Belsare Ketaki D, Horn Thomas, Ruff Anna Joëlle, Martinez Ronny, Magnusson Anders, Holtmann Dirk, Schrader Jens, Schwaneberg Ulrich
Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
Biochemical Engineering Group, DECHEMA Research Institute, Theodor-Heuss-Allee 25, 60486 Frankfurt am Main, Germany.
Protein Eng Des Sel. 2017 Feb;30(2):119-127. doi: 10.1093/protein/gzw072. Epub 2016 Dec 22.
Directed evolution is a powerful method to optimize enzyme properties for application demands. Interesting targets are P450 monooxygenases which catalyze the stereo- and regiospecific hydroxylation of chemically inert C-H bonds. Synthesis employing P450s under cell-free reaction conditions is limited by low total turnover numbers, enzyme instability, low product yields and the requirement of the expensive co-factor NADPH. Bioelectrocatalysis is an alternative to replace NADPH in cell-free P450-catalyzed reactions. However, natural enzymes are often not suitable for using non-natural electron delivery systems. Here we report the directed evolution of a previously engineered P450 CinA-10aa-CinC fusion protein (named P450cin-ADD-CinC) to use zinc/cobalt(III)sepulchrate as electron delivery system for an increased hydroxylation activity of 1,8-cineole. Two rounds of Sequence Saturation Mutagenesis (SeSaM) each followed by one round of multiple site-saturation mutagenesis of the P450 CinA-10aa-CinC fusion protein generated a variant (Gln385His, Val386Ser, Thr77Asn, Leu88Arg; named KB8) with a 3.8-fold increase in catalytic efficiency (28 µM min) compared to P450cin-ADD-CinC (7 µM min). Furthermore, variant KB8 exhibited a 1.5-fold higher product formation (500 µM µM P450) compared to the equimolar mixture of CinA, CinC and Fpr using NADPH as co-factor (315 µM µM P450). In addition, electrochemical experiments with the electron delivery system platinum/cobalt(III)sepulchrate showed that the KB8 variant had a 4-fold higher product formation rate (0.16 nmol (nmol) P450 min cm) than the P450cin-ADD-CinC (0.04 nmol (nmol) P450 min cm). In summary, the current work shows prospects of using directed evolution to generate P450 enzymes suitable for use with alternative electron delivery systems.
定向进化是一种根据应用需求优化酶特性的强大方法。有趣的目标是细胞色素P450单加氧酶，它能催化化学惰性C-H键的立体和区域特异性羟基化反应。在无细胞反应条件下使用细胞色素P450进行合成受到总周转数低、酶不稳定性、产物收率低以及需要昂贵的辅因子NADPH的限制。生物电催化是在无细胞细胞色素P450催化反应中替代NADPH的一种方法。然而，天然酶通常不适用于使用非天然电子传递系统。在此，我们报道了对先前工程化的细胞色素P450 CinA-10aa-CinC融合蛋白（命名为P450cin-ADD-CinC）进行定向进化，以使用锌/钴（III）sepulchrate作为电子传递系统，从而提高1,8-桉叶素的羟基化活性。对细胞色素P450 CinA-10aa-CinC融合蛋白进行两轮序列饱和诱变（SeSaM），随后进行一轮多位点饱和诱变，产生了一个变体（Gln385His、Val386Ser、Thr77Asn、Leu88Arg；命名为KB8），与P450cin-ADD-CinC（7 μM min）相比，其催化效率提高了3.8倍（28 μM min）。此外，与使用NADPH作为辅因子的CinA、CinC和Fpr的等摩尔混合物（315 μM μM P450）相比，变体KB8的产物形成量高1.5倍（500 μM μM P450）。此外，使用电子传递系统铂/钴（III）sepulchrate进行的电化学实验表明，KB8变体的产物形成速率（0.16 nmol（nmol）P450 min cm）比P450cin-ADD-CinC（0.04 nmol（nmol）P450 min cm）高4倍。总之，目前的工作展示了利用定向进化产生适用于替代电子传递系统的细胞色素P450酶的前景。
