Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, People's Republic of China.
Appl Microbiol Biotechnol. 2018 Jan;102(2):559-567. doi: 10.1007/s00253-017-8568-y. Epub 2017 Nov 27.
With increasing concerns in sustainable development, biocatalysis has been recognized as a competitive alternative to traditional chemical routes in the past decades. As nature's biocatalysts, enzymes are able to catalyze a broad range of chemical transformations, not only with mild reaction conditions but also with high activity and selectivity. However, the insufficient activity or enantioselectivity of natural enzymes toward non-natural substrates limits their industrial application, while directed evolution provides a potent solution to this problem, thanks to its independence on detailed knowledge about the relationship between sequence, structure, and mechanism/function of the enzymes. A proper high-throughput screening (HTS) method is the key to successful and efficient directed evolution. In recent years, huge varieties of HTS methods have been developed for rapid evaluation of mutant libraries, ranging from in vitro screening to in vivo selection, from indicator addition to multi-enzyme system construction, and from plate screening to computation- or machine-assisted screening. Recently, there is a tendency to integrate directed evolution with metabolic engineering in biosynthesis, using metabolites as HTS indicators, which implies that directed evolution has transformed from molecular engineering to process engineering. This paper aims to provide an overview of HTS methods categorized based on the reaction principles or types by summarizing related studies published in recent years including the work from our group, to discuss assay design strategies and typical examples of HTS methods, and to share our understanding on HTS method development for directed evolution of enzymes involved in specific catalytic reactions or metabolic pathways.
随着可持续发展的关注度不断提高，生物催化在过去几十年中已被视为传统化学路线的一种有竞争力的替代方法。作为自然界的生物催化剂，酶能够催化广泛的化学转化，不仅具有温和的反应条件，而且具有高活性和选择性。然而，天然酶对非天然底物的活性或对映选择性不足限制了它们的工业应用，而定向进化提供了一种有效的解决方案，这得益于它不依赖于对酶的序列、结构和机制/功能之间关系的详细了解。适当的高通量筛选 (HTS) 方法是成功和高效定向进化的关键。近年来，已经开发出了各种高通量筛选方法，用于快速评估突变文库，从体外筛选到体内选择，从指示剂添加到多酶系统构建，从平板筛选到计算或机器辅助筛选。最近，在生物合成中，定向进化与代谢工程相结合的趋势越来越明显，使用代谢物作为 HTS 指标，这意味着定向进化已经从分子工程转变为过程工程。本文旨在根据近年来发表的相关研究，包括我们小组的工作，按反应原理或类型对 HTS 方法进行分类综述，讨论测定设计策略和 HTS 方法的典型例子，并分享我们对特定催化反应或代谢途径中酶的定向进化的 HTS 方法开发的理解。
