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J Biotechnol. 2016 Jan 20;218:34-40. doi: 10.1016/j.jbiotec.2015.11.021. Epub 2015 Nov 27.

Expression of human CYP27A1 in B. megaterium for the efficient hydroxylation of cholesterol, vitamin D3 and 7-dehydrocholesterol.

Author information

1
Universität des Saarlandes, Institut für Biochemie, Campus B2.2, 66123 Saarbrücken, Germany.
2
Universität des Saarlandes, Institut für Biochemie, Campus B2.2, 66123 Saarbrücken, Germany. Electronic address: ritabern@mx.uni-saarland.de.

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.

KEYWORDS:

7-Dehydrocholesterol; Bacillus megaterium; CYP27A1; Cholesterol; Cytochrome P450; Vitamin D3

PMID:
26638999
DOI:
10.1016/j.jbiotec.2015.11.021
[Indexed for MEDLINE]

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