Article Figures & Data
Figures
- FIG. 1.
Putative biosynthetic pathway of PUFAs in Mortierella alpina 1S-4. OA, oleic acid; LA, linoleic acid; ALA, α-linolenic acid; GLA, γ-linolenic acid; SDA, stearidonic acid; EDA, n-9 eicosadienoic acid; DGLA, dihomo-γ-linolenic acid; ETA, n-3 eicosatetraenoic acid; MA, Mead acid. Open and black arrows indicate elongase and desaturase reactions, respectively.
- FIG. 2.
Construction of binary vector pBIG3ura5, pBIG3ura5s, pBIG3ura5sω3x1, and pBIG3ura5sω3x2 for M. alpina 1S-4 transformation. The 1-kb M. alpina his H4.1 and his 550 promoter fragment was derived from strain CBS 528.72. The 700-bp fragment containing the trpC transcription terminator region was derived from Aspergillus nidulans. HPH, hygromycin B phosphotransferase gene; ura5, orotate phosphoribosyl transferase gene of M. alpina 1S-4; NPTIII, neomycin phosphotransferase III gene; TrfA, TrfA locus, which produces two proteins that promote replication of the plasmid; ColEI ori, ColEI origin of replication; oriV, pRK2 origin of replication; RB, right border; LB, left border; ω3, ω3-desaturase gene.
- FIG. 3.
(A) Effect of cocultivation temperature on transformation of M. alpina 1S-4. An Agrobacterium culture at an OD660 of 1.5 was used for cocultivation. The spore suspension (100 μl) and bacterial cell suspension (100 μl) were mixed and then incubated on an IM agar plate containing 200 μM of acetosyringone for 2 days. (B) Effect of cocultivation duration on transformation of M. alpina 1S-4. An Agrobacterium culture at an OD660 of 1.5 was used for cocultivation. The spore suspension (100 μl) and bacterial cell suspension (100 μl) were mixed and then incubated on an IM agar plate containing 200 μΜ of acetosyringone at 23°C. (C) Effect of concentration of A. tumefaciens cell suspension on transformation of M. alpina 1S-4. The spore suspension (100 μl) and bacterial cell suspension (100 μl) at the indicated concentrations (OD660) were mixed and then incubated on an IM agar plate containing 200 μM acetosyringone for 5 days. Other conditions were the same as described in Materials and Methods.
- FIG. 4.
Southern blot analysis of stable transformants. LB, left border; RB, right border. (A) Genomic DNA (10 μg) was digested with EcoRI and XbaI. (B) Genomic DNA (10 μg) was digested with EcoRI and SpeI. (C) Genomic DNA (10 μg) was digested with XhoI and XbaI. (D) Genomic DNA (10 μg) was digested with SpeI and XbaI. Hybridization was performed using a 0.6-kb DNA probe covering the ura5 gene. The white arrow in panel A indicates the size corresponding to an intact 2.3-kb ura5 gene cassette. The endogenous ura5 gene is indicated by black arrows in panels A, B, C, and D. Molecular size markers (kb) are indicated on the right.
- FIG. 5.
Comparison of fatty acid composition among transformants. Cultivation was performed in 4 ml GY medium with shaking at 120 strokes/min for 2 days at 28°C and for a further 16 days at 12°C. ▪, EPA; shaded bar, other n-3 PUFAs (α-linolenic acid, stearidonic acid, and n-3 eicosatetraenoic acid); (▒), AA; ▩, other n-6 PUFAs (linoleic acid; γ-linolenic acid, and dihomo-γ-linolenic acid); □, other PUFAs.
Tables
- TABLE 1.
Fatty acid production by transformants
Strain Dry cellsa (g/liter) Total fatty acidsa EPA (20:5) yield mg/g g/liter mg/g g/liter Mortierella alpina 1S-4 (wild) 10.6 177.7 1.89 10.9 0.12 pBIG3ura5s no. 1 (control) 10.2 143.8 1.47 8.2 0.08 pBIG3ura5sω3x1 no. 1 10.7 170.1 1.82 44.2 0.47 pBIG3ura5sω3x1 no. 4 10.8 133.6 1.44 37.9 0.41 pBIG3ura5sω3x1 no. 8 10.8 153.1 1.65 31.3 0.34 pBIG3ura5sω3x1 no. 10 11.2 180.8 2.02 37.4 0.42 pBIG3ura5sω3x2 no. 1 11.3 124.9 1.41 46.3 0.52 pBIG3ura5sω3x2 no. 9 11.1 137.3 1.52 45.8 0.51 pBIG3ura5sω3x2 no. 11 10.3 101.5 1.04 42.8 0.44 pBIG3ura5sω3x2 no. 13 10.9 163.5 1.78 62.8 0.68 pBIG3ura5sω3x2 no. 14 11.0 172.0 1.89 60.6 0.67 pBIG3ura5sω3x2 no. 16 11.5 169.4 1.94 57.0 0.65 ↵ a Mycelia were precultured for 2 days at 28°C and then cultured for 16 days at 12°C.
