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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus megaterium / enzymology*
  • Bacillus megaterium / genetics
  • Bacillus megaterium / metabolism
  • Calcifediol / metabolism
  • Cholecalciferol / metabolism*
  • Cholestanetriol 26-Monooxygenase / biosynthesis*
  • Cholestanetriol 26-Monooxygenase / chemistry
  • Cholestanetriol 26-Monooxygenase / genetics
  • Cholesterol / analogs & derivatives
  • Cholesterol / biosynthesis
  • Cholesterol / metabolism*
  • Dehydrocholesterols / metabolism*
  • Genetic Vectors
  • Humans
  • Hydroxycholesterols / metabolism
  • Hydroxylation
  • Mitochondria / enzymology
  • Mitochondria / metabolism
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics


  • 26-hydroxy-7-dehydrocholesterol
  • Dehydrocholesterols
  • Hydroxycholesterols
  • Recombinant Proteins
  • Cholecalciferol
  • 27-hydroxycholesterol
  • Cholesterol
  • 25-hydroxy-7-dehydrocholesterol
  • 7-dehydrocholesterol
  • CYP27A1 protein, human
  • Cholestanetriol 26-Monooxygenase
  • Calcifediol