A system for the continuous directed evolution of biomolecules.
A system for the continuous directed evolution of biomolecules.
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DOI：
10.1038/nature09929
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发表时间：
2011-04-28
期刊：
影响因子：
64.8
通讯作者：
Liu, David R.
中科院分区：
文献类型：
--
作者：
Esvelt, Kevin M.;Carlson, Jacob C.;Liu, David R.
实验室进化已经产生了许多具有所需特性的生物分子，但单轮突变、基因表达、筛选或选择以及复制通常需要数天或更长时间，并且需要频繁的人为干预。由于进化的成功取决于执行的总轮数，因此连续快速地执行实验室进化的方法可以显着提高其有效性。虽然研究人员加速了进化周期中的各个步骤，但之前连续定向进化的唯一例子是乔伊斯的里程碑式研究，乔伊斯通过体外复制周期不断进化出RNA连接酶核酶，不幸的是，这种复制周期无法轻易适应其他生物分子。在这里，我们描述了一个系统，该系统能够实现基因编码分子的连续定向进化，这些分子可以与大肠杆菌中的蛋白质生产相关联。在噬菌体辅助连续进化 (PACE) 过程中，进化基因通过修改后的噬菌体生命周期以依赖于目标活性的方式从宿主细胞转移到宿主细胞。在无需人工干预的情况下，PACE 的一天内可以发生数十轮进化。使用 PACE，我们进化出了 T7 RNA 聚合酶，可以识别不同的启动子，用 A 而不是 G 启动转录本，并用 C 启动转录本。在一个例子中，PACE 在 8 天的时间内执行了 200 轮蛋白质进化。在其中两个案例中，从检测不到的活性水平开始，在 PACE 不到一周的时间内就出现了具有三种目标活性的酶。在所有这三种情况下，PACE 进化的聚合酶活性都超过或相当于其野生型启动子上的野生型 T7 RNAP，提高了数百倍。通过大大加速实验室进化，PACE 可以为其他棘手的定向进化问题提供解决方案，并解决有关分子进化的新问题。
Laboratory evolution has generated many biomolecules with desired properties, but a single round of mutation, gene expression, screening or selection, and replication typically requires days or longer with frequent human intervention. Since evolutionary success is dependent on the total number of rounds performed, a means of performing laboratory evolution continuously and rapidly could dramatically enhance its effectiveness. While researchers have accelerated individual steps in the evolutionary cycle, the only previous example of continuous directed evolution was the landmark study of Joyce, who continuously evolved RNA ligase ribozymes with an in vitro replication cycle that unfortunately cannot be easily adapted to other biomolecules. Here we describe a system that enables the continuous directed evolution of gene-encoded molecules that can be linked to protein production in E. coli. During phage-assisted continuous evolution (PACE), evolving genes are transferred from host cell to host cell through a modified bacteriophage life cycle in a manner that is dependent on the activity of interest. Dozens of rounds of evolution can occur in a single day of PACE without human intervention. Using PACE, we evolved T7 RNA polymerases that recognize a distinct promoter, initiate transcripts with A instead of G, and initiate transcripts with C. In one example, PACE executed 200 rounds of protein evolution over the course of eight days. Starting from undetectable activity levels in two of these cases, enzymes with each of the three target activities emerged in less than one week of PACE. In all three cases, PACE-evolved polymerase activities exceeded or were comparable to that of the wild-type T7 RNAP on its wild-type promoter, representing improvements of up to several hundred-fold. By greatly accelerating laboratory evolution, PACE may provide solutions to otherwise intractable directed evolution problems and address novel questions about molecular evolution.
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影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
DOI：
10.1073/pnas.262420099
发表时间：
2002-12-24
影响因子：
11.1
作者：
Baker, K;Bleczinski, C;Cornish, VW
通讯作者：
Cornish, VW
影响因子：
5.4
作者：
Makeyev, EV;Bamford, DH
通讯作者：
Bamford, DH
影响因子：
3.7
作者：
NELSON, FK;FRIEDMAN, SM;SMITH, GP
通讯作者：
SMITH, GP
影响因子：
64.8
作者：
Wang HH;Isaacs FJ;Carr PA;Sun ZZ;Xu G;Forest CR;Church GM
通讯作者：
Church GM