The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes.,Angewandte Chemie International Edition

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The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes.
Angewandte Chemie International Edition ( IF 16.1 ) Pub Date : 2019-07-02 , DOI: 10.1002/anie.201901491
Ge Qu ₁ , Aitao Li ₂ , Carlos G Acevedo-Rocha ₃ , Zhoutong Sun ₁ , Manfred T Reetz _{1,

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Affiliation

Directed evolution of stereo‐, regio‐, and chemoselective enzymes constitutes a unique way to generate biocatalysts for synthetically interesting transformations in organic chemistry and biotechnology. In order for this protein engineering technique to be efficient, fast, and reliable, and also of relevance to synthetic organic chemistry, methodology development was and still is necessary. Following a description of early key contributions, this review focuses on recent developments. It includes optimization of molecular biological methods for gene mutagenesis and the design of efficient strategies for their application, resulting in notable reduction of the screening effort (bottleneck of directed evolution). When aiming for laboratory evolution of selectivity and activity, second‐generation versions of Combinatorial Active‐Site Saturation Test (CAST) and Iterative Saturation Mutagenesis (ISM), both involving saturation mutagenesis (SM) at sites lining the binding pocket, have emerged as preferred approaches, aided by in silico methods such as machine learning. The recently proposed Focused Rational Iterative Site‐specific Mutagenesis (FRISM) constitutes a fusion of rational design and directed evolution. On‐chip solid‐phase chemical gene synthesis for rapid library construction enhances library quality notably by eliminating undesired amino acid bias, the future of directed evolution?

中文翻译：

方法论发展在选择性酶的定向进化中的关键作用。

立体，区域和化学选择性酶的定向进化构成了一种生成生物催化剂的独特方法，可用于有机化学和生物技术领域中有趣的合成转化。为了使这种蛋白质工程技术高效，快速和可靠，并且与合成有机化学有关，方法学的开发仍然是必要的。在描述了早期的主要贡献之后，本文将重点介绍最新的发展。它包括针对基因诱变的分子生物学方法的优化和有效应用策略的设计，从而显着减少了筛选工作（定向进化的瓶颈）。当针对选择性和活性的实验室进化时，结合主动口袋饱和度测试（CAST）和迭代饱和诱变（ISM）的第二代版本都涉及结合口袋内衬位点的饱和诱变（SM），在计算机等计算机方法的帮助下，它们已成为首选方法学习。最近提出的聚焦理性迭代定点诱变（FRISM）构成了理性设计和定向进化的融合。用于快速构建文库的片上固相化学基因合成可通过消除不需要的氨基酸偏倚（定向进化的未来）显着提高文库质量。最近提出的聚焦理性迭代定点诱变（FRISM）构成了理性设计和定向进化的融合。用于快速构建文库的片上固相化学基因合成可通过消除不需要的氨基酸偏倚（定向进化的未来）显着提高文库质量。最近提出的聚焦理性迭代定点诱变（FRISM）构成了理性设计和定向进化的融合。用于快速构建文库的片上固相化学基因合成可通过消除不需要的氨基酸偏倚（定向进化的未来）显着提高文库质量。

更新日期：2019-07-02

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