Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
PLoS One. 2013 Aug 8;8(8):e70516. doi: 10.1371/journal.pone.0070516. eCollection 2013.
Escherichia coli K-12 and B strains are among the most frequently used bacterial hosts for production of recombinant proteins on an industrial scale. To improve existing processes and to accelerate bioprocess development, we performed a detailed host analysis. We investigated the different behaviors of the E. coli production strains BL21, RV308, and HMS174 in response to high-glucose concentrations. Tightly controlled cultivations were conducted under defined environmental conditions for the in-depth analysis of physiological behavior. In addition to acquisition of standard process parameters, we also used DNA microarray analysis and differential gel electrophoresis (Ettan(TM) DIGE). Batch cultivations showed different yields of the distinct strains for cell dry mass and growth rate, which were highest for BL21. In addition, production of acetate, triggered by excess glucose supply, was much higher for the K-12 strains compared to the B strain. Analysis of transcriptome data showed significant alteration in 347 of 3882 genes common among all three hosts. These differentially expressed genes included, for example, those involved in transport, iron acquisition, and motility. The investigation of proteome patterns additionally revealed a high number of differentially expressed proteins among the investigated hosts. The subsequently selected 38 spots included proteins involved in transport and motility. The results of this comprehensive analysis delivered a full genomic picture of the three investigated strains. Differentially expressed groups for targeted host modification were identified like glucose transport or iron acquisition, enabling potential optimization of strains to improve yield and process quality. Dissimilar growth profiles of the strains confirm different genotypes. Furthermore, distinct transcriptome patterns support differential regulation at the genome level. The identified proteins showed high agreement with the transcriptome data and suggest similar regulation within a host at both levels for the identified groups. Such host attributes need to be considered in future process design and operation.
大肠杆菌 K-12 和 B 菌株是工业规模生产重组蛋白最常使用的细菌宿主之一。为了改进现有工艺并加速生物工艺开发，我们进行了详细的宿主分析。我们研究了大肠杆菌生产菌株 BL21、RV308 和 HMS174 对高葡萄糖浓度的不同反应。在明确的环境条件下进行严格控制的培养，以深入分析生理行为。除了获取标准工艺参数外，我们还使用 DNA 微阵列分析和差异凝胶电泳（ Ettan(TM) DIGE）。批处理培养显示出不同菌株在细胞干重和生长速率方面的产量不同，其中 BL21 的产量最高。此外，由于过量葡萄糖供应，K-12 菌株的乙酸产量比 B 菌株高得多。转录组数据分析表明，在三个宿主共有的 3882 个基因中，有 347 个基因发生了显著变化。这些差异表达的基因包括参与运输、铁获取和运动的基因。对蛋白质组图谱的分析还额外揭示了所研究的宿主之间存在大量差异表达的蛋白质。在所研究的 38 个斑点中，包括参与运输和运动的蛋白质。这项全面分析的结果提供了三个被研究菌株的完整基因组图片。确定了用于有针对性的宿主修饰的差异表达组，如葡萄糖转运或铁获取，从而能够优化菌株以提高产量和工艺质量。菌株的不同生长曲线证实了不同的基因型。此外，不同的转录组模式支持基因组水平的差异调节。鉴定的蛋白质与转录组数据高度一致，并表明在鉴定的组中，在宿主内的两个水平上存在相似的调节。在未来的工艺设计和操作中需要考虑这些宿主属性。
