Determination of concentrations of intracellular acyl-CoA esters

The concentrations of intracellular M-CoA and MM-CoA in the relevant strains at 48 h and 72 h of incubation were extracted following the method described by Lu et al. (2016) and determined by LC–MS/MS. For each time point, samples of 25 mL were harvested. One milliliter of culture was collected to quantify the dry cell weight. The liquid cell sample of remaining culture was transferred into a precooled tube containing quenching and extraction solution (acetonitrile/methanol/0.1% glacial acetate at a volume ratio of 45:45:10, − 20 °C). The extraction was performed by repetitive vortexing and cooling on ice for 15 min and centrifuged to collect the supernatant (12,000 rpm, 4 °C, 3 min). Samples were analyzed using an Atlantis BEH C18 (1.7 µm, 2.1 × 100 mm, Waters Co., Milford, MA) on a triple quadrupole MS (Waters). The mobile phase was acetonitrile with 50 mM ammonium hydrogen carbonate (solvent A), 0.1% ammonium hydroxide (solvent B), ddH₂O (solvent C) and 0.1% ammonium hydroxide-acetonitrile (solvent D). Elution was performed as follows: 0–3 min 20% A 5% B 75% C, 3–3.5 min 20% A 10% B 30% C 40% D, 3.5–5 min 20% A 80% D, 5–7 min 20% A 5% B 75% C. Quantification was detected in the multiple reaction monitoring mode (MRM) with the m/z parent > m/z daughter (M-CoA 854 > 347, MM-CoA 868 > 361).