17 gene (sdd17m), was constructed and introduced into the M. alpina S14 uracil auxotroph using the ATMT method. As a result, five stable transformants were randomly selected and used for further studies. The selected transformants and the host strain S14 were cultivated in GY medium at 28 C, and the time course of their fatty acid production and composition were analyzed (Fig. 5). All transformants showed accumulation of ETA, which was not detected in the host strain S14 at normal temperature. Transformant #7 exhibited the highest ETA content (24.9% of total fatty acids on day 10) among all transformants. The trace amount of ALA and SDA was detected in transformants. DISCUSSION The oleaginous filamentous fungus M. alpina 1S-4 is known as an industrial strain that produces arachidonic acid (ARA) commercially (19). Up until now, we have succeeded in the industrial production of PUFAs using M. alpina 1S-4 and its mutant strains (21,22). This strain is also known to accumulate considerable
amounts of EPA, when cultivated at low temperatures, which cause the expression of the endogenous u3-desaturase gene and increase the activity of the gene product (33). Based on these observations, we hypothesized that M. alpina has a potential of u3-PUFAs accumulation and employed this fungus as a host strain to produce a rare u3-PUFA: ETA. M. alpina S14 is a D5-desaturation activity-defective mutant (23) which was derived from M. alpina 1S-4 by treating parental spores with a chemical mutagen, N-methyl-N0 -nitro-N-nitrosoguanidine (MNNG), as described previously (34). M. alpina S14 also exhibits a higher accumulation of DGLA, a precursor of ETA, than strain 1S-4 (24). Therefore, we surmised that this strain would be suitable for evaluation of endogenous u3- and heterologous D17-desaturase gene expression, which both can be converted to DGLA to ETA. First, we succeeded in obtaining some ura5 mutants derived from the strain S14, and selected S14-2 as the host strain for transformation. We performed transformation with the uracil auxotroph, strain S14-2, with the endogenous u3-desaturase gene, and observed the improvement of fatty acid composition of transformants, which showed considerable production of ETA (Fig. 4). Other rare u3-PUFAs such as SDA were also accumulated in the transformants. This result indicates that it is possible to obtain some transformants producing other unique PUFAs by constructing a transformation system with other useful mutants derived from M. alpina 1S-4. Thus, we demonstrated potency of M. alpina as an ETA source via overexpression of the endogenous u3-desaturase gene. For industrial production, however, low-temperature cultivation is not suitable because of its high running cost compared to normaltemperature cultivation. Therefore, we subsequently tested the transformation with sdd17m gene which enables the strain to produce u3-PUFAs at a normal room temperature. For the introduction of the sdd17m gene, we used the ATMT method (20) instead
Please cite this article in press as: Okuda, T., et al., Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.014
OMEGA-3 EICOSATETRAENOIC ACID PRODUCTION
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Other fatty acids Saturated fatty acids OA LA GLA DGLA ARA ETA
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FIG. 5. Time course of growth, fatty acid production, and composition of the M. alpina 1S-4 wild-type strain, S14 host strain and its transformants expressing the sdd17m gene. All strains were cultivated in 4 mL of the GY liquid medium at 28 C for 3, 7, and 10 days. The data are shown as mean SD from 3 individual experiments. For other all abbreviations, see the legend of Fig. 1.
of the microprojectile bombardment method which often causes undesired mutations in host cells and results in unstable transformants because of the physical damage by high pressure employed to transfer plasmid DNA, and random multiple integrations of plasmids into the chromosomal DNA. The ATMT method results in a single-copy integration of T-DNA at a random location in chromosomal DNA and the increased transformation frequency under mild conditions for M. alpina cells. Moreover, application of stronger promoters, not the conventional histone promoter, is expected to lead to the further improvements in ETA production. We assessed the transformation with sdd17m gene controlled by a strong promoter SSA2 p into uracil auxotrophic S14 using the ATMT method, in order to produce ETA at normal temperature. As a result, we observed that some transformants exhibited ETA production at 28 C (Fig. 5), and confirmed that the Sdd17 protein was functional in M. alpina S14. On the other hand, we found that the activity of Sdd17 to convert DGLA to ETA might be lower than that of ARA to EPA because residual accumulation of DGLA was observed in the transformants while efficient conversion of ARA to EPA was reported (32). It was also suggested that the D15-desaturation activity of Sdd17 for C18 substrates is extremely low because ALA and SDA accumulation were hardly detected in transformants (Fig. 5). Additional expression of heterologous D15-desaturase and endogenous u3-desaturase might lead to enhanced ETA biosynthesis and productivity. In addition, this study is the first report to show that the SSA2 promoter can be used for modification of the PUFA production in M. alpina. Recently, several useful promoters of M. alpina have been newly identified. By applying them, further efficient production of u3-PUFAs might be possible using M. alpina. As mentioned in the introduction section, there are few reports of ETA production in the recent past. This report demonstrates the potency of M. alpina as a practical source of ETA. Establishment of a
stable supply of ETA by using M. alpina will lead to subsequent studies of physiological function of ETA and may result in expansion of its applications in medical and pharmaceutical fields. ACKNOWLEDGMENTS This work was partially supported by Institute for Fermentation, Osaka, Japan (IFO) and the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN) to J.O.; Advanced Low Carbon Technology Research and Development Program (ALCA) to E.S.; and Grants-in-Aid for Scientific Researches (no. 22380051) from Ministry of Education, Culture, Sports, Science, and Technology, Japan to E.S. References 1. Horrobin, D. F.: Nutritional and medical importance of g-linolenic acid, Prog. Lipid. Res., 31, 163e194 (1992). 2. Romieu, I., Tellez-Rojo, M. M., Lazo, M., Manzano-Patino, A., CortezLugo, M., Julien, P., Belanger, M. C., Hernandez-Avila, M., and Holguin, F.: Omega-3 fatty acid prevents heart rate variability reductions associated with particulate matter, Am. J. Respir. Crit. Care Med., 172, 1534e1540 (2005). 3. von Schacky, C.: Cardiovascular disease prevention and treatment, Prostaglandins Leukot. Essent. Fatty Acids, 81, 193e198 (2009). 4. Simopoulos, A. P.: The importance of the ratio of omega-6/omega-3 essential fatty acids, Biomed. Pharmacother., 56, 365e379 (2002). 5. Balk, E. M., Lichtenstein, A. H., Chung, M., Kupelnick, B., Chew, P., and Lau, J.: Effects of omega-3 fatty acids on serum markers of cardiovascular disease risk: a systematic review, Atherosclerosis, 189, 19e30 (2006). 6. Schacky, C. v and Harris, W. S.: Cardiovascular benefits of omega-3 fatty acids, Cardiovasc. Res., 73, 310e315 (2007). 7. Yates, C. M., Calder, P. C., and Ed Rainger, G.: Pharmacology and therapeutics of omega-3 polyunsaturated fatty acids in chronic inflammatory disease, Pharmacol. Ther., 141, 272e282 (2014). 8. Lucas, M., Mirzaei, F., O’Reilly, E. J., Pan, A., Willett, W. C., Kawachi, I., Koenen, K., and Ascherio, A.: Dietary intake of n-3 and n-6 fatty acids and the
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Please cite this article in press as: Okuda, T., et al., Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.014
Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4 Tomoyo Okuda,1 Akinori Ando,1, 2 Hiroaki Negoro,1 Hiroshi Kikukawa,1 Takaiku Sakamoto,1 Eiji Sakuradani,3 Sakayu Shimizu,1, 4 and Jun Ogawa1, 2, * Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan,1 Research Unit for the Physiological Chemistry, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan,2 Institute of Technology and Science, The University of Tokushima, 2-1 Minami-josanjima, Tokushima 770-8506, Japan,3 and Department of Bioscience and Biotechnology, Faculty of Bioenvironmental Science, Kyoto Gakuen University, 1-1 Nanjo-otani, Sogabe-cho, Kameoka, Kyoto 621-8555, Japan4 Received 10 November 2014; accepted 16 January 2015 Available online xxx
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 u3desaturase gene or the heterologous Saprolegnia diclina D17 (sdd17m) desaturase gene were overexpressed in M. alpina S14, a D5-desaturation activity-defective mutant derived from M. alpina 1S-4. M. alpina S14 transformants introduced with the endogenous u3-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. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Omega-3 polyunsaturated fatty acids; Lipid-producing fungus; Mortierella alpina; Rare fatty acid; Eicosatetraenoic acid]
Omega-3 polyunsaturated fatty acids (u3-PUFAs) are found in natural sources. Each u3-PUFA and its derivative have been reported to have some particular physiological function. For example, Eicosapentaenoic acid (EPA, 20:5u3) and docosahexaenoic acid (DHA, 20:6u3) now typically derived from a natural marine source and supplied sufficiently, have been known as precursors of the eicosanoids of signaling molecules including prostaglandins, thromboxanes, and leukotrienes (1). Recently, EPA and DHA have attracted much attention for the beneficial effects on human health in reducing the risk for sudden death caused by cardiac arrhythmias and all-cause mortality in patients with known coronary heart disease (2e4). In addition, there are some reports suggestive of effects on prevention of rheumatoid arthritis and asthma (5e7). Alpha-linolenic acid (ALA, 18:3u3), which is contained in natural oils from higher plants such as linseed and soya, has been also reported to contribute to prevention several diseases, such as coronary heart disease and depressive illness (8,9). Furthermore, u3PUFA derivatives were also important as anti-inflammatory lipid mediators and candidates of anti-influenza agent (10,11). Therefore, the demand for u3-PUFAs is rapidly increasing in the pharmaceutical, medical and nutritional fields.
* Corresponding author at: Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan. Tel.: þ81 75 753 6115; fax: þ81 75 753 6113. E-mail addresses: [email protected] (H. Kikukawa), [email protected] (T. Sakamoto), [email protected] (J. Ogawa).
EPA, DHA, and ALA, which can be prepared from natural sources, have been well studied because of their sufficient natural supply. On the other hand, there are few reports on the other u3-PUFAs, such as stearidonic acid (SDA, 18:4u3), docosapentaenoic acid (DPA, 22:5u3), and eicosatetraenoic acid (ETA, 20:4u3), because their supply has been limited to date. These minor u3-PUFAs have been expected to possess beneficial function as precursors of bioactive substances as well as EPA and so on, therefore sufficient supply of such rare PUFAs has been required for elucidation of their physiological function. Recently, SDA has been reported to be accumulated in Echium plantagineum (12), and the development of its purification method is now under way. In addition, the SDAaccumulating soybean derived by the molecular breeding has been commoditized already (13). DPA also has been reported to be present in several natural oils produced by earless seal (14), Cyanea capillata, and Mola mola (15). Thus, sources of SDA and DPA are being developed, therefore research on their physiological function will be advancing in the near future. On the other hand, ETA is hard to find in nature. In addition, there are only few reports of ETA production at a low level by overexpression of D6 desaturase gene in E. plantagineum (16), by mutation in Mortierella alpina (17), and molecular breeding in Arabidopsis thaliana (18). Although ETA has been expected to show beneficial effects on human health, the detailed bioactivity of ETA has remained almost unknown because of lack of its sources. Therefore establishment of a stable supply of ETA is needed for subsequent studies of physiological function of ETA
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.01.014
Please cite this article in press as: Okuda, T., et al., Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.014
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followed by expansion of its applications in medical and pharmaceutical fields. Mortierella alpina 1S-4, an oleaginous fungus, is known as an industrial strain that produces arachidonic acid (ARA, 20:4u6) commercially (19). To date, we have reported considerable accumulation of EPA by overexpressing endogenous u3-desaturase gene in M. alpina 1S-4 as a novel alternative source of u3-PUFAs (20). We have also succeeded in the industrial production of various kinds of PUFAs by using mutants derived from M. alpina 1S4 through chemical mutagenesis (21,22). M. alpina S14 is a D5desaturation activity-defective mutant derived from M. alpina 1S4, after treating the parental spores with a chemical mutagen (23) (Fig. 1). The strain S14 produces only a trace (about 1%) amount of ARA, and the ratio of dihomo-g-linolenic acid (DGLA, 18:4u6) to total fatty acids is markedly high, accounting for as much as 43% (24) and has been applied to the industrial production of DGLA (25). ETA can be biosynthesized by u3- (e.g., similar to D17-) desaturation from DGLA; therefore, we hypothesized that this strain would be a good host strain for ETA production by expressing u3- or D17desaturase gene. In this study, we aimed to evaluate ETA production using expression of the endogenous u3-desaturase gene and the heterologous D17-desaturase gene in M. alpina S14. MATERIALS AND METHODS Strain, media, and growth conditions M. alpina S14, a D5-desaturation activity-defective mutant derived from M. alpina 1S-4 (24), was maintained on Potato Dextrose Agar medium (Difco, MI, USA), and the uracil auxotrophs (ura5 strain) derived from M. alpina S14 on the PDA medium containing uracil (0.05 mg/ mL). GY medium consisting of 2% (w/v) glucose and 1% yeast extract was used for fatty acid composition analysis and for extracting genomic DNA. Czapek-Dox minimal medium consisting of 3% sucrose, 0.2% NaNO3, 0.1% KH2PO4, 0.05% KCl, 0.05% MgSO4$7H2O, and 0.001% FeSO4$7H2O, pH 6.0 was used for the sporulation of the fungi. SC medium was used as a uracil-free synthetic medium, which contained 5 g of Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate (Difco), 1.7 g of (NH4)2SO4, 20 g of glucose, 20 mg of adenine, 30 mg of tyrosine, 1 mg of methionine, 2 mg of arginine, 2 mg of histidine, 4 mg of lysine, 4 mg of tryptophan, 5 mg of threonine, 6 mg of isoleucine, 6 mg of leucine, and 6 mg of phenylalanine per liter. Solid media contained 1.5% agar. Escherichia coli DH5a cells were used for DNA manipulation and cultivated at 37 C with vigorous shaking at 300 rpm. Isolation of uracil auxotrophs of M. alpina S14 Isolation of uracil auxotrophs was performed as described previously (26). Mutant S14 was incubated on Czapek-Dox agar medium at 28 C for 1 month, and allowed to sporulate at 12 C for 1 month. Spores of S14 were harvested from the surface of Czapek-Dox (2.6 108 spores/225 cm2); 2.6 107 spores were spread on a GY agar medium containing 1.0 mg/mL of 5-fluoroorotic acid (5-FOA) and 0.05 mg/mL of uracil. By means of 5-FOA positive selection, uracil auxotrophs that acquired 5-FOA resistance could be isolated. Fatty acid analysis All strains were inoculated in GY medium and then the culture was carried out at 12 C or 28 C with reciprocal shaking (120 strokes/min) for
a desired period. The mycelia were harvested by suction filtration and dried at 120 C. The dried cells were weighed and transmethylated with 10% methanolic HCl and dichloromethane at 55 C for 2 h, containing 0.2 mg of n-tricosanoic acid as an internal standard. The resultant fatty acid methylesters were extracted with nhexane, concentrated and then analyzed using gas chromatography. Isolation of the ura5 genomic gene of uracil auxotrophs of S14 The ura5 genomic gene was amplified using forward primer ura5upF (50 TTTCTGATGTGTCTCCCACC-30 ) and reverse primer ura5downR (50 -TTCCAACAGAACCTTCCCTCG-30 ) with uracil auxotrophic S14 genomic DNA as the template. A 700-bp PCR product was cloned into the pUC118 vector using Reagent Set for Mighty Cloning Kit (Takara, Shiga, Japan). DNA sequencing Nucleotide sequences were identified using the dideoxy chain termination method by means of a CEQ Dye Terminator Cycle Sequencing kit (Beckman Coulter Inc., CA, USA) and an automated sequencer DNA Analysis System CEQ 8000 (Beckman Coulter Inc.). The sequence data were analyzed in the GenetyxWindows software, ver. 11.0.3 (Software Development, Japan). Construction of a transformation vector for M. alpina S14 ura5L strain Transformation vectors pSDura5u3 and pSDura5u32 were constructed by the modification of pSDura5 z (27,28). The u3-desaturase gene was amplified using a forward primer, w3F2PciI (50 -GGGAATATTAAGCTTACATGTCCCC-30 ) and a reverse primer, w3R2BamHI (50 -GCCGGATCCAAATTGTTAATGCTTG-30 ) at 56 C with M. alpina 1S-4 cDNA as a template. The 2 primers contained a PciI and a BamHI site, respectively (underlined). About 1.3-kb of PCR product was ligated to the pT7 Blue T-Vector (Novagen, Darmstadt, Germany), resulting in construction of a plasmid named pT7u3. We checked its sequence. The u3-desaturase gene was digested with PciI and BamHI, followed by ligation into pBlueshtp treated with NcoI and BamHI to construct pBluesu3 (29). The u3-desaturase expression unit including a promoter and a terminator was cut out by EcoRI from pBluesu3 and ligated into pSDura5 digested with the same enzyme to generate pSDura5u3 and pSDura5u32 (Fig. 2). The latter plasmid possessed two u3-desaturase expression units. Saprolegnia diclina D17 desaturase gene (sdd17m) was synthesized with optimized codon usage to reflect the codon bias of M. alpina 1S-4 (obtained from the Kazusa database; http://www.kazusa.or.jp/codon/), with additional SpeI and BamHI restriction enzyme sites at the 50 and 30 ends, respectively. The sdd17m expression cassette, with the SSA2 promoter (30) and SdhB terminator (19), was generated by fusion PCR with XbaI and NheI restriction sites at the 50 and 30 ends of the cassette, respectively. This cassette was then digested with XbaI and NheI and ligated into pBIG35ZhSSA2psdd17m, which had been digested with same restriction enzymes (Fig. 2). Transformation of M. alpina by microprojectile bombardment A spore suspension from the M. alpina S14 ura5 strain was freshly prepared from cultures growing on Czapek-Dox agar medium supplemented with 0.05 mg/mL uracil; the suspension was filtered through Miracloth (Calbiochem) and spread on a uracilfree SC medium. A PDS-1000/He Particle Delivery System (Bio-Rad Laboratories Inc., CA, USA) was used for the transformation. Tungsten particles (0.4 mm in diameter) coated with pSDura5, pSDura5u3 and pSDura5u32 were prepared according to the manufacturer’s manual. Bombardment was performed in the plate placed on the device under a helium pressure of 1100 psi (7580 kPa). After the bombardment, the plate was incubated at 28 C (3e6 days) (28). Transformation of M. alpina using the ATMT method The spore suspension from the M. alpina S14 ura5 strain was prepared as mentioned at the description of microprojectile bombardment method. Transformation of the M. alpina S14 ura5 strain with pBIG35ZhSSA2psdd17m was performed using the Agrobacterium tumefaciens-mediated transformation (ATMT) method described previously (20) with slight modification. Initially, A. tumefaciens C58C1 was transformed with each vector via electroporation as
COOH 16:0
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FIG. 1. A biosynthetic pathway of polyunsaturated fatty acids (PUFA) in the mutant strain S14 derived from Mortierella alpina 1S-4. OA, oleic acid; LA, linoleic acid; GLA, g-linolenic acid; DGLA, dihomo-g-linolenic acid; ARA, arachidonic acid; ALA, a-linolenic acid; SDA, stearidonic acid; ETA, u3 eicosatetraenoic acid; EPA, u3 eicosapentaenoic acid.
Please cite this article in press as: Okuda, T., et al., Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.014
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his p
FIG. 2. Vector constructs used for expression of (A) u3-desaturase and (B) sdd17m in M. alpina S14. u3, Mortierella alpina u3-desaturase; hisH 4.1p, M. alpina histone H4.1 promoter; trpC t, Aspergillus nidulans trpC transcription terminator; rDNA, M. alpina 1S-4 18S rDNA fragment; bla, ampicillin resistance gene; ura5, orotate phosphoribosyl transferase gene of M. alpina; SSA2 p, M. alpina SSA2 promoter; SdhB t, M. alpina SdhB transcription terminator; Sdd17m, codon-optimized D17 fatty acid desaturase gene from S. diclina; NPTIII, neomycin phosphotransferase III gene; TrfA, TrfA locus, which produces 2 proteins that promote replication of the plasmid; ColEI ori, ColEI origin of replication; oriV, pRK2 origin of replication; RB, right border; LB, left border.
described previously (31) and its transformants were isolated on LB-Mg agar plates supplemented with kanamycin (20 mg/mL), ampicillin (50 mg/mL) and rifampicin (50 mg/mL). A. tumefaciens transformants were cultivated in 100 mL of MM supplemented with kanamycin (20 mg/mL) and ampicillin (50 mg/mL) at 28 C for 48 h with shaking (120 rpm). Bacterial cells were harvested by centrifugation at 8000 g, washed once with fresh IM, and then diluted to an optical density of 660 nm (OD660) of 0.1e0.2 in 10 mL of fresh IM. After pre-incubation for 12e16 h at 28 C with shaking (300 rpm) to an OD660 of 1.5e2.0, 100 mL of the bacterial cell suspension was mixed with an equal volume of a spore suspension (108 spores/mL) from the M. alpina 1S-4 ura5 strain, and then spread on membranes (Whatman 50 Hardened Circles 70 mm, Whatman International Ltd., UK) which were put on cocultivation media (IM in 1.5% agar) and incubated at 23 C for 5 days. After cocultivation, the membranes were transferred to uracil-free SC agar plates that contained 0.03% Nile blue A (SigmaeAldrich Japan) to discriminate between fungal colonies and the white color of the membrane. After 2 days of incubation at 28 C, hyphae from visible fungal colonies were transferred to fresh uracil-free SC agar plates; this process was repeated 3 times to obtain candidates. Integration of the vector into the chromosome of the host strain was verified by PCR, as described previously (28).
RESULTS Isolation and characterization of uracil auxotrophs of M. alpina S14 M. alpina S14, a D5-desaturase defective mutant derived from M. alpina 1S-4, was used as the host strain for heterologous gene expression. The strain S14 accumulates a higher amount of DGLA, a precursor for ETA, than does the wildtype strain 1S-4. To develop a transformation system using M. alpina S14, we obtained 4 uracil auxotrophic S14 mutants (S14-1, 2, 3, and 6) that grow on the SC medium with uracil, but not on uracil-free SC medium (Fig. 3A). We evaluated these S14 uracil auxotrophs as a host strain for molecular breeding based on growth and fatty acid production and composition. As a result, we found that all 4 uracil auxotrophs showed vigorous growth and as much fatty acid production and composition as the wild-type strain S14 (data not shown). To assess whether the homologous ura5 gene was suitable as a selective marker for uracil auxotrophic mutants, the ura5 gene in the genome of these mutants was sequenced (26). As shown in Fig. 3B, in case of a mutant S14-2, the mutation of base substitution was observed in the ura5 gene: the substitution of G for A and G for T were observed at the þ211 and þ212 nucleotide positions, leading to amino acid replacement, G 71 I. A base-pair change was detected in that of S14-3: the substitution of A to G at þ1 caused the deficiency of start codon. S14-1 and -6 were found to have no mutations point in their ura5 gene. Consequently, the strain S14-2 was used as a host strain for transformation in subsequent studies because of the evident multiple mutational points in its ura5 gene.
Transformation of the M. alpina S14 uracil auxotroph with pSDura5u332 and fatty acid analysis We performed transformation of the M. alpina S14 uracil auxotroph with pSDura5u32 using the microprojectile bombardment method and selected one stable transformant (u3#1). Subsequently, we evaluated ETA production of the 1S-4 wild-type strain, S14 host strain, and the transformant u3#1 during cultivation at 12 C (Fig. 4). The amount of ETA in u3#1 remarkably increased with the elapse of cultivation time compared to wild-type 1S-4 and host S14. The ETA contents of u3#1 reached 42.1% in the total fatty acids, while those of wild-type 1S-4 and S14 were just 0.5% and 13.1%: ETA content of u3#1 was up to 84.2-fold and 3.2-fold higher compared to wild-type 1S-4 and S14, respectively. ALA and SDA
(A) wild
wild
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FIG. 3. Characterization of uracil auxotrophic strains of M. alpina S14. (A) Growth of M. alpina 1S-4, S14 and uracil auxotrophic S14 on SC medium with or without uracil. (B) A mutation site of the ura5 gene in M. alpina uracil auxotrophic S14.
Please cite this article in press as: Okuda, T., et al., Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.014
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FIG. 4. Time course of growth, fatty acid production, and composition of the M. alpina 1S-4 wild-type strain, S14 host strain, and its transformant u3#1 overexpressing u3desaturase gene. All strains were cultivated in 10 mL of the GY liquid medium at 12 C for 5, 7, 9, and 11 days. The data are shown as mean SD from 3 individual experiments. For all other abbreviations, see the legend of Fig. 1.
contents of u3#1 were up to 3.7% and 14.2% of total fatty acid on Day 5. In contrast, no accumulation of u3-PUFAs including ETA was observed in u3#1 when cultivated at 28 C (data not shown). Transformation of the M. alpina S14 uracil auxotroph with pBIG35Zhsdd17m and fatty acid analysis In order to produce ETA in M. alpina S14 at a normal temperature, we used the S. diclina fatty acid D17-desaturase gene (sdd17m). S. diclina is an oleaginous microorganism producing u3-PUFAs at a normal temperature, and Sdd17 is able to catalyze a desaturation at u3 position of DGLA resulting in ETA biosynthesis (32) as well as endogenous u3 desaturase in M. alpina. The expression vector pBIG35ZhSSA2psdd17m (Fig. 2), carrying the codon-optimized sdd17 gene (sdd17m), was constructed and introduced into the M. alpina S14 uracil auxotroph using the ATMT method. As a result, five stable transformants were randomly selected and used for further studies. The selected transformants and the host strain S14 were cultivated in GY medium at 28 C, and the time course of their fatty acid production and composition were analyzed (Fig. 5). All transformants showed accumulation of ETA, which was not detected in the host strain S14 at normal temperature. Transformant #7 exhibited the highest ETA content (24.9% of total fatty acids on day 10) among all transformants. The trace amount of ALA and SDA was detected in transformants. DISCUSSION The oleaginous filamentous fungus M. alpina 1S-4 is known as an industrial strain that produces arachidonic acid (ARA) commercially (19). Up until now, we have succeeded in the industrial production of PUFAs using M. alpina 1S-4 and its mutant strains (21,22). This strain is also known to accumulate considerable
amounts of EPA, when cultivated at low temperatures, which cause the expression of the endogenous u3-desaturase gene and increase the activity of the gene product (33). Based on these observations, we hypothesized that M. alpina has a potential of u3-PUFAs accumulation and employed this fungus as a host strain to produce a rare u3-PUFA: ETA. M. alpina S14 is a D5-desaturation activity-defective mutant (23) which was derived from M. alpina 1S-4 by treating parental spores with a chemical mutagen, N-methyl-N0 -nitro-N-nitrosoguanidine (MNNG), as described previously (34). M. alpina S14 also exhibits a higher accumulation of DGLA, a precursor of ETA, than strain 1S-4 (24). Therefore, we surmised that this strain would be suitable for evaluation of endogenous u3- and heterologous D17-desaturase gene expression, which both can be converted to DGLA to ETA. First, we succeeded in obtaining some ura5 mutants derived from the strain S14, and selected S14-2 as the host strain for transformation. We performed transformation with the uracil auxotroph, strain S14-2, with the endogenous u3-desaturase gene, and observed the improvement of fatty acid composition of transformants, which showed considerable production of ETA (Fig. 4). Other rare u3-PUFAs such as SDA were also accumulated in the transformants. This result indicates that it is possible to obtain some transformants producing other unique PUFAs by constructing a transformation system with other useful mutants derived from M. alpina 1S-4. Thus, we demonstrated potency of M. alpina as an ETA source via overexpression of the endogenous u3-desaturase gene. For industrial production, however, low-temperature cultivation is not suitable because of its high running cost compared to normaltemperature cultivation. Therefore, we subsequently tested the transformation with sdd17m gene which enables the strain to produce u3-PUFAs at a normal room temperature. For the introduction of the sdd17m gene, we used the ATMT method (20) instead
Please cite this article in press as: Okuda, T., et al., Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.014
OMEGA-3 EICOSATETRAENOIC ACID PRODUCTION
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Other fatty acids Saturated fatty acids OA LA GLA DGLA ARA ETA
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FIG. 5. Time course of growth, fatty acid production, and composition of the M. alpina 1S-4 wild-type strain, S14 host strain and its transformants expressing the sdd17m gene. All strains were cultivated in 4 mL of the GY liquid medium at 28 C for 3, 7, and 10 days. The data are shown as mean SD from 3 individual experiments. For other all abbreviations, see the legend of Fig. 1.
of the microprojectile bombardment method which often causes undesired mutations in host cells and results in unstable transformants because of the physical damage by high pressure employed to transfer plasmid DNA, and random multiple integrations of plasmids into the chromosomal DNA. The ATMT method results in a single-copy integration of T-DNA at a random location in chromosomal DNA and the increased transformation frequency under mild conditions for M. alpina cells. Moreover, application of stronger promoters, not the conventional histone promoter, is expected to lead to the further improvements in ETA production. We assessed the transformation with sdd17m gene controlled by a strong promoter SSA2 p into uracil auxotrophic S14 using the ATMT method, in order to produce ETA at normal temperature. As a result, we observed that some transformants exhibited ETA production at 28 C (Fig. 5), and confirmed that the Sdd17 protein was functional in M. alpina S14. On the other hand, we found that the activity of Sdd17 to convert DGLA to ETA might be lower than that of ARA to EPA because residual accumulation of DGLA was observed in the transformants while efficient conversion of ARA to EPA was reported (32). It was also suggested that the D15-desaturation activity of Sdd17 for C18 substrates is extremely low because ALA and SDA accumulation were hardly detected in transformants (Fig. 5). Additional expression of heterologous D15-desaturase and endogenous u3-desaturase might lead to enhanced ETA biosynthesis and productivity. In addition, this study is the first report to show that the SSA2 promoter can be used for modification of the PUFA production in M. alpina. Recently, several useful promoters of M. alpina have been newly identified. By applying them, further efficient production of u3-PUFAs might be possible using M. alpina. As mentioned in the introduction section, there are few reports of ETA production in the recent past. This report demonstrates the potency of M. alpina as a practical source of ETA. Establishment of a
stable supply of ETA by using M. alpina will lead to subsequent studies of physiological function of ETA and may result in expansion of its applications in medical and pharmaceutical fields. ACKNOWLEDGMENTS This work was partially supported by Institute for Fermentation, Osaka, Japan (IFO) and the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN) to J.O.; Advanced Low Carbon Technology Research and Development Program (ALCA) to E.S.; and Grants-in-Aid for Scientific Researches (no. 22380051) from Ministry of Education, Culture, Sports, Science, and Technology, Japan to E.S. References 1. Horrobin, D. F.: Nutritional and medical importance of g-linolenic acid, Prog. Lipid. Res., 31, 163e194 (1992). 2. Romieu, I., Tellez-Rojo, M. M., Lazo, M., Manzano-Patino, A., CortezLugo, M., Julien, P., Belanger, M. C., Hernandez-Avila, M., and Holguin, F.: Omega-3 fatty acid prevents heart rate variability reductions associated with particulate matter, Am. J. Respir. Crit. Care Med., 172, 1534e1540 (2005). 3. von Schacky, C.: Cardiovascular disease prevention and treatment, Prostaglandins Leukot. Essent. Fatty Acids, 81, 193e198 (2009). 4. Simopoulos, A. P.: The importance of the ratio of omega-6/omega-3 essential fatty acids, Biomed. Pharmacother., 56, 365e379 (2002). 5. Balk, E. M., Lichtenstein, A. H., Chung, M., Kupelnick, B., Chew, P., and Lau, J.: Effects of omega-3 fatty acids on serum markers of cardiovascular disease risk: a systematic review, Atherosclerosis, 189, 19e30 (2006). 6. Schacky, C. v and Harris, W. S.: Cardiovascular benefits of omega-3 fatty acids, Cardiovasc. Res., 73, 310e315 (2007). 7. Yates, C. M., Calder, P. C., and Ed Rainger, G.: Pharmacology and therapeutics of omega-3 polyunsaturated fatty acids in chronic inflammatory disease, Pharmacol. Ther., 141, 272e282 (2014). 8. Lucas, M., Mirzaei, F., O’Reilly, E. J., Pan, A., Willett, W. C., Kawachi, I., Koenen, K., and Ascherio, A.: Dietary intake of n-3 and n-6 fatty acids and the
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Please cite this article in press as: Okuda, T., et al., Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.014
