Process development for the production of 15β-hydroxycyproterone acetate using Bacillus megaterium expressing CYP106A2 as whole-cell biocatalyst

Flora M. Kiss, Marie Therese Lundemo, Josef Zapp, John Woodley, Rita Bernhardt

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Abstract

Background: CYP106A2 from Bacillus megaterium ATCC 13368 was first identified as a regio- and stereoselective 15 beta-hydroxylase of 3-oxoΔ4-steroids. Recently, it was shown that besides 3-oxo-Δ4-steroids, 3-hydroxy-Δ5-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 6 β, 7 a/β, 9a, 11a and 15a 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, 15 β-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.
Original languageEnglish
JournalMicrobial Cell Factories
Volume14
Issue number28
Number of pages13
ISSN1475-2859
DOIs
Publication statusPublished - 2015

Bibliographical note

This is an Open Access article distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Keywords

  • Bacillus megaterium
  • Whole-cell biocatalyst
  • Steroid hydroxylase
  • Cyproterone acetate
  • Bioreactor
  • Cyclodextrin

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