Institute of Biotechnology 2, Forschungszentrum Jülich GmbH, Germany.
Chembiochem. 2010 Mar 22;11(5):713-21. doi: 10.1002/cbic.200900706.
Cytochrome P450 monooxygenases are of outstanding interest for the synthesis of pharmaceuticals and fine chemicals, due to their ability to hydroxylate C--H bonds mainly in a stereo- and regioselective manner. CYP106A2 from Bacillus megaterium ATCC 13368, one of only a few known bacterial steroid hydroxylases, enables the oxidation of 3-keto-4-ene steroids mainly at position 15. We expressed this enzyme together with the electron-transfer partners bovine adrenodoxin and adrenodoxin reductase in Escherichia coli. Additionally an enzyme-coupled cofactor regeneration system was implemented by expressing alcohol dehydrogenase from Lactobacillus brevis. By studying the conversion of progesterone and testosterone, the bottlenecks of these P450-catalyzed hydroxylations were identified. Substrate transport into the cell and substrate solubility turned out to be crucial for the overall performance. Based on these investigations we developed a new concept for CYP106A2-catalyzed steroid hydroxylations by which the productivity of progesterone and testosterone conversion could be increased up to 18-fold to yield an absolute productivity up to 5.5 g L(-1) d(-1). Product extraction with absorber resins allowed the recovery of quantitative amounts of 15beta-OH-progesterone and 15beta-OH-testosterone and also the reuse of the biocatalyst.
细胞色素 P450 单加氧酶因其能够主要以立体和区域选择性方式羟化 C--H 键，而对药物和精细化学品的合成具有重要意义。来自巨大芽孢杆菌 ATCC 13368 的 CYP106A2 是少数已知的细菌甾体羟化酶之一，能够主要在 15 位氧化 3-酮-4-烯甾体。我们在大肠杆菌中表达了这种酶，以及电子转移伙伴牛肾上腺皮质酮和肾上腺皮质酮还原酶。此外，通过表达短乳杆菌的醇脱氢酶，还实现了酶偶联辅酶再生系统。通过研究孕酮和睾酮的转化，可以确定这些 P450 催化羟化反应的瓶颈。底物向细胞内的输送和底物的溶解度对整体性能至关重要。基于这些研究，我们开发了一种新的 CYP106A2 催化甾体羟化概念，通过该概念，孕酮和睾酮转化率的生产力可提高 18 倍，达到 5.5 g L(-1) d(-1)的绝对生产力。用吸收树脂进行产物萃取可定量回收 15β-OH-孕酮和 15β-OH-睾酮，并可重复使用生物催化剂。
