Fame32

Lipids were extracted from freeze-dried cells (around 20 mg) by transmethylation described by Browse et al. [18 (link)]. The methylated FA (FAMEs) were then analyzed using gas chromatography equipped with a flame ionization detector (GC-FID, Varian 3900, Walnut Creek, CA, USA) and a Varian FactorFour vf-23 ms column where the bleed specification at 260 °C is 3 pA (30 m, 0.25 mm, 0.25 μm).
The FAMEs were identified via comparisons with commercial standards (FAME32, Supelco, Bellefonte, PA, USA) and quantified using the internal standard, 100 μg of commercial dodecanoic acid (Sigma-Aldrich, St. Louis, MO, USA). Commercial standards of OCFAs (Odd Carbon Straight Chains Kit containing 9 FAs, OC9, Supelco, Bellefonte, PA, USA,) were converted into their FAMEs using the same method employed with the yeast samples. They were then analyzed by GC to identify the OCFAs from the yeast samples. For each data point, we used three biological replicates and calculated average and standard deviation values.

Lipid content of the cells was determined by quantifying their FA fraction, as previously described [59 (link)]. Cells from-10 mL culture samples were collected in pre-weighed tubes, washed twice in water (for cultures in sugar-based medium), or twice successively in a mixture of 0.5% BSA and 0.9% NaCl and once in water (for oily medium), and resuspended in 1 mL water. After freeze-drying for 24 h at − 55 °C (Alpha 1-2Dplus, Bioblock Scientific), samples in their tubes were weighed and stored at − 20 °C. The difference in tube weights represented the mass of the cells found in 10-mL cultures and permitted calculation of the biomass in g/L.
Fatty acid methyl esters (FAMES) were recovered from 10 to 20 mg aliquots of freeze-dried cells (2 to 4 mg in samples collected at 4.5 h), using a hot methanol–H₂SO₄ method, adapted from Browse et al. [79 (link)], and analyzed by gas chromatography on a Varian 430 equipped with a flame-ionization detector and a FactorFour vf-23 ms column. FAMES were identified by comparison to commercial standards (FAME32; Supelco); an internal FA standard (100 µg C12:0 from Sigma) was added to each sample prior to transesterification to enable FA quantification in the analyzed aliquots.
Lipid content is expressed with respect to DCW: lipid content of 1% DCW = 10 mg FA per g of dry cell.

The fatty acids (FAs) in 15-mg aliquots of freeze-dried cells were converted into methyl esters using the method described in Browse et al. [34 (link), 30 (link)]. FA methyl esters were analyzed by gas chromatography (GC) on a Varian 3900 equipped with a flame ionization detector and a Varian Factor Four vf-23 ms column, for which the bleed specification at 260°C was 3 pA (30 m, 0.25 mm, 0.25 μm). FAs were identified by comparing their GC patterns to those of commercial FA methyl ester standards (FAME32; Supelco) and quantified using the internal standard method, which involved the addition of 50 mg of commercial C17:0 (Sigma). Total lipid extractions were obtained from 100-mg samples (expressed in terms of CDW, as per Folch et al. [16 ]). Briefly, yeast cells were spun down, washed with water, freeze dried, and then resuspended in a 2:1 chloroform/methanol solution and vortexed with glass beads for 20 min. The organic phase was collected and washed with 0.4 mL of 0.9% NaCl solution before being dried at 60°C overnight and weighed to quantify lipid production.