Programming cells by multiplex genome engineering and accelerated evolution.
Programming cells by multiplex genome engineering and accelerated evolution.
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DOI：
10.1038/nature08187
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发表时间：
2009-08-13
期刊：
影响因子：
64.8
通讯作者：
Church GM
中科院分区：
文献类型：
--
作者：
Wang HH;Isaacs FJ;Carr PA;Sun ZZ;Xu G;Forest CR;Church GM
在自然界生物中发现的基因组多样性的广度使种群能够适应不同的环境。然而，基因组多样性很难在实验室中产生，新的表型也不容易在实际的时间尺度上出现。尽管体外和定向进化方法已经创造了具有有用改变表型的遗传变体，但这些方法仅限于对单个基因的费力和连续操作，并且不用于基因网络或基因组的平行和连续定向进化。在这里，我们描述了多路自动化基因组工程（法师）的大规模编程和进化的细胞。法师同时靶向染色体上的许多位置，用于在单个细胞中或跨细胞群体进行修饰，从而产生组合的基因组多样性。由于该过程是循环的和可扩展的，我们构建了使法师技术自动化的原型装置，以促进快速和连续产生一组不同的遗传变化（错配、插入、缺失）。我们应用法师优化大肠杆菌中1-脱氧-d-木酮糖-5-磷酸（DXP）生物合成途径，以过量生产工业上重要的类异戊二烯番茄红素。DXP途径中的24个遗传组分使用合成DNA的复杂池同时进行修饰，每天产生超过43亿个组合基因组变体。我们分离出了在3天内番茄红素产量增加5倍以上的变体，这是对现有代谢工程技术的重大改进。我们的多元化方法包括在进化的背景下，通过加快具有新的和改进的特性的生物体的设计和进化的工程。
The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-d-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.