定向进化在蛋白质工程中的应用研究进展

祁延萍; 朱晋; 张凯; 刘彤; 王雅婕

doi:10.12211/2096-8280.2022-025

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定向进化在蛋白质工程中的应用研究进展

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- 定向进化在蛋白质工程中的应用研究进展
- Recent development of directed evolution in protein engineering
- 合成生物学 2022年3卷第6期页码：1081-1108
- 作者机构：
  
  1.西湖大学工学院，浙江杭州 310024
  2.西湖大学合成生物学与生物智造中心，浙江杭州 331712
- 作者简介：
  
  [ "祁延萍（1995—），女，博士研究生。研究方向为酿酒酵母代谢工程。E-mail：qiyanping@westlake.edu.cn" ]
  [ "王雅婕（1989—），女，西湖大学特聘研究员，博士生导师。研究方向为化学-酶偶联协同催化体系的构建、酶定向进化、代谢工程等。E-mail：wangyajie@westlake.edu.cn" ]
- 基金信息：
  
  西湖大学王雅婕实验室人才引进专项(103110456022101)
- DOI：10.12211/2096-8280.2022-025
  中图分类号：
- 收稿：2022-01-12，
  
  修回：2022-01-20，
  
  纸质出版：2022-12-31
- 稿件说明：
移动端阅览
祁延萍，朱晋，张凯，刘彤，王雅婕. 定向进化在蛋白质工程中的应用研究进展［J］. 合成生物学， 2022， 3（6）： 1081-1108

QI Yanping， ZHU Jin， ZHANG Kai， LIU Tong， WANG Yajie. Recent development of directed evolution in protein engineering［J］. Synthetic Biology Journal， 2022， 3（6）： 1081-1108
祁延萍，朱晋，张凯，刘彤，王雅婕. 定向进化在蛋白质工程中的应用研究进展［J］. 合成生物学， 2022， 3（6）： 1081-1108 DOI： 10.12211/2096-8280.2022-025.

QI Yanping， ZHU Jin， ZHANG Kai， LIU Tong， WANG Yajie. Recent development of directed evolution in protein engineering［J］. Synthetic Biology Journal， 2022， 3（6）： 1081-1108 DOI： 10.12211/2096-8280.2022-025.

摘要

定向进化旨在通过基因多样化和突变体库筛选的迭代循环，加速实现在胞内或胞外进行的自然进化过程。近年来，因其强大的功能而被广泛应用于酶工程当中。本文概述了近十年助力定向进化发展的最新技术，包括胞外和胞内高效构建基因突变体库的方法、高通量筛选突变体库的方法、连续定向进化策略、自动化生物合成平台助力定向进化的策略、计算机技术辅助定向进化的应用实例。为了阐述定向进化在酶工程中的应用价值，本文着重讨论了利用定向进化技术对酶进行改造的代表性案例，其中包括改善酶在有机溶剂中的耐受性、提高酶的热稳定性、增强天然酶对非天然底物的催化能力、提高酶催化化学反应的选择性（包括区域选择性、立体选择性和对映选择性）以及拓展酶催化的反应类型。最后，本文对定向进化在未来可能遇到的挑战及应用前景进行了归纳总结。

Abstract

Directed evolution aims to accelerate the natural evolution process

in vitro

in vivo

through iterative cycles of genetic diversification and screening or selection. It has been one of the most solid and widely used tools in protein engineering. This review outlines the representative methods developed in the past 10 years that increase the throughput of directed evolution

including

in vitro

and

in vivo

gene diversification methods

high-throughput selection and screening methods

continuous evolution strategies

automation-assisted evolution strategies

and AI-assisted protein engineering. To illustrate the significant applications of directed evolution in protein engineering

this review subsequently discusses some remarkable cases to show how directed evolution was used to improve various properties of enzymes

such as the tolerance to elevated temperature or organic solvent

the activities on non-nativ

e substrates

and chemo-

regio-

stereo-

and enantio-selectivities. In addition

directed evolution has also been widely used to expand the biocatalytic repertories by engineering enzymes with abiotic activities. In addition to the native enzymes

directed evolution has also been used to engineer

de novo

designed enzymes and artificial metalloenzymes with activities comparable to or exceeding the ones of the native enzymes. Finally

this review has pointed out that further improving the efficiency and effectiveness of directed evolution remains challenging. Some advanced continuous evolution and high throughput screening strategies have been succesfully demonstrated in improving the throughput of directed evolutions extensively

but they have been limited to engineering certain protein targets. To resolve those issues

continuously improved computational modeling tools and machine learning strategies can assist us to create a smaller but more accurate library to enhance the probabilities of discovering variants with improved properties. Additionally

laboratorial automation platforms coupled with advanced screening and selection techniques also have great potential to extensively explore the protein fitness landscape by evolving multiple targets continuously in a high throughput manner.

关键词

Keywords

references

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