Kiss Flora M, Lundemo Marie T, Zapp Josef, Woodley John M, Bernhardt Rita
Institute of Biochemistry, University of Saarland, Campus B 2.2, 66123, Saarbruecken, Germany.
CAPEC-PROCESS, Department of Chemical and Biochemical Engineering, Technical University of Denmark, DK-2800, Lyngby, Denmark.
Microb Cell Fact. 2015 Mar 5;14:28. doi: 10.1186/s12934-015-0210-z.
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.
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.
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.
巨大芽孢杆菌ATCC 13368中的CYP106A2最初被鉴定为3-氧代-Δ4-甾体的区域和立体选择性15β-羟化酶。最近研究表明，除了3-氧代-Δ4-甾体外，3-羟基-Δ5-甾体以及二萜和三萜也可作为这种生物催化剂的底物。它对15β位具有高度选择性，但6β、7α/β、9α、11α和15α位也被描述为羟基化的靶点。基于广泛的底物谱和羟化能力，它是生产人类药物代谢物和药物前体的优秀候选者。
在本研究中，我们展示了在体外和体内条件下，CYP106A2对合成睾酮衍生物醋酸环丙孕酮的转化。使用过表达CYP106A2的巨大芽孢杆菌全细胞系统，获得了足够量的产物用于通过核磁共振光谱进行结构解析。产物被鉴定为15β-羟基醋酸环丙孕酮，这是主要的人体代谢物。由于该产物具有药物研究价值，我们的目标是通过提高底物浓度来强化该过程，并将反应从摇瓶放大到生物反应器，以证明其高效、绿色且具有成本效益的生产方式。使用台式生物反应器和重组巨大芽孢杆菌系统，成功实现了发酵和转化过程。为了提高未来工业应用的产量和产物滴度，解决了反应的主要瓶颈问题。使用2-羟丙基-β-环糊精，在400 mL规模下，以1 mM底物浓度实现了98%的有效生物转化，产物生成量为0.43 g/L。
在此我们描述了在全细胞系统中使用CYP106A2酶，成功将醋酸环丙孕酮的转化从摇瓶放大到生物反应器。底物被转化为其主要的人体代谢物15β-羟基醋酸环丙孕酮，这是一种极具吸引力的候选药物，因为它保留了抗雄激素活性，但与母体化合物相比，孕激素特性显著降低。该过程的优化使总转化率从55%提高到98%，产物生成量为0.43 g/L，接近工业生产要求和未来大规模应用。
