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首页> 外文期刊>Applied Microbiology >Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity
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Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity

机译：巨大芽孢杆菌重组蛋白生产过程中质粒的极性固定导致种群异质性

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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 viv o 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.
机译：在过去的20年中，系统地开发了巨大芽孢杆菌，用于以克/升为单位的重组蛋白生产。使用的基于质粒的表达系统使用木糖控制的启动子。使用绿色荧光蛋白作为模型产品在单细胞水平上进行蛋白质生产分析，揭示了细胞培养的异质性，其特征是生产力较低的细菌占很大比例。由于巨大芽孢杆菌的巨大大小，通过延时显微镜和流式细胞术很容易分析亚群中这种双稳态行为。细胞培养的异质性并非仅由质粒丢失引起：相反，在指数增长阶段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。但是，体内和体外实验表明，在产生大量蛋白质的条件下，质粒保留在细胞极之一处。此外，发现具有积累的质粒和高蛋白产生的细胞停止了细胞分裂。结果，培养物的总蛋白产量主要通过具有足够质粒拷贝数的亚群实现。根据我们的实验数据，我们提出了一个模型，通过该模型可以在蛋白质生产条件下通过极性固定控制多拷贝质粒的分布。因此，出现了具有波动的质粒丰度的细胞系，导致群体异质性。我们的结果提供了对芽孢杆菌属中基于质粒的重组蛋白生产过程中细胞异质性机制的初步见解。

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