title: Process development for the production of 15β-hydroxycyproterone acetate using Bacillus megaterium expressing CYP106A2 as whole-cell biocatalyst.
abstract: BACKGROUND
  CYP106A2 from Bacillus megaterium ATCC 13368 was first identified as a regio- and stereoselective 15β-hydroxylase of 3-oxo-∆4-steroids. Recently, it was shown that besides 3-oxo-∆4-steroids, 3-hydroxy-∆5-steroids as well as di- and triterpenes can also serve as substrates for this biocatalyst. It is highly selective towards the 15β position, but the 6β, 7α/β, 9α, 11α and 15α positions have also been described as targets for hydroxylation. Based on the broad substrate spectrum and hydroxylating capacity, it is an excellent candidate for the production of human drug metabolites and drug precursors.
  
  
  RESULTS
  In this work, we demonstrate the conversion of a synthetic testosterone derivative, cyproterone acetate, by CYP106A2 under in vitro and in vivo conditions. Using a Bacillus megaterium whole-cell system overexpressing CYP106A2, sufficient amounts of product for structure elucidation by nuclear magnetic resonance spectroscopy were obtained. The product was characterized as 15β-hydroxycyproterone acetate, the main human metabolite. Since the product is of pharmaceutical interest, our aim was to intensify the process by increasing the substrate concentration and to scale-up the reaction from shake flasks to bioreactors to demonstrate an efficient, yet green and cost-effective production. Using a bench-top bioreactor and the recombinant Bacillus megaterium system, both a fermentation and a transformation process were successfully implemented. To improve the yield and product titers for future industrial application, the main bottlenecks of the reaction were addressed. Using 2-hydroxypropyl-β-cyclodextrin, an effective bioconversion of 98% was achieved using 1 mM substrate concentration, corresponding to a product formation of 0.43 g/L, at a 400 mL scale.
  
  
  CONCLUSIONS
  Here we describe the successful scale-up of cyproterone acetate conversion from shake flasks to bioreactors, using the CYP106A2 enzyme in a whole-cell system. The substrate was converted to its main human metabolite, 15β-hydroxycyproterone acetate, a highly interesting drug candidate, due to its retained antiandrogen activity but significantly lower progestogen properties than the mother compound. Optimization of the process led to an improvement from 55% to 98% overall conversion, with a product formation of 0.43 g/L, approaching industrial process requirements and a future large-scale application.
authors.affiliation: Institute of Biochemistry, University of Saarland, Campus B 2.2, 66123, Saarbruecken, Germany. flora.marta.kiss@gmail.com.
authors.forename: Flora M
authors.initials: FM
authors.lastname: Kiss
authors.affiliation: CAPEC-PROCESS, Department of Chemical and Biochemical Engineering, Technical University of Denmark, DK-2800, Lyngby, Denmark. mande@kt.dtu.dk.
authors.forename: Marie T
authors.initials: MT
authors.lastname: Lundemo
authors.affiliation: Institute of Pharmaceutical Biology, University of Saarland, Campus B 2.2, 66123, Saarbruecken, Germany. j.zapp@mx.uni-saarland.de.
authors.forename: Josef
authors.initials: J
authors.lastname: Zapp
authors.affiliation: CAPEC-PROCESS, Department of Chemical and Biochemical Engineering, Technical University of Denmark, DK-2800, Lyngby, Denmark. jw@kt.dtu.dk.
authors.forename: John M
authors.initials: JM
authors.lastname: Woodley
authors.affiliation: Institute of Biochemistry, University of Saarland, Campus B 2.2, 66123, Saarbruecken, Germany. ritabern@mx.uni-sarland.de.
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D000073738:2-Hydroxypropyl-beta-cyclodextrin; D001410:Bacillus megaterium / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D017373:Cyproterone Acetate / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013237:Stereoisomerism; D013379:Substrate Specificity; D047392:beta-Cyclodextrins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D047392:beta-Cyclodextrins; D000073738:2-Hydroxypropyl-beta-cyclodextrin; D017373:Cyproterone Acetate; D003577:Cytochrome P-450 Enzyme System; C517094:15beta-hydroxylase CYP106A2, Bacillus megaterium