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A system for the continuous directed evolution of biomolecules

机译：生物分子连续定向进化的系统

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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. Because evolutionary success is dependent on the total number of rounds performed2, a means of performing laboratory evolution continuously and rapidly could dramatically enhance its effectiveness. Although researchers have accelerated individual steps in the evolutionary cycle, the only previous example of continuous directed evolution was the landmark study of Wright and 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 Escherichia 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 polymerase (RNAP) variants that recognize a distinct promoter, initiate transcripts with ATP instead of GTP, and initiate transcripts with CTP. In one example, PACE executed 200 rounds of protein evolution over the course of 8 days. Starting from undetectable activity levels in two of these cases, enzymes with each of the three target activities emerged in less than 1 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.
机译：实验室的发展已经产生了许多具有所需特性的生物分子，但是在人类的频繁干预下，单轮突变，基因表达，筛选或选择以及复制通常需要几天或更长时间。由于进化的成功取决于所进行的回合总数2，因此连续而快速地进行实验室进化的方法可以大大提高其有效性。尽管研究人员已经加快了进化周期中的各个步骤，但先前连续定向进化的唯一例子是Wright和Joyce的里程碑式研究，他们以体外复制周期连续进化了RNA连接酶核酶，不幸的是，这种复制过程无法轻易地适应其他生物分子。在这里，我们描述了一个系统，该系统可使基因编码的分子持续定向进化，而该分子可与大肠杆菌中的蛋白质生产相关联。在噬菌体辅助的连续进化（PACE）过程中，进化的基因通过修饰的噬菌体生命周期以依赖于目的活性的方式从宿主细胞转移到宿主细胞。无需人工干预，PACE的一天就可以进行数十轮进化。使用PACE，我们进化了可识别不同启动子的T7 RNA聚合酶（RNAP）变体，用ATP代替GTP起始转录本，并用CTP起始转录本。在一个示例中，PACE在8天的过程中执行了200轮蛋白质进化。在其中两种情况下，从无法检测到的活性水平开始，在不到1周的PACE中出现了具有三种目标活性的酶。在所有这三种情况下，PACE进化的聚合酶活性在其野生型启动子上均超过或可与野生型T7 RNAP媲美，最多可提高数百倍。通过大大加快实验室的发展，PACE可以为原本难以解决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。

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  • 来源
    《Nature》 |2011年第7344期|p.499-503|共5页
  • 作者

    Kevin M. Esvelt; Jacob C. Carlson; David R. Liu;

  • 作者单位

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA;

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge,Massachusetts 02138, USA;

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge,Massachusetts 02138, USA,Howard Hughes Medical Institute, Cambridge, Massachusetts 02138, USA;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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