2.4 Quantification of free fatty acids
All strains were precultured in YPD medium for 24 h and then transferred to Delft minimal medium containing 20 g/L glucose and specific amino acids or nucleotides at 30℃, 220 rpm. After cultivation for 48 h, FFA were determined according to the previous method[19]. In brief, 100 μL sample was taken for FFA extraction. Then, the 100 μL ddH2O, 10 μL 40% tetrabutylammonium hydroxide and 200 μL dichloromethane (200 mM methyl iodide as methyl donor and 100 mg/L pentadecanoic acid as an internal standard) were successively added to the sample. The mixtures were shaken for 30 minutes at 1,600 rpm in whirlpool mixer, and centrifugation was performed at 3,000 g for 10 minutes to promote phase separation. The 200 μL dichloromethane was transferred to GC bottles containing glass insert. The samples were dried at room temperature for 4 h, and re-suspended with 200 μL N-hexane. Finally, the samples were analyzed by gas chromatography. Specific procedures is shown by predecessors [19].