Pentadecanoic acid

FFAs were simultaneously extracted and methylated by dichloromethane containing methyl iodide as methyl donor52 (link). Since the FFAs were secreted and cell culture formed an emulsion (Supplementary Fig. 4c), the cell culture should be mixed well before sample taking. Cell cultures from shake flask were diluted twofold with water and those from bioreactor were diluted 10-fold. Briefly, 200 μl aliquots of cell culture dilutions were taken into glass vials from 72 h incubated cultures, then 10 μl 40% tetrabutylammonium hydroxide (base catalyst) was added immediately followed by addition of 200 μl dichloromethane containing 200 mM methyl iodide as methyl donor and 100 mg l⁻¹ pentadecanoic acid as an internal standard. The mixtures were shaken for 30 min at 1,400 r.p.m. by using a vortex mixer, and then centrifuged at 5,000g to promote phase separation. A 160 μl dichloromethane layer was transferred into a GC vial with glass insert, and evaporated 4 h to dryness. The extracted methyl esters were resuspended in 160 μl hexane and then analysed by gas chromatography (Focus GC, ThermoFisher Scientific) equipped with a Zebron ZB-5MS GUARDIAN capillary column (30 m × 0.25 mm × 0.25 μm, Phenomenex) and a DSQII mass spectrometer (ThermoFisher Scientific). The GC program was as follows: initial temperature of 40 °C, hold for 2 min; ramp to 130 °C at a rate of 30 °C per minute, then raised to 280 °C at a rate of 10 °C per min and hold for 3 min. The temperature of inlet, mass transfer line and ion source were kept at 280, 300 and 230 °C, respectively. The injection volume was 1 μl. The flow rate of the carrier gas (helium) was set to 1.0 ml min⁻¹, and data were acquired at full-scan mode (50–650 m/z). Final quantification was performed using the Xcalibur software.
For alkane and fatty alcohol quantification, cell pellets were collected from 5 ml (fatty alcohol) or 10 ml (alkane) cell culture and then freeze dried for 48 h. Metabolites were extracted by 2:1 chloroform:methanol solution53 (link), which contained hexadecane (alkanes) and pentadecanol (fatty alcohols) as internal standards. The extracted fraction was dried by rotary evaporation and dissolved in hexane (alkanes) or ethyl acetate (fatty alcohols). Quantification of fatty alcohols and alkanes was performed on the same GC–MS system as used for fatty acid analysis. The GC program for alkane analysis was as follows: initial temperature of 50 °C, hold for 5 min; then ramp to 140 °C at a rate of 10 °C per min and hold for 10 min; ramp to 310 °C at a rate of 15 °C per min and hold for 7 min. The GC program for fatty alcohol quantification was as follow: initial temperature of 45 °C hold for 2.5 min; then ramp to 220 °C at a rate of 20 °C per min and hold for 2 min; ramp to 300 °C at a rate of 20 °C per min and hold for 5 min. The temperature of inlet, mass transfer line and ion source were kept at 250, 300 and 230 °C, respectively. The flow rate of the carrier gas (helium) was set at 1.0 ml min⁻¹, and data were acquired at full-scan mode (50–650 m/z). Final quantification was performed with Xcalibur software.
The extracellular glucose, ethanol and organic acid concentrations were determined by high-performance liquid chromatography analysis. To that end, a 1 ml broth sample was filtered through a 0.2 μm syringe filter and analysed on an Aminex HPX-87G column (Bio-Rad) on an Ultimate 3000 HPLC (Dionex Softron GmbH). The column was eluted with 5 mM H₂SO₄ at a flow rate of 0.6 ml min⁻¹ at 45 °C for 26 min.

Biodistribution of 15‐[p‐iodophenyl]‐3‐[R,S]‐methyl pentadecanoic acid (¹²⁵I‐BMIPP) and 2‐fluorodeoxyglucose (¹⁸F‐FDG) was determined as described previously.^{14 (link)–15 (link)} Mice received intravenous injections of ¹²⁵I‐BMIPP (5 kBq) and ¹⁸F‐FDG (100 kBq) via the lateral tail vein in a volume of 100 μL. ¹²⁵I‐BMIPP was a gift from Nihon Medi‐Physics Co Ltd, and ¹⁸F‐FDG was obtained from batches prepared for clinical PET imaging at Gunma University. The animals were euthanized 2 hours after injection. The isolated tissues were weighed and counted in a well‐type gamma counter (ARC‐7001; Aloka). Each experiment was performed at least twice.

Lyophilized yeast cells were disrupted by freeze-thawing in hydrochloric acid and extracted for fatty acids using the chloroform-methanol method, dried by nitrogen blowing, and then methylated by adding 2 mL of 1% (v/v) sulfuric acid-methanol for 60 min in 80 °C [16 (link)]. Fatty acid profiles were analyzed as fatty acid methyl esters (FAMEs) by gas chromatography-mass spectrometry (GC-MS; GCMS-QP2010 Ultra, Shimadzu, Kyoto, Japan). Pentadecanoic acid (C15:0) served as an internal standard, and relative quantification was based on the ratio of each fatty acid to the internal standard peak area. The temperature program was as previously detailed [17 (link)]. The transformation efficiency of the mutants was calculated using the substrate conversion rate: Rate of substrate conversion (%) = 100 × [(product)/(product + substrate)], from three independent experiments.