Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Werck-Reichhart D, Feyereisen R (2000) Cytochromes P450: a success story. Genome Biol 1:3003.1–3003.9
Wu ZL, Qiao J, Zhang ZG, Guengerich FP, Liu Y, Pei XQ (2009) Enhanced bacterial expression of several mammalian cytochrome P450 s by codon optimization and chaperone coexpression. Biotechnol Lett. doi:10.1007/s10529-009-0059-5
Lee SY (1996) High cell-density culture of Escherichia coli. Trends Biotechnol 14:98–105
Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421
Friedberg T, Pritchard MP, Bandera M, Hanlon SP, Yao D, Mclaughlin LA, Ding S, Burchell B, Wolf CR (1999) Merits and limitations of recombinant models for the study of human P450-mediated drug metabolism and toxicity: an intralaboratory comparison. Drug Metab Rev 31:523–544
Gillam EMJ (1998) Human cytochrome P450 enzymes expressed in bacteria: reagents to probe molecular interactions in toxicology. Clin Exp Pharmacol Physiol 25:877–886
Guengerich FP (2002) Cytochrome P450 enzymes in the generation of commercial products. Nat Rev Drug Discov 1:359–366
Parikh A, Gillam EMJ, Guengerich FP (1997) Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat Biotechnol 15:784–788
Zhang JD, Li AT, Yang Y, Xu JH (2010) Sequence analysis and heterologous expression of a new cytochrome P450 monooxygenase from Rhodococcus sp. for asymmetric sulfoxidation. Appl Microbiol Biotechnol 85:615–624
Roberts GA, Grogan G, Greter A, Flitsch SL, Turner NJ (2002) Identification of a new class of cytochrome P450 from a Rhodococcus sp. J Bacteriol 184:3898–3908
Roberts GA, Celik A, Hunter DJ, Ost TW, White JH, Chapman SK, Turner NJ, Flitsch SL (2003) A self-sufficient cytochrome P450 with a primary structural organization that includes a flavin domain and a [2Fe–2S] redox center. J Biol Chem 278:48914–48920
Liu L, Schmid RD, Urlacher VB (2006) Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319. Appl Microbiol Biotechnol 72:876–882
Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239:2370–2378
Li AT, Zhang JD, Xu JH, Lu WY, Lin GQ (2009) Isolation of Rhodococcus sp. ECU0066: a new sulfide monooxygenase producing strain for asymmetric sulfoxidation. Appl Environ Microbiol 75:551–556
Farinas ET, Schwaneberg U, Glieder A, Arnold FH (2001) Directed evolution of a cytochrome P450 monooxygenase for alkane oxidation. Adv Synth Catal 343:601–606
Gustafsson MCU, Roitel O, Marshall KR, Noble MA, Chapman SK, Pessegueiro A, Vail RB, Homann MJ, Hanna I, Zaks A (2005) Preparative synthesis of drug metabolites using human cytochrome P450 s 3A4, 2C9 and 1A2 with NADPH-P450 reductase expressed in Escherichia coli. J Ind Microbiol Biotechnol 32:67–74
Simon P, Sven MR, Vlada BU (2007) Development of a fed-batch process for the production of the cytochrome P450 monooxygenase CYP102A1 from Bacillus megaterium in E. coli. J Biotechnol 129:481–488
Choi JH, Lee SJ, Lee SJ, Lee SY (2003) Enhanced production of insulin like growth factor I fusion protein in Escherichia coli by coexpression of the down-regulated genes identified by transcriptome profiling. Appl Environ Microb 69:4737–4742
Endo T, Koizumi S (2001) Microbial conversion with cofactor regeneration using genetically engineered bacteria. Adv Synth Catal 343:521–526
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 20773038 and 20902023), Ministry of Science and Technology, P.R. China (Grant Nos. 2009CB724706 and 2009ZX09501-016), China National Special Fund for State Key Laboratory of Bioreactor Engineering (Grant No. 2060204) and Shanghai Leading Academic Discipline Project (No. B505).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Zhang, JD., Li, AT. & Xu, JH. Improved expression of recombinant cytochrome P450 monooxygenase in Escherichia coli for asymmetric oxidation of sulfides. Bioprocess Biosyst Eng 33, 1043–1049 (2010). https://doi.org/10.1007/s00449-010-0429-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00449-010-0429-3