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Strategy for directing combinatorial genome engineering in Escherichia coli

机译：指导大肠杆菌中组合基因组工程的策略

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We describe a directed genome-engineering approach that combines genome-wide methods for mapping genes to traits [Warner JR. Reeder PJ, Karimpour-Fard A, Woodruff LBA, Gill RT (2010) Nat Biotechnol 28:856-862] with strategies for rapidly creating combinatorial ribosomal binding site (RBS) mutation libraries containing billions of targeted modifications [Wang HH, et al. (2009) Nature 460:894-898]. This approach should prove broadly applicable to various efforts focused on improving production of fuels, chemicals, and Pharmaceuticals, among other products. We used bar-coded promoter mutation libraries to map the effect of increased or decreased expression of nearly every gene in Escherichia coli onto growth in several model environments (cellulosic hydroly-sate, low pH, and high acetate). Based on these data, we created and evaluated RBS mutant libraries (containing greater than 100,000,000 targeted mutations), targeting the genes identified to most affect growth. On laboratory timescales, we successfully identified a broad range of mutations (>25 growth-enhancing mutations confirmed), which improved growth rate 10-200% for several different conditions. Although successful, our efforts to identify superior combinations of growth-enhancing genes emphasized the importance of epistatic interactions among the targeted genes (synergistic, antagonistic) for taking full advantage of this approach to directed genome engineering.
机译：我们描述了一种直接的基因组工程方法，该方法结合了全基因组方法来将基因定位到性状[Warner JR。 Reeder PJ，Karimpour-Fard A，Woodruff LBA，Gill RT（2010）Nat Biotechnol 28：856-862]，其策略是快速创建包含数十亿个靶向修饰的组合核糖体结合位点（RBS）突变文库[Wang HH等，2009年。 （2009）Nature 460：894-898]。这种方法应该广泛适用于致力于改善燃料，化学制品和药品等产品生产的各种努力。我们使用条形码启动子突变库来绘制大肠杆菌中几乎每个基因的表达增加或减少对几种模型环境（纤维素水解物，低pH和高乙酸盐）中生长的影响。基于这些数据，我们创建并评估了RBS突变体文库（包含超过1亿个目标突变），以鉴定出对生长有最大影响的基因为目标。在实验室的时间尺度上，我们成功地鉴定出了广泛的突变（已确认> 25个可增强生长的突变），在多种不同条件下，它们可将生长速率提高10-200％。尽管成功，但我们为确定生长增强基因的优良组合所做的努力强调了目标基因（协同，拮抗）之间的上位相互作用对于充分利用这种方法进行定向基因组工程的重要性。

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