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

Abstract

BackgroundCYP106A2 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.ResultsIn 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.ConclusionsHere 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.

Process limitations of a whole-cell P450 catalyzed reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli

“…The initial rate of experiments performed in bioreactors was shown to be higher compared to experiments performed in shake flasks. This has also been observed previously and is believed to be due to enhanced mixing in the stirred tank reactor (Kiss et al 2015). The effect in this case could alternatively be attributed to the cells originating from different fermentation batches, stressing the importance of standardizing the expression to get identical specific activity throughout batches in order to design the resting cell transformation properly.…”

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

“…This progress can directly be of use for efficient and timesaving production of human drug metabolites. High yields and conversion rates can already be achieved by using corresponding human (Rushmore et al, 2000;Vail et al, 2005;Schroer et al, 2010;Geier et al, 2012;Schiffer et al, 2015) or suitable nonhuman (Taylor et al, 1999;Otey et al, 2006;Sawayama et al, 2009;Reinen et al, 2011;Di Nardo and Gilardi, 2012;Kiss et al, 2015;Ren et al, 2015) P450 enzymes in a whole-cell system to produce respective metabolites. The majority of published bacterial P450 enzymes used for the conversion of drugs are mutants of CYP102A1 (BM3) from Bacillus megaterium.…”

Whole cell biocatalysts: essential workers from Nature to the industry

“…VLB120, with the cells doubling the specific activity and presenting high glucose yields when compared with growing cells. Kiss et al (2015) could also efficiently scale-up the conversion of cyproterone acetate to its main human metabolite, 15b-hydroxycyproterone acetate, by Bacillus megaterium ATCC 13368 which contains the enzyme CYP106A2. Product formation reached 0.43 g L À1 and no significant differences could be observed between growing and resting cells.…”