Genome-scale analysis of anti-metabolite directed strain engineering
Genome-scale analysis of anti-metabolite directed strain engineering
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DOI：
10.1016/j.ymben.2007.10.002
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发表时间：
2008-03-01
影响因子：
8.4
通讯作者：
Gill, Ryan T.
中科院分区：
文献类型：
--
作者：
Bonomo, Jeanne;Lynch, Michael D.;Gill, Ryan T.
经典的菌株工程方法此前一直受到传统测序技术低通量的限制。在这里，我们应用了一种新的基因组学技术，即文库富集标量分析 (SCALE)，来测量 4300 万个大肠杆菌基因组文库克隆富集模式，这些模式是通过使用三种天冬氨酸抗代谢物进行生长选择而产生的。我们的目标是评估获得基因组规模的富集模式将在多大程度上提供通过使用传统测序无法合理获得的菌株工程见解。我们确定，与传统测序显示的区域数量（1-3 个区域）相比，SCALEs 方法鉴定出令人惊讶的大范围抗代谢物耐受区域（三种抗代谢物中的每一种都有 423、865 或 909 个区域）。基因组规模方法独特地实现了克隆的计算。通过在选择一段时间之前和之后提供基因组文库内所有克隆的浓度数据来确定适合度值。我们观察到了那个克隆。适合度值与克隆浓度值有很大差异，这是由于整体克隆的差异造成的。每个选择的适应度分布。最后，我们展示了许多最高的克隆。适应度在所有选择中重叠，表明对天冬氨酸代谢的抑制（而不是特定的抑制酶）主导了每个选择。我们的后续研究表明。证实了我们观察到的生长表型，并表明几个已识别克隆的细胞内氨基酸水平也发生了改变。这些结果表明，基因组规模的方法（例如 SCALE）可用于显着提高对经典菌株工程方法的理解。 (C) 2007 Elsevier Inc. 保留所有权利。
Classic strain engineering methods have previously been limited by the low-throughput of conventional sequencing technology. Here, we applied a new genomics technology, scalar analysis of library enrichments (SCALEs), to measure 43 million Escherichia coli genomic library clone enrichment patterns resulting from growth selections employing three aspartic-acid anti-metabolites. Our objective was to assess the extent to which access to genome-scale enrichment patterns would provide strain-engineering insights not reasonably accessible through the use of conventional sequencing. We determined that the SCALEs method identified a surprisingly large range of antimetabolite tolerance regions (423, 865, or 909 regions for each of the three anti-metabolites) when compared to the number of regions (1-3 regions) indicated by conventional sequencing. Genome-scale methods uniquely enable the calculation of clone. fitness values by providing concentration data for all clones within a genomic library before and after a period of selection. We observed that clone. fitness values differ substantially from clone concentration values and that this is due to differences in overall clone. fitness distributions for each selection. Finally, we show that many of the clones of highest. fitness overlapped across all selections, suggesting that inhibition of aspartate metabolism, as opposed to specific inhibited enzymes, dominated each selection. Our follow up studies con. confirmed our observed growth phenotypes and showed that intracellular amino-acid levels were also altered in several of the identified clones. These results demonstrate that genome-scale methods, such as SCALEs, can be used to dramatically improve understanding of classic strain engineering approaches. (C) 2007 Elsevier Inc. All rights reserved.