饱和突变
合理设计
定向进化
蛋白质工程
突变
定向分子进化
合成生物学
计算生物学
瓶颈
生物
生化工程
计算机科学
遗传学
工程类
生物化学
酶
基因
突变体
嵌入式系统
作者
Danyang Li,Qi Wu,Manfred T. Reetz
出处
期刊:Methods in Enzymology
日期:2020-01-01
卷期号:: 225-242
被引量:51
标识
DOI:10.1016/bs.mie.2020.04.055
摘要
Directed evolution has emerged as the most productive enzyme engineering method, with stereoselectivity playing a crucial role when evolving mutants for application in synthetic organic chemistry and biotechnology. In order to reduce the screening effort (bottleneck of directed evolution), improved methods for the creation of small and smart mutant libraries have been developed, including the combinatorial active-site saturation test (CAST) which involves saturation mutagenesis at appropriate residues surrounding the binding pocket, and iterative saturation mutagenesis (ISM). Nevertheless, even CAST/ISM mutant libraries require a formidable screening effort. Thus far, rational design as the alternative protein engineering technique has had only limited success when aiming for stereoselectivity. Here, we highlight a recent methodology dubbed focused rational iterative site-specific mutagenesis (FRISM), in which mutant libraries are not involved. It makes use of the tools that were previously employed in traditional rational enzyme design, but, inspired by CAST/ISM, the process is performed in an iterative manner. Only a few predicted mutants need to be screened, a fast process which leads to the identification of highly enantioselective and sufficiently active mutants.
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