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. 2007 Sep 14;73(21):6965–6971. doi: 10.1128/AEM.01008-07

Heterologous Production of Dihomo-γ-Linolenic Acid in Saccharomyces cerevisiae▿

Hisashi Yazawa 1, Hitoshi Iwahashi 1, Yasushi Kamisaka 1, Kazuyoshi Kimura 1, Tsunehiro Aki 2, Kazuhisa Ono 2, Hiroshi Uemura 1,*
PMCID: PMC2074983  PMID: 17873077

Abstract

To make dihomo-γ-linolenic acid (DGLA) (20:3n-6) in Saccharomyces cerevisiae, we introduced Kluyveromyces lactis Δ12 fatty acid desaturase, rat Δ6 fatty acid desaturase, and rat elongase genes. Because Fad2p is able to convert the endogenous oleic acid to linoleic acid, this allowed DGLA biosynthesis without the need to supply exogenous fatty acids on the media. Medium composition, cultivation temperature, and incubation time were examined to improve the yield of DGLA. Fatty acid content was increased by changing the medium from a standard synthetic dropout medium to a nitrogen-limited minimal medium (NSD). Production of DGLA was higher in the cells grown at 15°C than in those grown at 20°C, and no DGLA production was observed in the cells grown at 30°C. In NSD at 15°C, fatty acid content increased up until day 7 and decreased after day 10. When the cells were grown in NSD for 7 days at 15°C, the yield of DGLA reached 2.19 μg/mg of cells (dry weight) and the composition of DGLA to total fatty acids was 2.74%. To our knowledge, this is the first report describing the production of polyunsaturated fatty acids in S. cerevisiae without supplying the exogenous fatty acids.

The polyunsaturated fatty acids (PUFAs) have been attracting considerable interest because of their important roles in human health and nutrition (3, 6, 11, 23, 25). Many PUFAs are essential and must be obtained from the diet, because mammals, including humans, cannot synthesize the so-called essential fatty acids, linoleic acid (LA) (18:2n-6) and α-linolenic acid (ALA) (18:3n-3).

Recently, a C20 PUFA with three double bonds, dihomo-γ-linolenic acid (DGLA) (20:3n-6), has attracted great interest because it has unique biological activities in addition to its importance as a precursor of a large family of anti-inflammatory eicosanoids, such as series 1 prostaglandins and thromboxanes (6, 9, 12, 17, 18, 20, 34, 39, 41, 45). C20 fatty acids such as DGLA (20:3n-6) are synthesized by sequential desaturation and elongation of dietary LA (18:2n-6) (3, 14, 24, 33). PUFA synthesis in mammals proceeds predominantly by a Δ6 desaturation pathway, in which the first step is the Δ6 desaturation of LA and ALA to yield γ-linolenic acid (GLA) (18:3n-6) and stearidonic acid (18:4n-3), respectively. Further fatty acid elongation and desaturation steps give rise to arachidonic acid (AA) (20:4n-6) via DGLA and eicosapentaenoic acid (EPA) (20:5n-3) via eicosatetraenoic acid (20:4n-3) (Fig. 1).

FIG. 1.

FIG. 1.

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Biosynthetic route for DGLA. The presumptive route for the formation of 18:3n-4 is indicated by dotted lines.

As the demand for these beneficial PUFAs has increased drastically in recent years, alternative sources of PUFAs that are more economical and sustainable become desirable. To reconstitute the long-chain PUFA biosynthetic pathways in a heterologous host, genes encoding Δ6 desaturase and elongase have been cloned from a variety of organisms, including higher plants, algae, mosses, fungi, nematodes, and humans (1, 2, 14, 15, 28, 30, 43, 46). One option is to modify the oilseed crops to produce PUFAs through genetic engineering techniques, and the other option is to produce them in well-studied microorganisms, such as Saccharomyces cerevisiae. Since S. cerevisiae has served as a model organism for the development of metabolic engineering strategies to produce certain metabolites (27), the concept of obtaining PUFAs from S. cerevisiae in commercial and sustainable quantities is particularly attractive.

The primary products of fatty acid biosynthesis in most organisms are 16- and 18-carbon compounds, and fatty acid desaturation is initiated by introducing a double bond at the Δ9 position of saturated fatty acids palmitic (16:0) and stearic (18:0) acids. S. cerevisiae contains a Δ9 desaturase (OLE1) capable of producing monounsaturated palmitoleic (16:1) and oleic (18:1) acids (40). In some organisms, the Δ9-unsaturated C18 fatty acid (oleic acid, 18:1) is subsequently desaturated to LA (18:2) by the introduction of a second double bond at the Δ12 position by Δ12 fatty acid desaturase (Fig. 1); however, S. cerevisiae is not capable of producing such a further unsaturated fatty acid.

Therefore, there is a need to genetically engineer the capacity to synthesize PUFAs in S. cerevisiae. Δ12 fatty acid desaturase genes, known as FAD2 genes, have been reported mainly from fungi and plants (4, 13, 26, 29, 35, 44). Very recently, based on the draft genome sequence of Kluyveromyces lactis, we also cloned Δ12 fatty acid desaturase from K. lactis (KlFAD2) and confirmed its Δ12 desaturase activity in S. cerevisiae (21).

Many groups have engineered production of PUFAs in S. cerevisiae (2, 7, 8), but since S. cerevisiae does not produce LA (18:2), this has required supplying fatty acids in the medium. Taking advantage of Δ12 fatty acid desaturase KlFAD2, we first arranged for endogenous production of n-6 PUFAs and then, additionally introducing Δ6 fatty acid desaturase and elongase genes, obtained production of GLA and DGLA. In this study, we report the influence of medium composition, temperature, and incubation time on production of PUFAs.

MATERIALS AND METHODS

Strains, media, and growth.

Plasmid manipulations were carried out using Escherichia coli DH5α (F− endA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 (ΔlacU169 φ80dlacZΔM15) grown in Luria-Bertani broth, supplemented with ampicillin. The S. cerevisiae strain YHU3046-4A (MATa leu2-3 leu2-112 ura3-52 his3::kamMX3) (21) was used for the transformation of plasmids containing KlFAD2, rat Δ6 fatty acid desaturase, and rat elongase (rELO1) genes. S. cerevisiae strains were grown in synthetic minimal medium, synthetic complete medium (SC), or synthetic complete dropout medium (37), depending on the selective pressure required to maintain the plasmids. Synthetic minimal medium or SC contained 0.17% (wt/vol) Bactoyeast nitrogen base without amino acids or ammonium sulfate (Difco), 2% (wt/vol) glucose, and 0.5% (wt/vol) ammonium sulfate. In nitrogen-limited minimal medium (NSD), the amount of ammonium sulfate was reduced to 0.1% and the concentration of glucose was increased to 10%.

Yeast cells were transformed by the method of Ito et al. (16). For the assay of fatty acids, YHU3046-4A transformants were precultured overnight at 30°C in SC and the resultant cultures were inoculated into 100 ml of SC or NSD at an optical density at 600 nm (OD600) of 0.2 in 300-ml Erlenmeyer flasks and reciprocally shaken at specified temperatures. Growth in liquid media was monitored by measuring turbidity of the cells at OD600.

Plasmid construction.

Standard techniques of DNA manipulation used in this study are described by Sambrook and Russell (36). KlFAD2 was expressed on a multicopy plasmid with a LEU2 marker under the control of the ADH1 promoter of S. cerevisiae. pL2199-1 contains the entire coding region of KlFAD2 under the ADH1 promoter (21). pL2308-7 was constructed from pL2199-1 by removing the (underlined) 41-bp HindIII-XbaI multiple cloning sequence (AAGCTTCCCGGGAATTCGTCGACTGGGATCCCTCGAGGAGCTCAGTCTAGA) of pL2199-1 to shorten the distance between the ADH1 promoter and the initiation site of KlFAD2. pL1177-2, a derivative of pVT100-U (42), is a corresponding empty vector with the ADH1 promoter and a LEU2 marker (21).

The rat Δ6 fatty acid desaturase gene was expressed under the control of the TDH3 promoter of S. cerevisiae on YEp352-GAP, a YEp352 (10) derivative that contains the promoter and the terminator of TDH3 (kindly provided by Takehiko Yokoo, AIST). The entire coding region of the rat Δ6 fatty acid desaturase gene was excised from its original plasmid (1) and inserted at the EcoRI cloning site of YEp352-GAP to construct pL2313-2. To make truncated Δ6 fatty acid desaturase genes, the original clone was digested with BstEII (located in codon 5) plus EcoNI (located 25 bp downstream of the termination codon), Ecl136II (located in codon 13) plus EcoNI, or FspI (located in codon 32) plus EcoNI, and the fragments were cloned at the PvuII, XhoI, or PvuII site of YEp352-GAP after blunting the restriction sites to construct pL2315-6, pL2314-136, or pL2316-7, respectively. Since the BstEII site is located in the fifth codon of the Δ6 desaturase gene, pL2315-6 made an in-frame fusion of the initiation codon of the TDH3 vector and the fifth codon of the Δ6 desaturase; the first 4 amino acids (aa) of the desaturase (MGKG) were replaced with the 6 artificial amino acids (MGTARA) derived from the cloning site. Likewise, pL2314-136 made an in-frame fusion in which the first 13 aa of the desaturase (MGKGGNQGEGSTE) were replaced with the 5 artificial amino acids (MGTAR) derived from the cloning site. In pL2316-7, the first 32 aa of the desaturase (MGKGGNQGEGSTELQAPMPTFRWEEIQKHNLR) were replaced with the 7 artificial amino acids (MGTARAG) derived from the cloning site.

rELO1 was cloned in both YEp and YCp plasmids. First, rELO1 was excised from pYES2/rELO1 (15) as a HindIII-XbaI fragment, and it was cloned at the HindIII-XbaI multiple cloning site of pL1177-2 (LEU2) to construct pL2118-1. Then, the entire expression unit, including the ADH1 promoter and terminator, was excised from pL2118-1 as an SphI fragment and it was cloned at the SmaI site of pRS313 (a single-copy plasmid with a HIS3 marker [38]) to construct pL2303-8.

Fatty acid analysis.

Total fatty acid contents were determined by gas chromatographic analysis as described previously (21). Fatty acid analysis was performed by the application of 100-μl aliquots to a gas chromatograph (GC2010; Shimadzu, Japan) equipped with a TC-70 capillary column (30-m by 0.25-mm inside diameter; GL Sciences, Japan) under temperature programming from 180 to 220°C at 4°C/min increments unless otherwise specified. Fatty acid composition was calculated based on the area of each peak, and the amount was determined by comparison with the methylheptadecanoate standard.

Gas chromatography-mass spectrometry (GC-MS) analysis of the fatty acid methyl esters was performed using a GCMS-QP5050A (Shimadzu, Japan) mass spectrometer linked to a gas chromatograph (GC-17A; Shimadzu) equipped with a DB-WAX column (30-m by 0.32-mm inside diameter; J&W Scientific, Inc., CA) as the sample inlet and operated in the electron impact mode at 70 eV.

RESULTS AND DISCUSSION

Construction of desaturase and elongase genes for expression in S. cerevisiae.

In S. cerevisiae monounsaturated fatty acids, palmitoleic (16:1) and oleic (18:1) acids are the major fatty acids, and no PUFAs are produced. Production of LA (18:2) from oleic acid (18:1) is catalyzed by a Δ12 fatty acid desaturase. We have recently isolated the Δ12 fatty acid desaturase gene from K. lactis (KlFAD2, pL2199-1) and confirmed its activity in S. cerevisiae (21). In our previous paper (21), we expressed it under the control of the ADH1 promoter, but the yield of LA was low and the amount of endogenous LA was 1.04 μg/mg of cells (dry weight) (DCW) (0.9% of total fatty acids) when the cells were grown at 15°C in NSD for 5 days. Since Δ12 desaturation is the first step for the production of PUFAs, high activity is important to obtain a good yield of final products. To improve the expression level of KlFAD2, we constructed pL2308-7 by removing the 41-bp HindIII-XbaI multiple cloning site of pL2199-1 to shorten the distance between the ADH1 promoter and the KlFAD2 initiation site. YHU3046-4A containing pL2308-7 produced 30.4 μg of LA per mg of cells (dry weight) (27.7% composition of total fatty acids) when the cells were grown at 15°C in NSD for 5 days, a 30-fold improvement in yield.

The rat Δ6 fatty acid desaturase gene was expressed under the control of the TDH3 promoter with a URA3 marker (pL2314-136). We constructed several plasmids containing different parts of the Δ6 desaturase. pL2313-2 (aa 1 to 444) contains the entire coding region. In pL2315-6 (aa 5 to 444), pL2314-136 (aa 14 to 444), and pL2316-7 (aa 33 to 444), the first 4, 13, and 32 aa of the desaturase, respectively, were replaced with short artificial amino acids derived from the cloning site. These plasmids were transformed into YHU3046-4A containing pL2308-7 (KlFAD2), and the Δ6 desaturase activity was examined. The desaturase activity (conversion rate from LA to GLA) of pL2315-6 (aa 5 to 444) was almost the same as that of pL2313-2 (aa 1 to 444), but the desaturase activity of pL2314-136 (aa 14 to 444) was 1.35-fold higher than that of pL2313-2 (aa 1 to 444). These results fit with the report that the first 17 aa of Δ6 desaturase are not required for its activity, at least in S. cerevisiae (1). On the other hand, the truncation of the first 32 aa (pL2316-7 [aa 33 to 444]) abolished the desaturase activity. Thus, pL2314-136 (aa 14 to 444) was used as the Δ6 desaturase for our study.

rELO1 was expressed on a single-copy plasmid with a HIS3 marker under the control of the ADH1 promoter of S. cerevisiae (pL2303-8). Rat has two elongase genes (rELO1 and rELO2), and rELO1 preferably catalyzes the elongation of mono- and polyunsaturated fatty acids of C16 to C20. Inagaki et al. (15) reported that overexpression of rELO1 from the GAL1 promoter in S. cerevisiae produced substantial amounts (26.5% of total fatty acids) of 18:1n-7 from 16:1n-7. Similarly, with overexpression of rELO1 on a multicopy plasmid from the ADH1 promoter (pL2118-1), the amount of 18:1n-7, an undesired end product, reached 26.4% of total fatty acids when the cells were grown in NSD for 5 days at 20°C. However, with rELO1 on a single-copy plasmid from the ADH1 promoter (pL2303-8), no 18:1n-7 was produced; in addition, using exogenously added GLA, a 17.4% conversion to DGLA was obtained.

Triple expression of KlFAD2, rat Δ6 fatty acid desaturase, and rELO1 genes in S. cerevisiae.

As described in the previous section, a single-copy HIS3 plasmid with rELO1 under the control of the ADH1 promoter (pL2303-8) was introduced into YHU3046-4A harboring a LEU2 plasmid with KlFAD2 under the control of the ADH1 promoter (pL2308-7) and a URA3 plasmid with the rat Δ6 fatty acid desaturase gene under the control of the TDH3 promoter (pL2314-136). The transformants were grown on synthetic dropout plates for the selection of respective plasmids. To improve productivity, we investigated several factors: medium, temperature, and incubation time.

The triple transformants of YHU3046-4A or the empty vector controls were grown at 15°C, 20°C, or 30°C for 10 days in SC Leu-His-Ura dropout medium with 2% glucose or NSD containing 10% glucose and 0.1% ammonium sulfate, and their fatty acid compositions were examined. NSD was examined because the carbon/nitrogen ratio affects the amount of lipids in the cell (22, 32). Prior to this experiment, we preliminarily examined the effect of carbon sources that result in oxidative metabolism, as PUFA production requires an active cytochrome system. Two percent pyruvate or two percent each of glycerol and lactate was used instead of glucose, but contrary to our expectation, levels of PUFA did not increase. Furthermore, one problem is that growth of S. cerevisiae on respiratory substrates is so poor that there likely would not be a gain in actual production. Thus, we focused on media with glucose as a carbon source. Figure 2A and B show the changes in OD600 and DCW per ml culture of triple transformants grown in SC or NSD at 15°C, 20°C, and 30°C. Except for the cells grown in SC at 15°C and 20°C, the OD and DCW levels remained at almost the same levels during the course of cultivation from day 3 to day 10, and the change in OD was almost parallel to that in DCW. At all temperatures, absolute amounts of fatty acid per ml of culture and contents of total fatty acids per mg of DCW were higher in cells grown in NSD than in those grown in SC at the late stage of cultivation (Fig. 2C and D). Lipid content (total fatty acids/DCW) was the highest at 30°C in NSD (Fig. 2D).

FIG. 2.

FIG. 2.

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Effects of medium, temperature, and cultivation time on cell growth and fatty acid production. YHU3046-4 strains harboring pL2308-7 (KlFAD2, a LEU2 marker), pL2314-136 (rat Δ6 fatty acid desaturase, a URA3 marker), and pL2303-8 (rELO1, a HIS3 marker) were grown in SC or NSD at 15°C, 20°C, and 30°C. Growth (A), DCW per ml culture (B), amount of total fatty acids (FA) per ml culture (C), and amount of total FA per DCW (D) are depicted.

Gas chromatographic analysis of total fatty acids in S. cerevisiae overexpressing the three genes at 15°C showed specific peaks of retention time at 4.33, 5.42, 5.77, and 7.45 min, whereas the cells harboring the empty vectors did not show these peaks (compare Fig. 3A and B). Comparison of the retention times of the newly yielded peaks with those of standards has assigned the peaks to Δ9,12-hexadecadienoic acid (16:2), LA (18:2n-6), GLA (18:3n-6), and DGLA (20:3n-6). Identification of GLA and DGLA was positively supported by definitive assignments of the compounds by GC-MS analyses. These compounds showed mass spectra identical to those of authentic standards (data not shown).

FIG. 3.

FIG. 3.

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Gas chromatogram of total fatty acids in S. cerevisiae overexpressing KlFAD2, rat Δ6 fatty acid desaturase, and rELO1 genes. YHU3046-4 strains harboring pL2308-7 (KlFAD2, a LEU2 marker), pL2314-136 (rat Δ6 fatty acid desaturase, a URA3 marker), and pL2303-8 (rELO1, a HIS3 marker) (A) and YHU3046-4 strains harboring empty vector plasmids pL1177-2 (a LEU2 marker), YEp352-GAP (a URA3 marker), and pRS313 (a HIS3 marker) (B) were grown at 15°C for 7 days in NSD without leucine, histidine, and uracil. In panel C, the upper line shows a gas chromatogram of the same sample as shown in panel A, but the components were separated under temperature programming at 0.25°C/min increments (180 to 220°C). The lower line indicates a gas chromatogram of an authentic 18:2, 18:3n-6, 18:3n-3, and 20:3n-6 mixture. 16:0, palmitic acid; 16:1, palmitoleic acid; 16:2, hexadecadaenoic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, LA; 18:3n-6, GLA; 20:3n-6, DGLA. Int., methylheptadecanoate (17:0) internal standard (250 nmol). The asterisks (retention time at 5.96 min in panel A and 8.11 min in panel C) indicate the possible peak of 18:3n-4 (see text). FID, flame ionization detector.

Since the retention time at 5.96 min (Fig. 3A) was close to that of ALA (C18:3n-3, 6.05 min), we examined the retention time of this peak in more detail by reducing the temperature gradient. As shown in Fig. 3C, the retention time of the peak of interest (8.11 min) was clearly different from that of ALA (8.27 min). GC-MS analysis of this product showed that its mass spectrum was very close to, but different from, those of 18:3n-6 and 18:3n-3 (data not shown). 18:3n-4, a by-product which would be produced by the subsequent Δ12 desaturation, Δ6 desaturation, and elongation of palmitoleic acid (16:1n-7) instead of oleic acid (18:1n-9) (Fig. 1), could be the most likely candidate, although we could not conclude this because the authentic 18:3n-4 was not available commercially.

Although the fatty acid content was the highest in the cells grown in NSD at 30°C, no DGLA was produced at this temperature in NSD or SC at any stage of cultivation. This evidently reflects a low activity of KlFad2p at 30°C (Table 1, lines 10 and 12); it was also reported that S. cerevisiae cells expressing the Arabidopsis thaliana FAD2 gene accumulated a large amount of C18:2 at 15°C but not at 28°C (5). In contrast to a low KlFad2p activity (6% conversion of oleic acid to LA) in S. cerevisiae at 30°C, a relatively high desaturase activity was observed when the original K. lactis strain was grown at 30°C (63%) for 5 days in NSD; at 20°C, KlFad2p activities for S. cerevisiae and K. lactis were similar (67% and 69% conversions). The strong 30°C effect is not understood; perhaps there are unstable interactions with other components, such as cytochrome b5 (19), at the higher temperature.

TABLE 1.

Fatty acid composition of total fatty acids in S. cerevisiae overexpressing KlFAD2, rat Δ6 fatty acid desaturase, and rELO1 genesa

Line Expressed genes Medium Temp (°C) Fatty acid content in μg/mg of cells (dry wt) (%)
Fatty acid content in μg/mg of cells (dry wt) (%)
14:0 16:0 16:1 16:2 18:0 18:1 18:2 18:3n-6 18:3n-4b 20:3n-6 Total
1 Vectors SC 15 0.54 ± 0.01 (1.36 ± 0.08) 4.29 ± 0.60 (10.86 ± 1.98) 20.39 ± 1.39 (51.37 ± 1.30) 0 (0) 1.30 ± 0.16 (3.27 ± 0.27) 13.14 ± 0.75 (33.13 ± 0.49) 0 (0) 0 (0) 0 (0) 0 (0) 49.16 ± 15.13
2 Triple genes SC 15 0.61 ± 0.04 (1.22 ± 0.11) 4.82 ± 0.16 (9.62 ± 1.11) 16.27 ± 2.75 (32.16 ± 0.68) 5.83 ± 0.89 (11.54 ± 0.06) 3.38 ± 0.59 (6.69 ± 0.17) 7.15 ± 1.79 (14.04 ± 1.46) 10.05 ± 1.23 (19.94 ± 0.53) 1.22 ± 0.11 (2.42 ± 0.15) 0.65 ± 0.12 (1.32 ± 0.43) 0.54 ± 0.05 (1.06 ± 0.05) 50.52 ± 7.50
3 Vectors NSD 15 0.76 ± 0.20 (0.89 ± 0.10) 4.74 ± 0.80 (5.59 ± 0.13) 42.15 ± 6.61 (49.79 ± 0.58) 0 (0) 2.93 ± 0.33 (3.47 ± 0.11) 34.01 ± 4.36 (40.26 ± 0.70) 0 (0) 0 (0) 0 (0) 0 (0) 84.59 ± 12.29
4 Triple genes NSD 15 0.55 ± 0.12 (0.68 ± 0.14) 4.88 ± 0.04 (6.12 ± 0.24) 17.16 ± 0.49 (21.48 ± 0.06) 11.36 ± 1.02 (14.20 ± 0.84) 6.16 ± 0.40 (7.71 ± 0.26) 9.36 ± 0.23 (11.72 ± 0.07) 21.26 ± 0.43 (26.62 ± 0.29) 2.83 ± 0.00 (3.54 ± 0.11) 4.15 ± 0.09 (5.20 ± 0.27) 2.19 ± 0.10 (2.74 ± 0.21) 79.89 ± 2.47
5 Vectors SC 20 0.47 ± 0.01 (1.43 ± 0.09) 4.37 ± 0.13 (13.35 ± 0.79) 15.27 ± 1.40 (46.54 ± 0.17) 0 (0) 1.68 ± 0.01 (5.13 ± 0.48) 11.02 ± 1.36 (33.55 ± 1.19) 0 (0) 0 (0) 0 (0) 0 (0) 32.80 ± 2.89
6 Triple genes SC 20 0.57 ± 0.05 (1.48 ± 0.04) 4.41 ± 0.49 (11.42 ± 0.07) 12.92 ± 1.49 (33.43 ± 0.04) 3.72 ± 0.45 (9.62 ± 0.04) 2.43 ± 0.40 (6.27 ± 0.31) 6.67 ± 0.76 (17.25 ± 0.06) 6.90 ± 0.87 (17.83 ± 0.16) 0.51 ± 0.01 (1.32 ± 0.13) 0.24 ± 0.05 (0.62 ± 0.06) 0.29 ± 0.05 (0.76 ± 0.22) 38.65 ± 4.52
7 Vectors NSD 20 0.56 ± 0.13 (0.92 ± 0.07) 4.48 ± 0.74 (7.35 ± 0.04) 29.49 ± 4.41 (48.46 ± 0.48) 0 (0) 2.61 ± 0.32 (4.30 ± 0.17) 23.76 ± 4.11 (38.96 ± 0.54) 0 (0) 0 (0) 0 (0) 0 (0) 60.90 ± 9.71
8 Triple genes NSD 20 0.62 ± 0.11 (0.83 ± 0.01) 5.52 ± 0.77 (7.37 ± 0.41) 19.54 ± 4.22 (25.90 ± 0.55) 9.28 ± 1.07 (12.43 ± 1.01) 5.15 ± 0.69 (6.88 ± 0.42) 11.63 ± 3.06 (15.35 ± 1.06) 18.77 ± 3.52 (24.95 ± 0.20) 1.52 ± 0.44 (2.01 ± 0.19) 1.83 ± 0.54 (2.40 ± 0.25) 1.42 ± 0.28 (1.88 ± 0.01) 75.30 ± 14.70
9 Vectors SC 30 0.96 ± 0.10 (1.72 ± 0.09) 8.95 ± 0.06 (16.03 ± 0.68) 24.09 ± 0.83 (43.10 ± 0.64) 0 (0) 3.41 ± 0.17 (6.10 ± 0.01) 18.50 ± 1.60 (33.06 ± 1.23) 0 (0) 0 (0) 0 (0) 0 (0) 55.93 ± 2.76
10 Triple genes SC 30 1.11 ± 0.01 (1.79 ± 0.11) 8.46 ± 0.32 (13.63 ± 0.48) 23.97 ± 1.49 (38.59 ± 0.42) 0 (0) 3.44 ± 0.29 (5.53 ± 0.07) 24.06 ± 1.78 (38.72 ± 0.03) 1.09 ± 0.65 (1.73 ± 0.92) 0 (0) 0 (0) 0 (0) 62.13 ± 4.54
11 Vectors NSD 30 1.95 ± 0.06 (1.81 ± 0.01) 14.63 ± 0.59 (13.62 ± 0.08) 50.16 ± 1.59 (46.69 ± 0.16) 0 (0) 4.54 ± 0.01 (4.23 ± 0.16) 36.16 ± 1.54 (33.66 ± 0.25) 0 (0) 0 (0) 0 (0) 0 (0) 107.44 ± 3.77
12 Triple genes NSD 30 2.08 ± 0.17 (1.88 ± 0.02) 14.36 ± 1.04 (12.95 ± 0.04) 46.69 ± 2.36 (42.16 ± 0.78) 0 (0) 4.71 ± 0.64 (4.24 ± 0.29) 41.19 ± 3.33 (37.16 ± 0.43) 1.79 ± 0.12 (1.61 ± 0.00) 0 (0) 0 (0) 0 (0) 110.83 ± 7.66
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a

YHU3046-4A cells expressing KlFAD2, rat Δ6 fatty acid desaturase, and rELO1 genes were grown for 7 days in SC or NSD at 15°C, 20°C, and 30°C as described in Materials and Methods. YHU3046-4A harboring pL2308-7 (KlFAD2, a LEU2 marker), pL2314-136 (rat Δ6 fatty acid desaturase, a URA3 marker), and pL2303-8 (rELO1, a HIS3 marker) was used as a triple transformant. YHU3046-4A harboring pL1177-2 (a LEU2 marker), YEp352-GAP (a URA3 marker), and pRS313 (a HIS3 marker) empty vectors was used as a control strain. Values are the means ± standard deviations of two experiments carried out using the independently obtained transformants.

b

Presumptive 18:3n-4 compound.

Figure 4 shows the production profiles of DGLA and its intermediates (LA and GLA) in YHU3046-4A expressing triple genes under various culture conditions. The relative production of DGLA per DCW was highly correlated with the amounts of other intermediates, and it was higher in the cells grown in NSD. Production of DGLA was better in the growth at 15°C than that at 20°C, and the highest value was observed on day 7 (Fig. 4A and B). Table 1 shows the fatty acid compositions of total fatty acids on day 7 under all conditions. Under the best condition (15°C), the yields of GLA and DGLA reached 2.83 μg/mg and 2.19 μg/mg DCW and their compositions were 3.54% and 2.74% of total fatty acids, respectively (Table 1, line 4).

FIG. 4.

FIG. 4.

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Time course of production of DGLA and its intermediates by YHU3046-4A expressing triple genes under various conditions. The YHU3046-4A transformants were grown in SC or NSD at 15°C and 20°C for the periods indicated, and the amounts of PUFAs were measured as described in Materials and Methods. Error bars represent standard deviations. FA, fatty acids.

Conclusions.

So far, many groups have attempted to produce PUFAs in S. cerevisiae. Beaudoin et al. (2) produced AA from exogenous LA and EPA from exogenous ALA in S. cerevisiae by using Caenorhabditis elegans elongase, Mortierella alpina Δ5 fatty acid desaturase, and borage Δ6 fatty acid desaturase. With exogenous LA as a substrate, the compositions of GLA, DGLA, and AA to total fatty acids reached 6.8%, 1.4%, and 0.25%, respectively. Domergue et al. (8) produced AA from exogenous LA and EPA from ALA in S. cerevisiae by using Physcomitrella patens Δ6-specific elongase and Phaeodactylum tricornutum Δ5 and Δ6 fatty acid desaturases. The contents of DGLA and AA were 1.7% and 0.17%, respectively, by using exogenous LA. Domergue et al. (7) also produced AA from exogenous LA by using algal Δ5 and Δ6 desaturases and moss Δ6 elongase and produced DGLA from exogenous LA by using human Δ6 desaturase and moss Δ6 elongase. In all cases, exogenous LA or ALA was added for the production of n-6 or n-3 PUFAs, respectively.

For PUFA production in A. thaliana, the simple introduction of desaturase and elongase genes was sufficient, because plants are able to produce LA and ALA. Qi et al. (31) produced substantial quantities of AA (6.6%) and EPA (3%) by introducing Δ9-specific elongase, Δ8 desaturase, and Δ5 desaturase. In the present work, we have obtained DGLA synthesis from the endogenous oleic acid of S. cerevisiae by additionally introducing Δ12 fatty acid desaturase, which converts oleic acid to LA. To our knowledge, this is the first case of S. cerevisiae producing DGLA in the absence of exogenous fatty acid, with GLA and DGLA at ca. 3% of fatty acid content, comparable to the values obtained in the systems of Beaudoin et al. (2) and Domergue et al. (8). The total lipid content of S. cerevisiae is not high (22) and may have to be increased for our long-term goal of producing PUFAs by use of transgenic S. cerevisiae in sustainable quantities.

Acknowledgments

We are grateful to Yoshihiro Yamamoto and Mitsuhiro Tomosugi for the GC-MS analysis of fatty acids, Takehiko Yokoo for the YEp352-GAP plasmid, and Dan Fraenkel for comments.

Footnotes

▿

Published ahead of print on 14 September 2007.

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