Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity
Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity
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DOI：
10.1128/aem.00807-15
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发表时间：
2015-09-01
影响因子：
4.4
通讯作者：
Jahn, Dieter
中科院分区：
文献类型：
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作者：
Muench, Karin M.;Mueller, Johannes;Jahn, Dieter
在过去的20年中，巨大芽孢杆菌已经被系统地开发用于克/升规模的重组蛋白生产。所采用的基于质粒的表达系统使用木糖控制的启动子。在单细胞水平上使用绿色荧光蛋白作为模型产物的蛋白质生产分析揭示了细胞培养物的异质性，其特征在于显著比例的低生产力细菌。由于B的巨大尺寸。在巨大芽孢杆菌中，亚群中观察到的这种增殖行为容易通过延时显微镜和流式细胞术分析。细胞培养异质性不是简单地由质粒丢失引起的：相反，在指数生长期检测到细胞分裂过程中质粒的不对称分布。多拷贝质粒通常随机分布在子细胞之间。然而，体内和体外实验表明，在强蛋白质生产的条件下，质粒保留在细胞两极之一。此外，发现具有积累的质粒和高蛋白质产量的细胞停止细胞分裂。因此，培养物的总蛋白质产量主要由具有足够质粒拷贝数的亚群实现。基于我们的实验数据，我们提出了一个模型，其中多拷贝质粒的分布控制在蛋白质生产条件下的极性固定。因此，出现了具有波动质粒丰度的细胞系，这导致群体异质性。我们的研究结果提供了初步的见解细胞异质性的机制，在基于质粒的重组蛋白生产芽孢杆菌物种。
During the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species.