Development of a fed-batch process for the production of the cytochrome P450 monooxygenase CYP102A1 from Bacillus megaterium in E. coli.
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.
Pflug
Simon
S
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Richter
Sven M
SM
Urlacher
Vlada B
VB
D001410:Bacillus megaterium / Q000201:enzymology*; D001426:Bacterial Proteins / Q000096:biosynthesis*; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D002452:Cell Count; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006899:Mixed Function Oxygenases / Q000096:biosynthesis*; D009251:NADPH-Ferrihemoprotein Reductase; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013053:Spectrophotometry
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases