定向进化在蛋白质工程中的应用研究进展

doi:10.12211/2096-8280.2022-025

摘要/Abstract

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

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

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.

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.

图/表 18

图1 蛋白质定向进化原理

Fig. 1 The principle of directed evolution

图2 基于CRISPR的体内突变方法［7］a—基于CRISPR-Cas9同源重组的突变；b—基于nCas9和DNA聚合酶I的突变；c—基于nCas9和碱基编辑器的突变

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

图3 噬菌体辅助连续进化过程［71］

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

表1 PACE系统改造蛋白实例

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]

图4 AlphaFold 2计算原理示意图

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

图5 Rosetta的常用功能［93］

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

图6 NOD催化硅烷和重氮乙酸乙酯的卡宾插入构建C—Si键

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

图7 含27个突变的转氨酶催化的西格列汀的合成及与化学催化合成的对比

Fig. 7 Improvement of the synthetic process of sitagliptin

图8 体外九酶级联催化合成依拉曲韦

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

图9 基于P450变体催化的不对称合成［106］

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

图10 P411Diane2催化的不对称C（sp3）—H氨基化

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

图11 P411-E10催化的苯乙炔与重氮乙酸乙酯的双卡宾转移

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

图12 P450ATRCase催化的分子内原子转移自由基加成

Fig. 12 P450ATRCase catalyzed intramolecular atom transfer radical cyclization

图 13 Aldolase催化的可逆羟醛缩合反应及β-羰基酮与氨基酸残基形成乙烯基酰胺的催化抑制机理

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

图 14 Diels-Alder酶催化的1，3-丁二烯氨基甲酸酯与二甲基丙烯酰胺的D-A反应

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

图 15 Biot-Ru-SAV催化的闭环烯烃复分解

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

图16 双金链霉亲和素催化的乙炔脲区域选择性分子内环化

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

图17 （a） Ir-Cyp119催化的柠檬烯的环丙化；（b） Ir-Cyp119催化的对映选择性、区域选择性C—H活化

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

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