Growth rates of the P. pastoris wild-type strain CBS7435 and knock-out strains aox1, aox1 arg4, his4, aox1 his4, ku70, ku70 gut1, das1, das1 das2 and the corresponding complemented arg4, his4 and gut1 mutant strains were defined by measuring optical density (OD595) of triplicate cultures during exponential growth phase. An overnight culture was inoculated with a single colony in corresponding media and diluted to a starting OD of ～0.15. The main cultures were grown in standard conditions using 50 ml of buffered minimal media BMD2% (D-glucose), BMG1% (glycerol) or BMM0.5% (methanol) in 250 ml baffled shake flasks (28°C, 120 rpm with 50mm amplitude). Amino acids were added to 40 μg/ml (histidine) or 50 μg/ml (arginine) as required for non-complemented auxotrophic strains.

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