Recent development of directed evolution in protein engineering

doi:10.12211/2096-8280.2022-025

Abstract

Abstract:

Directed evolution aims to accelerate the natural evolution process in vitro or 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-native 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.

Key words: directed evolution, protein engineering, enzymatic reaction, biocatalysis

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。

关键词: 定向进化, 蛋白质工程, 酶工程, 生物催化

Yanping QI, Jin ZHU, Kai ZHANG, Tong LIU, Yajie WANG. 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.

Figures/Tables 18

Fig. 1 The principle of directed evolution

Fig. 2 CRISPR-assisted in vivo mutagenesis[7]a—CRISPR-Cas9-HDR; b—Random mutagenesis induced by nCas9-E. coli DNA PolI (error-prone) hybrid proteins; c—Gene mutangenesis caused by nCas9-deaminase hybrid proteins

Fig. 3 Progress of phage-assisted continuous evolution (PACE)[71]

Tab. 1 Cases for engineering proteins through PACE

目标蛋白 Target protein	目标性状 Target phenotype	基因回路设计 Genetic circuit design
T7聚合酶	拓宽可识别的启动子范围^[71]
T7聚合酶	增强识别人工启动子的特异性^[72]
TALEN	DNA序列识别特异性^[73]
spCas9	拓宽可识别的PAM序列^[74]
胞嘧啶碱基编辑器(CBEs)	拓宽可编辑的基因序列范围（例如GC丰富的序列）^[75]
腺嘌呤碱基编辑器（ABEs)	提高与Cas结构域的兼容性和编辑活性^[76]
苏云金芽孢杆菌δ-内毒素	增强与毛滴虫的钙黏蛋白样受体结合亲和力^[77]
抗体、麦芽糖结合蛋白	增强目标蛋白可溶性表达^[78]
蛋白水解酶	提高水解酶催化活性及底物特异性^[79-80]
肉毒神经毒素	使肉毒神经毒素有可编程的特异性^[81]
氨酰-tRNA 合成酶	生产高活性和选择性的正交氨基酰-tRNA合成酶^[82]

Fig. 4 The information flow among the various components of AlphaFold 2[94]

Fig. 5 Some popular tasks that can be addressed in Rosetta (blue) and major systems that can be modeled (red)[93]

Fig. 6 NOD catalyzed carbene insertion to construct C—Si bond

Fig. 7 Improvement of the synthetic process of sitagliptin

Fig. 8 Nine-enzymes cascade catalyzed synthesis of islatravir in vitro

Fig. 9 Asymmetric synthesis catalyzed by P450 variants[106]

Fig. 10 P411Diane2 catalyzed asymmetric amination of primary, secondary and tertiary C(sp3)—H bonds via nitrene insertion

Fig. 11 P411-E10 catalyzed double carbene transfer of phenylacetylene with EDA

Fig. 12 P450ATRCase catalyzed intramolecular atom transfer radical cyclization

Fig. 13 Aldolase-catalyzed reversible aldol condensation reaction and the catalytic inhibition mechanism of β-carbonyl ketones with amino acid residues to form vinyl amides

Fig. 14 DA_20_10 catalyzed D-A reaction of 1,3-butadiene carbamate and dimethylacrylamide

Fig. 15 Biot-Ru-SAV catalyzed closed-loop olefin metathesis

Fig. 16 Regioselective intramolecular cyclization of ethynylphenylurea catalyzed by dual gold Sav-SOD catalysis

Fig. 17 (a) Ir-Cyp119 catalyzed cyclopropanation of limonene; (b) Ir-Cyp119 catalyzed enantioselective and site-selective C—H activation

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