Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts
| Autor: | Haitham A. Hussain, John M. Ward |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Cytochrome
Hydroxylation medicine.disease_cause Applied Microbiology and Biotechnology Streptomyces Bacterial Proteins Cytochrome P-450 Enzyme System RNA Transfer Coumarins Escherichia coli medicine Ferredoxin Expression vector Ecology biology Streptomyces coelicolor Physiology and Biotechnology biology.organism_classification Molecular biology Recombinant Proteins Biochemistry Codon usage bias Oxygenases biology.protein Ferredoxins bacteria Heterologous expression Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 69:373-382 |
| ISSN: | 1098-5336 0099-2240 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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