Functional strains were cultured in YNB-LEU containing galactose to induce the promoters. After reaching an OD600 = 1, 20 mL of the culture was collected and pelleted and washed twice with 100 mM potassium phosphate buffer at pH = 6.5 to prepare resting cells and then transferred to 250 mL GL-45 Erlenmeyer flask (Chemglass Life Sciences). Biotransformation buffer used was 20 mL potassium phosphate buffer with 100 μL linoleic acid solution (5% v/v with 0.2% tween-80). The flasks were sealed with GL-45 open top cap and parafilms (Chemglass Life Sciences) and incubated at 30°C on an orbital shaker (250 rpm) for 3 days.
Headspace samples of the cultures were determined by Agilent 6890 N GC-FID system (Agilent) equipped with Agilent J&W DB-WAX column (30 m × 0.25 mm × 0.25 μm, Agilent). 1 mL SampleLock syringe (Hamilton) was used to draw out the headspaces of the 20 mL cultures to inject into GC system. GC settings were: carrier gas: helium; column flow: 2.0 ml/min; splitless; inject temperature: 230°C The analyzing temperature program used was: 50-230°C in 18 min; 230°C for 2 min. Product identification was carried out by comparing with authentic standards and benzoaldehyde was used as internal control for quantification.
Headspace samples of the cultures were determined by Agilent 6890 N GC-FID system (Agilent) equipped with Agilent J&W DB-WAX column (30 m × 0.25 mm × 0.25 μm, Agilent). 1 mL SampleLock syringe (Hamilton) was used to draw out the headspaces of the 20 mL cultures to inject into GC system. GC settings were: carrier gas: helium; column flow: 2.0 ml/min; splitless; inject temperature: 230°C The analyzing temperature program used was: 50-230°C in 18 min; 230°C for 2 min. Product identification was carried out by comparing with authentic standards and benzoaldehyde was used as internal control for quantification.