title: Coexpression of redox partners increases the hydrocortisone (cortisol) production efficiency in CYP11B1 expressing fission yeast Schizosaccharomyces pombe.
abstract: Cytochromes P450 play a vital role in the steroid biosynthesis pathway of the adrenal gland. An example of an essential P450 cytochrome is the steroid 11beta-hydroxylase CYP11B1, which catalyses the conversion of 11-deoxycorticol to hydrocortisone. However, despite its high biotechnological potential, this enzyme has so far been unsuccessfully employed in present-day biotechnology due to a poor expression yield and inherent protein instability. In this study, CYP11B1 was biotransformed into various strains of the yeast Schizosaccharomyces pombe, all of which also expressed the electron transfer proteins adrenodoxin and/or adrenodoxin reductase - central components of the mitochondrial P450 system - in order to maximise hydrocortisone production efficiency in our proposed model system. Site-directed mutagenesis of CYP11B1 at positions 52 and 78 was performed in order to evaluate the impact of altering the amino acids at these sites. It was found that the presence of an isoleucine at position 78 conferred the highest 11beta-hydroxylation activity of CYP11B1. Coexpression of adrenodoxin and adrenodoxin reductase appeared to further increase the 11beta-hydroxylase activity of the enzyme (3.4 fold). Adrenodoxin mutants which were found to significantly enhance enzyme efficiency in other cytochromes in previous studies were also tested in our system. It was found that, in this case, the wild type adrenodoxin was more efficient. The new fission yeast strain TH75 coexpressing the wild type Adx and AdR displays high hydrocortisone production efficiency at an average of 1mM hydrocortisone over a period of 72h, the highest value published to date for this biotransformation. Finally, our research shows that pTH2 is an ideal plasmid for the coexpression of the mitochondrial electron transfer counterparts, adrenodoxin and adrenodoxin reductase, in Schizosaccharomyces pombe, and so could serve as a convenient tool for future biotechnological applications.
authors.affiliation: Institute of Biochemistry, P.O. Box 151150, Saarland University, D-66041 Saarbrücken, Germany.
authors.forename: Tarek
authors.initials: T
authors.lastname: Hakki
authors.forename: Silvia
authors.initials: S
authors.lastname: Zearo
authors.forename: Călin-Aurel
authors.initials: CA
authors.lastname: Drăgan
authors.forename: Matthias
authors.initials: M
authors.lastname: Bureik
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D000325:Adrenodoxin / Q000737:chemistry / Q000378:metabolism; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D004583:Electrons; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D006854:Hydrocortisone / Q000096:biosynthesis*; D006900:Hydroxylation; D008928:Mitochondria / Q000201:enzymology; D008958:Models, Molecular; D010084:Oxidation-Reduction; D012568:Schizosaccharomyces / Q000166:cytology / Q000201:enzymology*; D013252:Steroid 11-beta-Hydroxylase / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
chemical_list: D000325:Adrenodoxin; D013252:Steroid 11-beta-Hydroxylase; D005287:Ferredoxin-NADP Reductase; D006854:Hydrocortisone