Department of Biochemistry, Saarland University, Campus B2.2, 66123 Saarbruecken, Germany.
Department of Biochemistry, Saarland University, Campus B2.2, 66123 Saarbruecken, Germany.
Biochim Biophys Acta Proteins Proteom. 2018 Jan;1866(1):11-22. doi: 10.1016/j.bbapap.2017.07.011. Epub 2017 Aug 2.
CYP106A2 from Bacillus megaterium ATCC13368, was identified in the 1970s as one of the first bacterial steroid hydroxylases responsible for the conversion of progesterone to 15β-hydroxyprogesterone. Later on it has been proven to be a potent hydroxylase of numerous 3-oxo-Δ as well as 3-hydroxy-Δ-steroids and has recently also been characterized as a regioselective allylic bacterial diterpene hydroxylase. The main hydroxylation position of CYP106A2 is thought to be influenced by the functional groups at C3 position in the steroid core leading to a favored 15β-hydroxylation of 3-oxo-Δ-steroids and 7β-hydroxylation of 3-hydroxy-Δ-steroids. However, in some cases the hydroxylation is not strictly selective, resulting in the formation of undesired side-products. To overcome the unspecific hydroxylations or, on the contrary, to gain more of these products in case they are of industrial interest, rational protein design and directed evolution have been successfully performed to shift the stereoselectivity of hydroxylation by CYP106A2. The subsequently obtained hydroxylated steroid and terpene derivatives are especially useful as drug metabolites and drug precursors for the pharmaceutical industry, due to their diverse biological properties and hardship of their chemical synthesis. As a soluble prokaryotic P450 with broad substrate spectrum and hydroxylating capacity, CYP106A2 is an outstanding candidate to establish bioconversion processes. It has been expressed with respectable yields in Escherichia coli and Bacillus megaterium and was applied for the preparative hydroxylation of several steroids and terpenes. Recently, the application of the enzyme was assessed under process conditions as well, depicting a successfully optimized process development and getting us closer to industrial scale process requirements and a future large scale application. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.
从巨大芽孢杆菌 ATCC13368 中鉴定出的 CYP106A2 是上世纪 70 年代发现的首批负责将孕酮转化为 15β-羟基孕酮的细菌甾体羟化酶之一。后来证明，它是许多 3-氧代-Δ 以及 3-羟基-Δ-甾体的有效羟化酶，最近还被表征为具有区域选择性的烯丙基细菌二萜羟化酶。CYP106A2 的主要羟化位置被认为受甾体核心 C3 位的官能团影响，导致 3-氧代-Δ-甾体优先进行 15β-羟化，3-羟基-Δ-甾体优先进行 7β-羟化。然而，在某些情况下，羟化并非严格选择性的，导致形成不需要的副产物。为了克服非特异性羟化，或者相反，在它们具有工业兴趣的情况下获得更多产物，可以通过合理的蛋白质设计和定向进化来改变 CYP106A2 的羟化立体选择性。随后获得的羟基化甾体和萜烯衍生物由于其多样的生物学性质和化学合成的困难，特别可用作药物代谢物和药物前体，用于制药工业。作为一种具有广泛底物谱和羟化能力的可溶性原核 P450，CYP106A2 是建立生物转化过程的杰出候选者。它在大肠杆菌和巨大芽孢杆菌中以可观的产量表达，并已应用于几种甾体和萜烯的制备性羟化。最近，该酶在过程条件下的应用也得到了评估，展示了成功优化的过程开发，并使我们更接近工业规模过程要求和未来的大规模应用。本文是主题为“细胞色素 P450 生物多样性和生物技术”的特刊的一部分，由 Erika Plettner、Gianfranco Gilardi、Luet Wong、Vlada Urlacher 和 Jared Goldstone 编辑。
