Almost 10-fold higher amount of procollagen II mRNA was detected in the cultures with quasi-continuous feeding of methanol compared to the pulse method. Also the expression of the translation factor EF3, which is an indicator of protein synthesis, was higher with constant feeding. Generally, in the case of such optimization approaches, and not limited to shake flasks studies, the quantitative analysis of mRNAs, especially for C-P4H and collagen in our case, seems to be a good indicator for the quality of the cultivation.