Improved expression of recombinant cytochrome P450 monooxygenase in Escherichia coli for asymmetric oxidation of sulfides.
Escherichia coli BL21 as production strain for the production of cytochrome P450 monooxygenase (P450SMO) from Rhodococcus sp. in high yields was developed. The expression was first optimized with a series of flask experiments testing several key parameters for their influence on the expression level and enzyme activity. The optimal process parameters found in the flask experiments were verified in a cultivation process in a 5-L bioreactor. Glycerol proved to be superior over glucose as carbon source. Low dissolved oxygen (DO) concentration (<10%) during expression was found to be critical for active P450s production, resulting in expression level of 400 nM for P450SMO. Intact cells were used to establish an efficient bioconversion system for the production of sulfoxidation product. With p-chlorothioanisole as a representative substrate, the desired product (S-sulfoxide) was afforded with 99% ee and highest production of 130 mg/L within 12 h.
Zhang
Jian-Dong
JD
Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, People's Republic of China.
Li
Ai-Tao
AT
Xu
Jian-He
JH
D002248:Carbon Monoxide / Q000737:chemistry; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D005285:Fermentation; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology / Q000379:methods*; D010100:Oxygen / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012240:Rhodococcus / Q000378:metabolism; D012997:Solvents; D013440:Sulfides / Q000737:chemistry*; D013696:Temperature
D011994:Recombinant Proteins; D012997:Solvents; D013440:Sulfides; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D005947:Glucose; D010100:Oxygen