Cell growth and Aox enzymatic activity assays

The knockout strains were pre-grown in YPD media to OD₆₀₀ of 2–8. Then the cells were harvested by centrifugation at 3000 g for 5 minutes and washed three times with sterile water. To examine growth, cell suspensions were spotted 5 μL in three dilutions (0.1, 0.01 and 0.001 OD₆₀₀) onto YND, YNG and YNM medium, respectively. The strains were grown for 2 days on YND and YNG while 3 days on YNM.

The colorimetrical assay was used to measure Aox enzymatic activity. Cells were re-suspended in 500 μL YNB supplemented with different carbon sources in 96 deep well plates to reach OD₆₀₀ of 1.0. After 6 hours culture on a shaker (200 rpm, 30°C), 1 mL reaction buffer was added. After incubation for 20 min, 100 μL mixer was transferred into another 96-well plate and scanned. The reaction buffer for colorimetrical assay including 0.05% (w/v) O-dianisidine, 0.15% (w/v) CTAB, 1% (v/v) methanol, 3 U/mL HRP, and 100 mmol/L potassium phosphate buffer (pH 7.0).