title: Functionalized PHB granules provide the basis for the efficient side-chain cleavage of cholesterol and analogs in recombinant Bacillus megaterium.
abstract: BACKGROUND
  Cholesterol, the precursor of all steroid hormones, is the most abundant steroid in vertebrates and exhibits highly hydrophobic properties, rendering it a difficult substrate for aqueous microbial biotransformations. In the present study, we developed a Bacillus megaterium based whole-cell system that allows the side-chain cleavage of this sterol and investigated the underlying physiological basis of the biocatalysis.
  
  
  RESULTS
  CYP11A1, the side-chain cleaving cytochrome P450, was recombinantly expressed in the Gram-positive soil bacterium B. megaterium combined with the required electron transfer proteins. By applying a mixture of 2-hydroxypropyl-β-cyclodextrin and Quillaja saponin as solubilizing agents, the zoosterols cholesterol and 7-dehydrocholesterol, as well as the phytosterol β-sitosterol could be efficiently converted to pregnenolone or 7-dehydropregnenolone. Fluorescence-microscopic analysis revealed that cholesterol accumulates in the carbon and energy storage-serving poly(3-hydroxybutyrate) (PHB) bodies and that the membrane proteins CYP11A1 and its redox partner adrenodoxin reductase (AdR) are likewise localized to their surrounding phospholipid/protein monolayer. The capacity to store cholesterol was absent in a mutant strain devoid of the PHB-producing polymerase subunit PhaC, resulting in a drastically decreased cholesterol conversion rate, while no effect on the expression of the recombinant proteins could be observed.
  
  
  CONCLUSIONS
  We established a whole-cell system based on B. megaterium, which enables the conversion of the steroid hormone precursor cholesterol to pregnenolone in substantial quantities. We demonstrate that the microorganism's PHB granules, aggregates of bioplastic coated with a protein/phospholipid monolayer, are crucial for the high conversion rate by serving as substrate storage. This microbial system opens the way for an industrial conversion of the abundantly available cholesterol to any type of steroid hormones, which represent one of the biggest groups of drugs for the treatment of a wide variety of diseases.
authors.affiliation: Centre for Human and Molecular Biology, Institute of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. a.gerber@mx.uni-saarland.de.
authors.forename: Adrian
authors.initials: A
authors.lastname: Gerber
authors.affiliation: Centre for Human and Molecular Biology, Institute of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. mic.kl@web.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Kleser
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, 38106, Braunschweig, Germany. r.biedendieck@tu-bs.de.
authors.forename: Rebekka
authors.initials: R
authors.lastname: Biedendieck
authors.affiliation: Centre for Human and Molecular Biology, Institute of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. ritabern@mx.uni-saarland.de.
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
authors.affiliation: Centre for Human and Molecular Biology, Institute of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. f.hannemann@mx.uni-saarland.de.
authors.forename: Frank
authors.initials: F
authors.lastname: Hannemann
mesh_terms: D000073738:2-Hydroxypropyl-beta-cyclodextrin; D001410:Bacillus megaterium / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D001711:Biotransformation; D002784:Cholesterol / Q000378:metabolism*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000096:biosynthesis; D006885:Hydroxybutyrates / Q000737:chemistry*; D011091:Polyesters / Q000737:chemistry*; D011284:Pregnenolone / Q000378:metabolism*; D000091402:Prohibitins; D062605:Quillaja Saponins / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis; D047392:beta-Cyclodextrins / Q000737:chemistry; D000094122:Polyhydroxybutyrates
chemical_list: D000073738:2-Hydroxypropyl-beta-cyclodextrin; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme; D006885:Hydroxybutyrates; D011091:Polyesters; D011284:Pregnenolone; D000091402:Prohibitins; D062605:Quillaja Saponins; D011994:Recombinant Proteins; D047392:beta-Cyclodextrins; C000717328:PHB protein, human; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates