We have shown previously by applying on-line monitoring sensors in shake flasks that the commonly used methanol feeding protocol for shake flask cultures of P. pastoris (two manual pulses of methanol per day) leads to long starvation phases between feeding pulses [15] . Starvation is obvious by a pO 2 peak after exhaustion of methanol, related to declining respiratory activity ( Figure 1A ). We concluded from this data that the methanol feeding in this shake flask process is not optimal. In the first step of optimization, the rapidly increasing pO 2 level after consumption of methanol was used as a signal to manually add a new methanol pulse. Each single methanol dose was the same as in the standard protocol and consequently much more methanol was added during the course of the cultivation. Although the procedure was laborious and difficult to reproduce manually, a 30% higher cell density was obtained and the product related mRNAs showed higher expression, such as the mRNAs encoding alcohol oxidase 1 (AOX), formaldehyde dehydrogenase, yeast PDI, type II procollagen chain and the C-P4H α(I) and PDI/β subunits ( Figure 1B) . Unexpectedly, the amount of collagen II was not increased, however (not shown).