Heterologous production of dihomo-gamma-linolenic acid in Saccharomyces cerevisiae

Abstract

To make dihomo-gamma-linolenic acid (DGLA) (20:3n-6) in Saccharomyces cerevisiae, we introduced Kluyveromyces lactis Delta12 fatty acid desaturase, rat Delta6 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 degrees C than in those grown at 20 degrees C, and no DGLA production was observed in the cells grown at 30 degrees C. In NSD at 15 degrees 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 degrees C, the yield of DGLA reached 2.19 microg/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.

FIG. 1.

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

FIG. 2.

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.

FIG. 3.

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.

FIG. 4.

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.