title: Expression of human CYP27A1 in B. megaterium for the efficient hydroxylation of cholesterol, vitamin D3 and 7-dehydrocholesterol.
abstract: In the current work the ability of Bacillus megaterium to take up hydrophobic substrates and efficiently express eukaryotic membrane proteins was utilized for establishing a CYP27A1-based biocatalyst. The human mitochondrial cytochrome P450CYP27A1 was co-expressed with its redox partners adrenodoxin reductase (Adr) and adrenodoxin (Adx). CYP27A1 could be localized at the cell's polyhydroxybutyrate (PHB) granules, carbon storage serving organelle-like vesicles that can take up cholesterol, resulting in bioreactor-like structures in B. megaterium . The resulting whole cell system allowed the efficient biotechnological conversion of the CYP27A1 substrates cholesterol, 7-dehydrocholesterol (7-DHC) and vitamin D3. After 48 h, nearly 100% of cholesterol was metabolized producing a final concentration of 113.14 mg/l 27-hydroxycholesterol (27-HC). Moreover, 70% of vitamin D3 was converted into 25-hydroxyvitamin D3 (25-OH-D3) with a final concentration of 80.81 mg/l. Also more than 97% of 7-DHC were found to be metabolized into two products, corresponding to 26/27-hydroxy-7-dehydrocholesterol (P1) and 25-hydroxy-7-dehydrocholesterol (P2). To our knowledge this is the first CYP27A1-based whole-cell system, allowing the efficient and low-cost production of pharmaceutically interesting metabolites of this enzyme from relatively cheap substrates.
authors.affiliation: Universität des Saarlandes, Institut für Biochemie, Campus B2.2, 66123 Saarbrücken, Germany.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Ehrhardt
authors.affiliation: Universität des Saarlandes, Institut für Biochemie, Campus B2.2, 66123 Saarbrücken, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Gerber
authors.affiliation: Universität des Saarlandes, Institut für Biochemie, Campus B2.2, 66123 Saarbrücken, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Hannemann
authors.affiliation: Universität des Saarlandes, Institut für Biochemie, Campus B2.2, 66123 Saarbrücken, Germany. Electronic address: ritabern@mx.uni-saarland.de.
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002112:Calcifediol / Q000378:metabolism; D002762:Cholecalciferol / Q000378:metabolism*; D053493:Cholestanetriol 26-Monooxygenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002784:Cholesterol / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism*; D003684:Dehydrocholesterols / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D006888:Hydroxycholesterols / Q000378:metabolism; D006900:Hydroxylation; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D000091402:Prohibitins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: C587260:26-hydroxy-7-dehydrocholesterol; D003684:Dehydrocholesterols; D006888:Hydroxycholesterols; C000717328:PHB protein, human; D000091402:Prohibitins; D011994:Recombinant Proteins; D002762:Cholecalciferol; C076996:27-hydroxycholesterol; D002784:Cholesterol; C587259:25-hydroxy-7-dehydrocholesterol; C016705:7-dehydrocholesterol; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; D002112:Calcifediol
keywords: 7-Dehydrocholesterol; Bacillus megaterium; CYP27A1; Cholesterol; Cytochrome P450; Vitamin D3