The starting point for our optimization approach was the generally applied recommended protocol for pulse feeding of methanol twice a day [11, 12] . This procedure is very different from the various cultivation techniques applied in fermentation processes which all are based on continuous addition of methanol. As model system we used the expression of recombinant human collagen II in P. pastoris, which has been described earlier [8] . The assembly of the triple-helical collagen is directly correlated with the hydroxylation of prolyl residues by C-P4H, which is coexpressed together with the collagen polypeptide chains.