Enhanced heterologous expression of two Streptomyces griseolus cytochrome P450s and Streptomyces coelicolor ferredoxin reductase as potentially efficient hydroxylation catalysts.
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.
Hussain
Haitham A
HA
Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom.
Ward
John M
JM
D001426:Bacterial Proteins; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005288:Ferredoxins / Q000235:genetics / Q000378:metabolism*; D006900:Hydroxylation; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D003374:Coumarins; D005288:Ferredoxins; D011994:Recombinant Proteins; C017299:7-ethoxycoumarin; D012343:RNA, Transfer; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C086254:P450SU1 protein, Streptomyces griseolus; C086255:P450SU2 protein, Streptomyces griseolus