Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, 52074, Aachen, Germany.
Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf, Wilhelm Johnen Strasse, 52426, Jülich, Germany.
Chemistry. 2021 Feb 5;27(8):2789-2797. doi: 10.1002/chem.202004471. Epub 2021 Jan 7.
The CompassR (computer-assisted recombination) rule enables, among beneficial substitutions, the identification of those that can be recombined in directed evolution. Herein, a recombination strategy is systematically investigated to minimize experimental efforts and maximize possible improvements. In total, 15 beneficial substitutions from Bacillus subtilis lipase A (BSLA), which improves resistance to the organic cosolvent 1,4-dioxane (DOX), were studied to compare two recombination strategies, the two-gene recombination process (2GenReP) and the in silico guided recombination process (InSiReP), employing CompassR. Remarkably, both strategies yielded a highly DOX-resistant variant, M4 (I12R/Y49R/E65H/N98R/K122E/L124K), with up to 14.6-fold improvement after screening of about 270 clones. M4 has a remarkably enhanced resistance in 60 % (v/v) acetone (6.0-fold), 30 % (v/v) ethanol (2.1-fold), and 60 % (v/v) methanol (2.4-fold) compared with wild-type BSLA. Molecular dynamics simulations revealed that attracting water molecules by charged surface substitutions is the main driver for increasing the DOX resistance of BSLA M4. Both strategies and obtained molecular knowledge can likely be used to improve the properties of other enzymes with a similar α/β-hydrolase fold.
CompassR（计算机辅助重组）规则允许在有益取代中识别那些可以在定向进化中重组的取代。在此，系统地研究了一种重组策略，以最小化实验工作量并最大化可能的改进。总共研究了来自枯草芽孢杆菌脂肪酶 A（BSLA）的 15 个有益取代，这些取代可提高对有机共溶剂 1,4-二恶烷（DOX）的抗性，以比较两种重组策略，即双基因重组过程（2GenReP）和基于 CompassR 的计算机指导重组过程（InSiReP）。值得注意的是，两种策略都产生了一种高度耐受 DOX 的变体 M4（I12R/Y49R/E65H/N98R/K122E/L124K），经过大约 270 个克隆的筛选后，其 DOX 抗性提高了 14.6 倍。与野生型 BSLA 相比，M4 在 60%（v/v）丙酮（6.0 倍）、30%（v/v）乙醇（2.1 倍）和 60%（v/v）甲醇（2.4 倍）中的抗性显著增强。分子动力学模拟表明，通过带电荷的表面取代吸引水分子是提高 BSLA M4 对 DOX 抗性的主要驱动力。这两种策略和获得的分子知识可能用于改善具有类似α/β-水解酶折叠的其他酶的性质。
