Laccase expression in P. pastoris. (A and B) Fermentation in a 500-mL bioreactor of P. pastoris transformants expressing the ChU-B mutant joined to the original (A) and the evolved (B) α-factor prepro-leader under the control of the PAOX1. After 22 h of batch phase, glycerol feed was maintained for 5 hours and thereafter the methanol induction started. Methanol was pumped to the culture so that a DO level ~4% was maintained throughout the fermentation. (C) 20-L fermentation in a 42-L bioreactor of a P. pastoris clone transformed with pPICZα*ChU-B. Fermentation comprised four steps: batch phase for 22 h, glycerol phase for 5 h, transition phase for 5 h to adapt the culture to grow on methanol, induction phase for 136 h. Black circles, laccase activity at the induction phase; black triangles, wet biomass; white squares, extracellular protein concentration.