Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (1): 42-51    DOI: 10.13523/j.cb.2009017
综述     
体内连续定向进化研究进展 *
察亚平1,朱牧孜2,李爽1,**()
1 华南理工大学生物科学与工程学院 广州 510006
2 广东省微生物研究所 广州 510070
Research Progress on In Vivo Continuous Directed Evolution
CHA Ya-ping1,ZHU Mu-zi2,LI Shuang1,**()
1 School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China
2 State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China
 全文: PDF(13868 KB)   HTML
摘要：

定向进化为合成生物学的发展提供了一种简单高效的工具,尤其在化学品合成和医药开发方面发挥着重要的作用。但是传统的定向进化技术存在操作繁琐、耗时和效率低的问题,不能满足大量突变文库的构建和筛选。近几年,一项将突变、翻译(进化非基因)、筛选和复制过程进行无缝连接的体内连续定向进化技术开始出现,该技术在噬菌体、细菌和真核细胞中均取得了突破性进展,极大地促进了定向进化技术的革新和应用。随着体内连续定向进化技术的不断发展,筛选方法和设备也不断改善。对体内连续定向进化技术、筛选方法和设备最新研究进展作一综述,并讨论当前面临的挑战和机遇。
关键词： 体内连续定向进化;  合成生物学;  高通量筛选;  进化设备    
Abstract:

Directed evolution provides a simple and high-efficiency tool for the development of synthetic biology, especially in the chemical synthesis and medicine. However, the traditional directed evolution technique has the problems of cumbersome operation, time-consuming and low-efficiency, which cannot satisfy the construction and screening of mass mutant libraries. In recent years, a technique about in vivo continuous directed evolution that seamlessly integrates mutation, translation (if the evolving molecules are not genes themselves), screening and replication processes into an uninterrupted cycle has emerged, which makes breakthrough in phage, bacteria and eukaryotic cells, greatly facilitating technological innovation of directed evolution. Simultaneously, with the development of in vivo continuous evolution technology, screening methods and evolutionary devices are also constantly improved. Here, this review is done to expound the latest research progress on continuous directed evolution techniques, screening methods and devices, and discuss the current challenges and opportunities.
Key words: In vivo continuous directed evolution    Synthetic biology    High-throughput screening    Evolutionary device
收稿日期: 2020-09-14 出版日期: 2021-02-09
ZTFLH:  Q819  
基金资助: * 国家自然科学基金(21878104);国家重点研发计划(2018YFA0901504)
通讯作者: 李爽     E-mail: shuangli@scut.edu.cn
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
察亚平
朱牧孜
李爽

引用本文:

察亚平, 朱牧孜, 李爽. 体内连续定向进化研究进展 *[J]. 中国生物工程杂志, 2021, 41(1): 42-51.

CHA Ya-ping, ZHU Mu-zi, LI Shuang. Research Progress on In Vivo Continuous Directed Evolution. China Biotechnology, 2021, 41(1): 42-51.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2009017    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I1/42

图1  噬菌体连续定向进化技术-PACE
图2  细菌连续定向进化技术-EvolvR
图3  真核细胞体内连续定向进化技术
定向进化 技术 突变位置 连续 诱变因素 突变速率/bp/代 优点 缺点 参考文献
噬菌体 PACE 靶基因
正交		 连续 M13聚合酶 2.3×10-3 进化速度快应用范围广 仅限于E. coli的细胞质 [14-24]
细菌 MAGE/GREACE 靶基因非
正交		 不连续 化学合成/dnaQ聚合酶 1.35×10-7
3×10-8		 自动整合至基因组 需人工设计突变文库,不连续 [25-26]
MMR/TM-MAGE/
pOPRTMAGE		 基因组 连续 敲除甲基修复基因mutS/mutL 1×10-8 可对细胞基因组进行突变和进化 无法针对特定基因进化 [27-29]
核糖体开关/双功能开关 基因组 连续 dnaQ聚合酶 - 对细胞基因组进行精准调控 无法针对特定基因进化 [30]
EvolvR 靶基因不
完全正交		 不连续 易错聚合酶 2.4×10-6 对特定基因
进化		 可编辑基因长度受限(≤350 bp) [32]
真核细胞 CRISPR-X 靶基因
非正交		 不连续 胞苷脱氨酶 1×10-3 特定类型碱基的突变 只能实现C到T的突变 [35]
ICE 靶基因正交 连续 易错逆转录酶 1.5×10-4 可体内连续
进化		 干扰细胞生长 [39]
OrthoRep 靶基因正交 连续 易错DNA聚合酶 1×10-5 可体内连续
进化		 系统设计复杂,细胞负担较大 [38]
表1  体内连续定向进化技术的比较
[1] Mills D R, Peterson R L, Spiegelman S. An extracellular darwinian experiment with a self-duplicating nucleic acid molecule. Proceedings of the National Academy of Sciences, 1967,58(1):217-224.
[2] Chen K, Arnold F H. Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proceedings of the National Academy of Sciences, 1993,90(12):5618-5622.
[3] Stemmer W P. Rapid evolution of a protein in vitro by DNA shuffling. Nature, 1994,370(6488):389-391.
doi: 10.1038/370389a0 pmid: 8047147
[4] Lutz S. Beyond directed evolution-semi-rational protein engineering and design. Current Opinion in Chemical Biology, 2010,21(6):734-743.
[5] Avoigt C, Kauffman S, Wang Z G. Rational evolutionary design: the theory of in vitro protein evolution. Evolutionary Protein Design, 2001,55:79-160.
[6] Arnold F H, Georgiou G. Directed enzyme evolution: screening and selection methods. Totowa: Humana Press, 2003: 1-2.
[7] Lang G I, Murray A W. Estimating the per-base-pair mutation rate in the yeast Saccharomyces cerevisiae. Genetics, 2008,178(1):67-82.
pmid: 18202359
[8] Camps M, Naukkarinen J, Johnson B P, et al. Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I. Proceedings of the National Academy of Sciences, 2003,100(17):9727-9732.
[9] Larrea A A, Lujan S A, Kunkel T A. SnapShot: DNA mismatch repair. Cell, 2010,141(4):730-731e1.
pmid: 20478261
[10] Morrison M S, Podracky C J, Liu D R. The developing toolkit of continuous directed evolution. Nature Chemical Biology, 2020,16(6):610-619.
[11] Badran A H, Liu D R. In vivo continuous directed evolution. Current Opinion in Chemical Biology, 2015,24:1-10.
pmid: 25461718
[12] D’Oelsnitz S, Ellington A. Continuous directed evolution for strain and protein engineering. Current Opinion in Chemical Biology, 2018,53:158-163.
[13] Husimi Y. Selection and evolution of bacteriophages in cellstat. Advances in Biophysics, 1989,25:1-43.
doi: 10.1016/0065-227x(89)90003-8 pmid: 2696338
[14] Esvelt K M, Carlson J C, Liu D R. A system for the continuous directed evolution of biomolecules. Nature, 2011,472(7344):499-503.
[15] Carlson J C, Badran A H, Guggiana-Nilo D A, et al. Negative selection and stringency modulation in phage-assisted continuous evolution. Nature Chemical Biology, 2014,10(3):216-222.
doi: 10.1038/NCHEMBIO.1453
[16] Dickinson B C, Packer M S, Badran A H, et al. A system for the continuous directed evolution of proteases rapidly reveals drug-resistance mutations. Nature Communications, 2014,5:5352.
[17] Packer M S, Rees H A, Liu D R. Phage-assisted continuous evolution of proteases with altered substrate specificity. Nature Communications, 2017,8(1):956.
[18] Badran A H, Guzov V M, Huai Q, et al. Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance. Nature, 2016,533(7601):58-63.
pmid: 27120167
[19] Wang T, Badran A H, Huang T P, et al. Continuous directed evolution of proteins with improved soluble expression. Nature Chemical Biology, 2018,14(10):972-980.
pmid: 30127387
[20] Bryson D, Fan C, Guo L T, et al. Continuous directed evolution of aminoacyl-tRNA synthetases. Nature Chemical Biology, 2017,13(2):1253-1260.
[21] Hubbard B P, Badran A H, Zuris J A, et al. Continuous directed evolution of DNA-binding proteins to improve TALEN specificity. Nature Methods, 2015,12(10):939-942.
pmid: 26258293
[22] Hu J H, Miller S M, Geurts M H, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature, 2018,556(7699):57-63.
[23] Miller S M, Wang T, Randolph P B, et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nature Biotechnology, 2020,38(4):471-481.
pmid: 32042170
[24] Thuronyi B W, Koblan L W, Levy J M, et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nature Biotechnology, 2019,37(9):1070-1079.
[25] Wang H H, Isaacs F J, Carr P A, et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature, 2009,460(7257):894-898.
pmid: 19633652
[26] Luan G, Cai Z, Li Y, et al. Genome replication engineering assisted continuous evolution (GREACE) to improve microbial tolerance for biofuels production. Biotechnology for Biofuels, 2013,6(1):137.
[27] Luan G D, Cai Z, Luan G, et al. Developing controllable hypermutable clostridium cells through manipulating its methyl-directed mismatch repair system. Protein Cell, 2013,4(11):854-862.
[28] Lennen R M, Nilsson Wallin A I, Pedersen M, et al. Transient overexpression of DNA adenine methylase enables efficient and mobile genome engineering with reduced off-target effects. Nucleic Acids Research, 2016,44(4):e36.
[29] Nyerges A, Csorgo B, Nagy I, et al. A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species. Proceedings of the National Academy of Sciences, 2016,113(9):2502-2507.
[30] Pham H L, Wong A, Chua N, et al. Engineering a riboswitch-based genetic platform for the self-directed evolution of acid-tolerant phenotypes. Nature Communications, 2017,8(1):411.
[31] Zhu L J, Li Y, Cai Z. Development of a stress-induced mutagenesis module for autonomous adaptive evolution of Escherichia coli to improve its stress tolerance. Biotechnology for Biofuels, 2015,8:93.
pmid: 26136829
[32] Halperin S O, Tou C J, Wong E B, et al. CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature, 2018,560(7717):248-252.
[33] Si T, Chao R, Min Y H, et al. Automated multiplex genome-scale engineering in yeast. Nature Communications, 2017,8:15187.
doi: 10.1038/ncomms15187 pmid: 28469255
[34] Garst A D, Bassalo M C, Pines G, et al. Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering. Nature Biotechnology, 2017,35(1):48-55.
[35] Hess G T, Fresard L, Han K, et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nature Methods, 2016,13(12):1036-1042.
[36] Ravikumar A, Arrieta A, Liu C C. An orthogonal DNA replication system in yeast. Nature Chemical Biology, 2014,10(3):175-177.
[37] Arzumanyan G, Gabriel K N, Ravikumar A, et al. Mutually orthogonal DNA replication systems in vivo. ACS Synthetic Biology, 2018,7(7):1722-1729.
[38] Ravikumar A, Arzumanyan G A, Obadi M K A, et al. Scalable, continuous evolution of genes at mutation rates above genomic error thresholds. Cell, 2018,175(7):1946-1957.
pmid: 30415839
[39] Crook N, Abatemarco J, Sun J, et al. In vivo continuous evolution of genes and pathways in yeast. Nature Communications, 2016,7:13051.
doi: 10.1038/ncomms13051 pmid: 27748457
[40] Romero P A, Arnold F H. Exploring protein fitness landscapes by directed evolution. Nature Reviews Molecular Cell Biology, 2009,10(12):866-876.
[41] Feist A M, Zielinski D C, Orth J D, et al. Model-driven evaluation of the production potential for growth-coupled products of Escherichia coli. Metabolic Engineering, 2010,12(3):173-186.
[42] Mohamed E T, Wang S, Lennen R M, et al. Generation of a platform strain for ionic liquid tolerance using adaptive laboratory evolution. Microbial Cell Factories, 2017,16(1):204.
[43] Caspeta L, Chen Y, Ghiaci P, et al. Altered sterol composition renders yeast thermotolerant. Science, 2014,346(6205):75-78.
pmid: 25278608
[44] Xie W P, Lv X M, Ye L D, et al. Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering. Metabolic Engineering, 2015,30:69-78.
pmid: 25959020
[45] Engström K, Nyhleén J, Sandström A G, et al. Directed evolution of an enantioselective lipase with broad substrate scope for hydrolysis of α-substituted esters. Journal of the American Chemical Society, 2010,132(20):7038-7042.
[46] Shepelin D, Hansen A S L, Lennen R, et al. Selecting the best: evolutionary engineering of chemical production in microbes. Genes, 2018,9(5):249.
[47] Burgard A P, Pharkya P, Maranas C D. Optknock: a bilevel programming framework for identifying gene knockout strategies for microbial strain optimization. Biotechnology and Bioengineering, 2003,84(6):647-657.
doi: 10.1002/bit.10803 pmid: 14595777
[48] Hassanpour N, Ullah E, Yousofshahi M, et al. Selection finder (SelFi): a computational metabolic engineering tool to enable directed evolution of enzymes. Metabolic Engineering Communications, 2017,4:37-47.
[49] Zhou S, Shanmugam K T, Ingram L O. Functional replacement of the Escherichia coli D-(-)-lactate dehydrogenase gene (ldhA) with the L-(+)-lactate dehydrogenase gene (ldhL) from Pediococcus acidilactici. Applied and Environmental Microbiology, 2003,69(4):2237-2244.
[50] Zhang X, Jantama K, Moore J C, et al. Production of L-alanine by metabolically engineered Escherichia coli. Applied and Environmental Microbiology, 2007,77(2):355-366.
[51] Shen C R, Lan E I, Dekishima Y, et al. Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli enabled by driving forces. Applied and Environmental Microbiology, 2011,77(9):2905-2915.
doi: 10.1128/AEM.03034-10 pmid: 21398484
[52] Jantama K, Haupt M J, Svoronos S A, et al. Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of Escherichia coli C that produce succinate and malate. Biotechnology and Bioengineering, 2008,99(5):1140-1153.
[53] Tai Y S, Xiong M, Jambunathan P, et al. Engineering nonphosphorylative metabolism to generate lignocellulose-derived products. Nature Chemical Biology, 2016,12(4):247-253.
pmid: 26854668
[54] Reyes L H, Gomez J M, Kao K C. Improving carotenoids production in yeast via adaptive laboratory evolution. Metabolic Engineering, 2014,21:26-33.
doi: 10.1016/j.ymben.2013.11.002 pmid: 24262517
[55] Minty J J, Lesnefsky A A, Lin F M, et al. Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli. Microbial Cell Factories, 2011,10(1):18.
[56] Zhuang Y, Yang G Y, Chen X, et al. Biosynthesis of plant-derived ginsenoside Rh2 in yeast via repurposing a key promiscuous microbial enzyme. Metabolic Engineering, 2017,42:25-32.
pmid: 28479190
[57] Cho S M. Optimizing tyrosine production in yeast via in vivo continuous evolution. Austin: The University of Texas at Austin, 2016.
[58] Tan Y M, Zhang Y, Han Y B, et al. Directed evolution of an α1, 3-fucosyltransferase using a single-cell ultrahigh-throughput screening method. Science Advances, 2019, 5(10): eaaw8451.
[59] Kortmann M, Mack C, Baumgart M, et al. Pyruvate carboxylase variants enabling improved Lysine production from glucose identified by biosensor-based high-throughput fluorescence-activated cell sorting screening. ACS Synthetic Biology, 2019,8(2):274-281.
doi: 10.1021/acssynbio.8b00510 pmid: 30707564
[60] Agresti J J, Antipov E, Abate A R, et al. Ultrahigh-throughput screening in drop-based microfluidics for directed evolution. Proceedings of the National Academy of Sciences, 2010,107(9):4004-4009.
[61] Saleski T E, Kerner A R, Chung M T, et al. Syntrophic co-culture amplification of production phenotype for high-throughput screening of microbial strain libraries. Metabolic Engineering, 2019,54:232-243.
[62] Monod J. La technique de culture continue: theorie et applications. Annales de l Institut Pasteur, 1950,79:390-410.
[63] De Crecy E, Jaronski S, Lyons B, et al. Directed evolution of a filamentous fungus for thermotolerance. BMC Biotechnology, 2009,9:74.
[64] Van Gerven N, Klein R D, Hultgren S J, et al. Bacterial amyloid formation: structural insights into curli biogensis. Trends in Microbiology, 2015,23(11):693-706.
[65] Hope E A, Amorosi C J, Miller A W, et al. Experimental evolution reveals favored adaptive routes to cell aggregation in yeast. Genetics, 2017,206(2):1153-1167.
doi: 10.1534/genetics.116.198895 pmid: 28450459
[66] Marliere P, Patrouix J, Doring V, et al. Chemical evolution of a bacterium’s genome. Angewandte Chemie International Edition, 2011,50(31):7109-7114.
[67] Bouzon M, Perret A, Loreau O, et al. A synthetic alternative to canonical one-carbon metabolism. ACS Synthetic Biology, 2017,6(8):1520-1533.
doi: 10.1021/acssynbio.7b00029 pmid: 28467058
[68] Jian X J, Guo X J, Wang J, et al. Microbial microdroplet culture system (MMC): an integrated platform for automated, high-throughput microbial cultivation and adaptive evolution. Biotechnology and Bioengineering, 2020,117(6):1724-1737.
doi: 10.1002/bit.27327 pmid: 32159223
[69] Wang J, Jian X J, Xing X H, et al. Empowering a methanol-dependent Escherichia coli via adaptive evolution using a high-throughput microbial microdroplet culture system. Frontiers in Bioengineering and Biotechnology, 2020,8:570.
[70] Horinouchi T, Minamoto T, Suzuki S, et al. Development of an automated culture system for laboratory evolution. Journal of Laboratory Automation, 2014,19(5):478-482.
doi: 10.1177/2211068214521417 pmid: 24526062
[71] Miller A W, Befort C, Kerr E O, et al. Design and use of multiplexed chemostat arrays. Journal of Visualized Experiments, 2013,72:e50262.
[1] 马宁,王汉杰. 光遗传学在细菌生产调控中的应用进展[J]. 中国生物工程杂志, 2021, 41(9): 101-109.
[2] 黄焕邦,吴洋,杨友辉,王兆官,齐浩. 基于古菌酪氨酰tRNA合成酶非天然氨基酸插入的研究进展[J]. 中国生物工程杂志, 2021, 41(9): 110-125.
[3] 郭芳,张良,冯旭东,李春. 植物源UDP-糖基转移酶及其分子改造*[J]. 中国生物工程杂志, 2021, 41(9): 78-91.
[4] 郭曼曼,田开仁,乔建军,李艳妮. 噬菌体重组酶系统在合成生物学中的应用*[J]. 中国生物工程杂志, 2021, 41(8): 90-102.
[5] 董曙馨,秦磊,李春,李珺. 利用转录因子工程重塑代谢网络实现细胞工厂高效生产[J]. 中国生物工程杂志, 2021, 41(4): 55-63.
[6] 栗波,王泽建,梁剑光,刘爱军,李海东. 等离子体作用结合氧限制模型选育利福霉素SV高产菌株 *[J]. 中国生物工程杂志, 2021, 41(2/3): 38-44.
[7] 范雁,杨淼,薛松. 基于光谱法-图像灰度法高通量筛选高效固定CO2的苯甲酸脱羧酶*[J]. 中国生物工程杂志, 2021, 41(11): 55-63.
[8] 郑义,郭世英,隋凤翔,杨骐羽,卫雅萱,李晓岩. 群体感应系统在合成生物学中的应用*[J]. 中国生物工程杂志, 2021, 41(11): 100-109.
[9] 郭二鹏, 张建志, 司同. 羊毛硫肽的高通量工程改造方法新进展 *[J]. 中国生物工程杂志, 2021, 41(1): 30-41.
[10] 常璐, 黄娇芳, 董浩, 周斌辉, 朱小娟, 庄英萍. 合成生物学改造微生物及生物被膜用于重金属污染检测与修复 *[J]. 中国生物工程杂志, 2021, 41(1): 62-71.
[11] 饶海密,梁冬梅,李伟国,乔建军,财音青格乐. 真菌芳香聚酮化合物的合成生物学研究进展*[J]. 中国生物工程杂志, 2020, 40(9): 52-61.
[12] 张玉婷,李伟国,梁冬梅,乔建军,财音青格乐. P450s在萜类合成方面的合成生物学研究进展 *[J]. 中国生物工程杂志, 2020, 40(8): 84-96.
[13] 王震,李霞,元英进. 微生物异源合成咖啡酸及其酯类衍生物研究进展 *[J]. 中国生物工程杂志, 2020, 40(7): 91-99.
[14] 孙青,刘德华,陈振. 甲醇的生物利用与转化*[J]. 中国生物工程杂志, 2020, 40(10): 65-75.
[15] 刘金丛,刘雪,於洪建,赵广荣. 微生物合成根皮素及其糖苷研究进展 *[J]. 中国生物工程杂志, 2020, 40(10): 76-84.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会