Global transcriptional analysis of metabolic burden due to plasmid maintenance in Escherichia coli DH5α during batch fermentation
Global transcriptional analysis of metabolic burden due to plasmid maintenance in Escherichia coli DH5α during batch fermentation
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DOI：
10.1016/j.enzmictec.2005.11.048
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发表时间：
2006-07-03
影响因子：
3.4
通讯作者：
Yap, Miranda Gek-Sim
中科院分区：
文献类型：
--
作者：
Ow, Dave Siak-Wei;Nissom, Peter Morin;Yap, Miranda Gek-Sim
大肠杆菌的DNA质粒是生产重组蛋白和代谢物的常见载体，在遗传疫苗和治疗方面具有潜在的治疗应用。然而，质粒的维持给宿主细胞带来了代谢负担，导致生长速度和细胞密度降低。在2 L的批量发酵中，携带7.3 kb NS3质粒的DH5 α细胞比不携带质粒的DH5 α细胞具有更低的特定生长速率（0.64 h（-1）比0.87 h(-1)）。在这项工作中，全球转录分析与蛋白质组学研究相结合，以评估质粒维持对基因表达的影响。质粒承载细胞在宿主上的转录表达分析显示生物合成/能量代谢基因下调、运输基因差异表达和热休克蛋白上调的总体趋势。在中心代谢途径中，大多数糖酵解基因表达下调，而戊糖磷酸途径表达差异较小。从蛋白质组学研究中鉴定的19种蛋白质的表达率与这些观察结果一致。我们的研究结果表明，质粒维持本身就扰乱了整体基因调控，并导致宿主中枢代谢途径的显著变化。这项工作有助于我们在基因表达水平上理解质粒代谢负荷，并可能有助于未来的代谢工程工作。(c) 2006爱思唯尔公司版权所有。
DNA plasmids of Escherichia coli are common vectors for recombinant protein and metabolite production and have potential therapeutic applications as genetic vaccines and therapeutics. However, plasmid maintenance imposes a metabolic burden on the host cells, resulting in reduced growth rate and cell density. In 2 L batch fermentation, DH5 alpha cells carrying a 7.3 kb NS3 plasmid had a lower specific growth rate than the non-plasmid-bearing host (0.64 h(-1) versus 0.87 h(-1)). In this work, global transcriptional analysis was combined with proteomics studies to evaluate the effect of plasmid maintenance on gene expression. Global transcriptional expression analysis of plasmid-bearing cells over host showed a general trend of downregulated biosynthetic/energy metabolism genes, differentially expressed transport genes and upregulated heat shock proteins. In the central metabolic pathways, most glycolytic genes were downregulated, while less expression difference was found in the pentose phosphate pathway. Expression ratios of 19 proteins identified from proteomics studies were consistent with these observations. Our findings suggest that plasmid maintenance alone perturbs global gene regulation, and leads to significant changes in central metabolic pathways in the host. This work contributes to our understanding of plasmid metabolic load at the gene expression level and could potentially aid in future metabolic engineering efforts. (c) 2006 Elsevier Inc. All rights reserved.