Statistical Analysis of the Benefits of Focused Saturation Mutagenesis in Directed Evolution Based on Reduced Amino Acid Alphabets,ACS Catalysis

当前位置： X-MOL 学术 › ACS Catal. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Statistical Analysis of the Benefits of Focused Saturation Mutagenesis in Directed Evolution Based on Reduced Amino Acid Alphabets
ACS Catalysis ( IF 11.3 ) Pub Date : 2019-07-19 00:00:00 , DOI: 10.1021/acscatal.9b02548
Aitao Li ₁ , Ge Qu ₂ , Zhoutong Sun ₂ , Manfred T. Reetz _{2,

3,

4}

Affiliation

Directed evolution of stereo-, regio-, and chemoselective enzymes has enriched the toolbox of synthetic organic chemistry. Among the different gene mutagenesis techniques, saturation mutagenesis (SM) at sites lining the enzyme’s binding pocket has emerged as a particularly viable approach to control selectivity and activity. Traditionally, NNK codon degeneracy encoding all 20 canonical amino acids is used, but as the size of the randomization site increases beyond a single residue, oversampling of transformants needed to ensure ≥95% library coverage rapidly reaches astronomical dimensions, impossible to screen in a practical manner. Therefore, many groups have been content with screening only a small segment of the designed protein sequence space, but this means that the best mutants will be missed. Alternatively, it has been shown that the use of highly reduced amino acid alphabets allows the generation of small and smart libraries requiring less screening. Here we address the question of which approach is more efficient. Two different enzyme types serve as model systems in stereoselective reactions, limonene epoxide hydrolase and P450-BM3. Equal numbers of transformants were screened for differently sized SM libraries that were constructed using NNK codon degeneracy and other codons corresponding to only one or just a few amino acids. Conversion as a rough indication of activity was used as the parameter in primary screening followed by GC-based ee-determination. Statistical analyses clearly show that it is more efficient to opt for rationally designed reduced amino acid alphabets because this approach results in a distinctly higher frequency of active mutants, stereoselectivity also being notably higher.

中文翻译：

基于减少的氨基酸字母的定向进化中集中饱和诱变的收益的统计分析

立体，区域和化学选择性酶的定向进化丰富了合成有机化学的工具箱。在不同的基因诱变技术中，位于酶结合口袋内衬的位点的饱和诱变（SM）已经成为控制选择性和活性的一种特别可行的方法。传统上，使用编码所有20个规范氨基酸的NNK密码子简并性，但是随着随机化位点的大小增加到超过单个残基，为了确保≥95％的文库覆盖率而需要过度转化的转化子会迅速达到天文尺寸，在实际应用中无法进行筛选方式。因此，许多小组只对筛选设计的蛋白质序列空间的一小部分感到满意，但这意味着将错过最佳的突变体。或者，已经表明，使用高度简化的氨基酸字母可以生成需要较少筛选的小型且智能的文库。在这里，我们讨论哪种方法更有效的问题。两种不同的酶在立体选择性反应中用作模型系统，柠檬烯环氧化物水解酶和P450-BM3。对于使用NNK密码子简并性和仅对应于一个或几个氨基酸的其他密码子构建的大小不同的SM文库，筛选了相同数量的转化子。转化作为活性的粗略指示被用作初步筛选，然后进行基于GC的ee测定的参数。

更新日期：2019-07-19

点击分享查看原文

点击收藏

阅读更多本刊新发论文本刊介绍/投稿指南