Heptadecanoic acid c17 0

Fatty acids were extracted as described by Tsen et al.⁴² , adopted from the classic Folch method^{43 (link)},using chloroform-methanol (2:1, volume to volume (v/v)) containing 0.01% butylated hydroxytoluene (Sigma-Aldrich, Oakville, Ontario, Canada), followed by methylation with methanolic HCl. Fatty acid methyl esters were then analyzed using an Agilent 6890 N (Agilent Technologies, Mississauga, ON, Canada) gas chromatograph equipped with a flame ionization detector. During the extraction and methylation, heptadecanoic acid (C17:0) was used as an internal standard (Sigma-Aldrich, Oakville, Ontario, Canada). Known fatty acid standards (Sigma-Aldrich, Oakville, Ontario, Canada) were used to identify the individual fatty acids in wheat samples. The level of each fatty acid was then calculated according to the corresponding peak area relative to the total area of total interested fatty acids, and considered as a percentage of the total fatty acids.

Lyophilized algal cells were grinded with liquid nitrogen, and total lipids were extracted by a solvent mixture of chloroform/methanol/0.75% aqueous NaCl solution (2:1:0.75, by volume). The chloroform layer containing lipid was extracted for lipid analysis. For total lipid quantification, lipid in chloroform was directly transesterificated with 1% (v/v) sulfuric acid in methanol at 85 °C for 2 h, and analyzed using gas chromatography–mass spectrometry (GC–MS) equipped with a DB-WAX capillary column (30 m × 0.25 mm × 0.25 μm) (Agilent, CA, USA). Heptadecanoic acid (C17:0, Sigma-Aldrich) was used as the internal standard. Helium was used as the carrier gas with the flow rate of 1.2 mL/min. The ion temperature and interface temperature were 200 °C and 240 °C, respectively. Samples were injected in split mode (5:1 split ratio) at an oven temperature of 45 °C with an injection volume of 1 μL. The oven temperature was raised to 150 °C at a rate of 15 °C min⁻¹, then to 240 °C at a rate of 6 °C min⁻¹ and held for 6 min. For TAG quantification, TAG was separated on a Silica gel 60 TLC plate (EMD Chemicals, Merck, Germany) using a mixture of hexane/tert-butylmethyl ether/acetic acid (80/20/2, by volume) as the mobile phase, then transesterificated and analyzed using above method.

To test whether MiPDAT had a substrate preference, LA, ALA, GLA, and ArA, the main FAs in M. incisa17 , were selected in this feeding experiment. The transgenic yeast was incubated with 200 μM FAs and 0.01% (v/v) tyloxapol (Sigma-Aldrich, Germany) as a surfactant in the induction medium described above and harvested at an optical density of 3.5. Approximately 150 mg of lyophilized yeast cells were used for lipid extraction and TLC analysis as described above. The recovery of TAG-containing fractions and the preparation of FA methyl esters (FAMEs) of the recovered TAG were according to the previously described method23 . Fifty micrograms of heptadecanoic acid (C17:0, Sigma-Aldrich, USA) was added as an internal standard.
GC-MS analysis was performed with an Agilent 7890-5975b system (Agilent Technologies, USA). The detailed GC-MS conditions are provided in the Supplemental Methods. The mole number of FAMEs was calculated with the peak area of the internal standard.

The total lipids were extracted from the 10 mg of lyophilized biomass with a chloroform-methanol (2:1 v/v) solvent mixture (Merck, Darmstadt, Germany) using a procedure similar to the Folch’s method [34 (link)]. Fatty acid methyl esters (FAMEs) were produced from the extracted lipid by a transesterification reaction. Briefly, methanol was added to the extracted lipid with sulfuric acid as a catalyst and a transesterification reaction was allowed to occur at 100°C for 10 minutes. After the reaction, 1 ml of deionized water was added and the organic phase was separated from water phase by centrifugation at 4000 rpm for 10 minutes. A total of 1 ml of chloroform containing 0.5 mg of heptadecanoic acid (C17:0; Sigma Aldrich, St. Louis, MO, USA) was added to each tube as an internal standard. The FAMEs in organic phase were analyzed by gas chromatography (HP5890, Agilent, Santa Clara, CA, USA) with a flame ionized detector (FID) and INNOWAX capillary column (Agilent, Santa Clara, United States, 30 m × 0.32 mm × 0.5 μm).

The resultant FAMEs were analyzed by gas chromatography using GC–2010 gas chromatograph (Shimadzu, Japan) equipped with a flame ionization (FID) and a split injection system (ratio of 1:30) and fitted with a capillary column (30m x 0.32mm inner diameter, 0.25μm film; Omegawax–320, Supelco Co., EUA). The injector and detector were operated at 260° and 280°C, respectively. The oven temperature was held at 40°C for 3min, temperature programmed at 2.5°C/min to 180°C, then temperature was programmed at 2.0°C/min to 210°C and then held for 25min. Helium was used as a carrier gas and column pressure was set to attain a carrier gas speed of 25.0cm/s.
Gas-chromatographic peaks of FAME of the samples were identified by comparison of the retention time data with that of standards. Heptadecanoic acid (C_17:0; Sigma Chemical Co.) was used as an internal standard for quantification.

Cellular fatty acids were extracted by incubating 10 mg of dried seeds of control and transformed plants or 50 mg of yeast powder and freeze-dried algae powder in 3 mL of 7.5% (w/v) KOH in methanol for saponification at 70 °C for 4 h. After the pH was adjusted to 2.0 with HCl, the fatty acid were subjected to methylesterification with 2 mL of 14% (w/v) boron trifluoride in methanol at 70 °C for 1.5 h. A phase separation was produced by adding 1 mL of 0.9% (w/v) NaCl and 4 mL of hexane. The upper phase was dried under a nitrogen gas flow and resuspended in 0.3 mL of acetic ether prior to GC analysis. An analysis of fatty acid methyl esters (FAME) was performed by GC-MS (gas chromatography–mass spectrometry, TurboMass, PerkinElmer, USA) equipped with a capillary column (BPX-70, 30 m × 0.25 mm × 0.25 μm). Hydrogen was used as the carrier gas at a flow rate of 1.0 mL/min. The injector and detector temperatures were held at 250 °C. The column oven was temperature-programmed from 100 to 190 °C at 15 °C/min, where the temperature was held for 1 min increased to 220 °C at 10 °C/min, and then held for 4 min. The total FA content was quantified using heptadecanoic acid (C17:0) (Sigma) as an internal standard added to samples prior to extraction.