A fed-batch process utilizing a pET-based expression system (pET28a+ derivative) and E. coli BL21(DE3) as production strain for the heterologous expression of recombinant cytochrome P450 monooxygenase CYP102A1 from Bacillus megaterium was developed. In a first step the expression was optimized during a series of flask experiments testing several parameters for their influence on the expression level, activity and solubility of the recombinant protein. The optimal process parameters found in the flask experiments were transferred to a cultivation process in a 5l (operating volume) bioreactor with a special focus on the feeding strategy and the aeration during expression. Glycerol feeding proved to be superior over glucose as carbon source since the formation of larger amounts of acetate was prevented. Expression levels exceeding 12,500nmoll(-1), corresponding to approximately 1.5gl(-1) of product in culture medium ( approximately 11% of CDW) could be demonstrated. The P450 enzyme showed high activity and high solubility. The findings now can be transferred to other enzyme variants and different P450 monooxygenases to increase production of recombinant proteins.