Multi-dimensional gene target search for improving lycopene biosynthesis in Escherichia coli
Multi-dimensional gene target search for improving lycopene biosynthesis in Escherichia coli
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DOI：
10.1016/j.ymben.2007.03.003
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发表时间：
2007-07-01
影响因子：
8.4
通讯作者：
Stephanopoulos, Gregory
中科院分区：
文献类型：
--
作者：
Jin, Yong-Su;Stephanopoulos, Gregory
鉴定对所需表型表现出不同作用模式的多个基因靶标是菌株改良的关键步骤。基于可追踪遗传扰动和化学计量建模的靶点识别方法以前曾用于挖掘推定的过表达和敲除靶点。大多数搜索方法都是顺序的，因此，它们可以探索的空间非常有限。在这项研究中，我们研究了一种多维搜索方法，通过采用正交搜索策略来评估由不同搜索方法确定的基因靶点的未知相互作用。为此，我们结合敲除和过表达基因的目标，确定通过系统和组合的方法，分别，以提高番茄红素生产大肠杆菌。具体地说，我们首先通过筛选大肠杆菌基因组文库来鉴定多个过表达靶标。大肠杆菌中的一个序列迭代的方式。如此鉴定的靶标确认了非甲羟戊酸途径中先前扩增的基因（dxs和idi）和一些调控基因（rpoS和app Y）。此外，该方法揭示了新的基因靶标（yjiD、ycg W、yhbL、purDH和yggT）。随后进行二维搜索，由此将选择的过表达靶标与通过化学计量建模预测的敲除靶标组合。构建了具有四种基因缺失背景（包括单（Δ gdhA或Δ aceE）、双（Δ JgdhA，Δ aceE）和三（Δ gdhA Δ aceE Δ fdhI）敲除）的单（rpoS、app Y、yjiD、ycg W和yhbL）、双（vjiD-ycgW）和三（yjiD-ycgW-yhbL）过表达的所有组合，并评价番茄红素产生。对这40种菌株跨越的代谢景观的研究鉴定了最佳工程菌株（T5（p）-dxs、T5（p）-idi、rrnB（p）-yjiD-ycgW、Delta gdh Delta aceE Delta fdhF、pACLYC），其在M9基本培养基中在具有5g/L葡萄糖的分批摇瓶中在24小时内积累了16，000 ppm（16 mg/g细胞）的番茄红素。(C)2007年爱思唯尔公司All rights reserved.
Identification of multiple gene targets that exhibit different modes of action toward a desired phenotype is a crucial step in strain improvement. Target identification methods based on traceable genetic perturbations and stoichiometric modeling have been employed before for the mining of putative overexpression and knock-out targets. Most search methods are sequential and, as such, quite limited in the space they can explore. In this study, we investigate a multi -dimensional search approach whereby unknown interactions of gene targets identified by different search methods are assessed by employing orthogonal search strategies. To this end, we combined knockout and overexpression gene targets, identified through systematic and combinatorial approaches, respectively, in order to improve lycopene production in Escherichia coli. Specifically, we first identified multiple overexpression targets by screening genomic libraries of E. coli in a sequential-iterative manner. Targets so identified confirmed previously amplified genes in the non-mevalonate pathway (dxs and idi) and some regulatory genes (rpoS and app Y). Additionally, this method revealed novel gene targets (yjiD, ycg W, yhbL, purDH, and yggT). A two-dimensional search was subsequently undertaken, whereby the selected overexpression targets were combined with the knock-out targets predicted by stoichiometric modeling. All combinations of single (rpoS, app Y, yjiD, ycg W, and yhbL), double (vjiD-ycgW) and triple (yjiD-ycgW-yhbL) overexpressions with four gene deletion backgrounds, including single (Delta gdhA, or Delta aceE), double (Delta JgdhA,Delta aceE), and triple (Delta gdhA Delta aceE Delta fdhI,) knockouts, were constructed and evaluated for lycopene production. Investigation of the metabolic landscape spanned by these 40 strains identified the best-engineered strain (T5(p)-dxs, T5(p)-idi, rrnB(p)-yjiD-ycgW,Delta gdh Delta aceE Delta fdhF, pACLYC), which accumulated 16,000 ppm (16 mg/g cell) of lycopene within 24 h in a batch shake flask with 5 g/L of glucose in M9 minimal medium. (C) 2007 Elsevier Inc. All rights reserved.