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Applied and Environmental Microbiology, January 2003, p. 373-382, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.373-382.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

Haitham A. Hussain and John M. Ward*

Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom

Received 19 June 2002/ Accepted 1 November 2002

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, University College London, Gower St., London WC1E 6BT, United Kingdom. Phone: 44-20-76792242. Fax: 44-20-76797193. E-mail: ward{at}biochemistry.ucl.ac.uk.

Applied and Environmental Microbiology, January 2003, p. 373-382, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.373-382.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kim, D.-H., Kim, K.-H., Kim, D.-H., Liu, K.-H., Jung, H.-C., Pan, J.-G., Yun, C.-H. (2008). Generation of Human Metabolites of 7-Ethoxycoumarin by Bacterial Cytochrome P450 BM3. Drug Metab. Dispos. 36: 2166-2170 [Abstract] [Full Text]  
  • Ghatge, M. S., Reynolds, K. A. (2005). The plmS2-Encoded Cytochrome P450 Monooxygenase Mediates Hydroxylation of Phoslactomycin B in Streptomyces sp. Strain HK803. J. Bacteriol. 187: 7970-7976 [Abstract] [Full Text]  


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