Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark, DK-2800 Kgs, Lyngby, Denmark.
Microb Cell Fact. 2012 Jul 16;11:94. doi: 10.1186/1475-2859-11-94.
Traditionally average values of the whole population are considered when analysing microbial cell cultivations. However, a typical microbial population in a bioreactor is heterogeneous in most phenotypes measurable at a single-cell level. There are indications that such heterogeneity may be unfavourable on the one hand (reduces yields and productivities), but also beneficial on the other hand (facilitates quick adaptation to new conditions--i.e. increases the robustness of the fermentation process). Understanding and control of microbial population heterogeneity is thus of major importance for improving microbial cell factory processes.
In this work, a dual reporter system was developed and applied to map growth and cell fitness heterogeneities within budding yeast populations during aerobic cultivation in well-mixed bioreactors. The reporter strain, which was based on the expression of green fluorescent protein (GFP) under the control of the ribosomal protein RPL22a promoter, made it possible to distinguish cell growth phases by the level of fluorescence intensity. Furthermore, by exploiting the strong correlation of intracellular GFP level and cell membrane integrity it was possible to distinguish subpopulations with high and low cell membrane robustness and hence ability to withstand freeze-thaw stress. A strong inverse correlation between growth and cell membrane robustness was observed, which further supports the hypothesis that cellular resources are limited and need to be distributed as a trade-off between two functions: growth and robustness. In addition, the trade-off was shown to vary within the population, and the occurrence of two distinct subpopulations shifting between these two antagonistic modes of cell operation could be distinguished.
The reporter strain enabled mapping of population heterogeneities in growth and cell membrane robustness towards freeze-thaw stress at different phases of cell cultivation. The described reporter system is a valuable tool for understanding the effect of environmental conditions on population heterogeneity of microbial cells and thereby to understand cell responses during industrial process-like conditions. It may be applied to identify more robust subpopulations, and for developing novel strategies for strain improvement and process design for more effective bioprocessing.
传统上，在分析微生物细胞培养物时，会考虑整个种群的平均值。然而，在大多数生物反应器中，典型的微生物种群在单细胞水平上具有多种表型，具有异质性。有迹象表明，这种异质性一方面可能是不利的（降低产量和生产力），但另一方面也可能是有利的（促进快速适应新条件，即增加发酵过程的稳健性）。因此，了解和控制微生物种群的异质性对于改进微生物细胞工厂的工艺非常重要。
在这项工作中，开发并应用了一种双报告系统，以在有氧培养条件下，在混合生物反应器中映射出酵母种群的生长和细胞适应性的异质性。该报告菌株是基于绿色荧光蛋白（GFP）在核糖体蛋白 RPL22a 启动子控制下的表达构建的，通过荧光强度水平可以区分细胞的生长阶段。此外，通过利用细胞内 GFP 水平和细胞膜完整性之间的强相关性，可以区分具有高和低细胞膜稳健性的亚群，从而能够耐受冻融应激。观察到生长和细胞膜稳健性之间存在强烈的负相关关系，这进一步支持了细胞资源有限的假设，需要在生长和稳健性这两个功能之间进行权衡分配的假设。此外，还表明这种权衡在种群内发生变化，可以区分在这两种拮抗的细胞操作模式之间转换的两个不同的亚群。
报告菌株能够在细胞培养的不同阶段映射出对冻融应激的种群异质性，包括生长和细胞膜稳健性。所描述的报告系统是一种非常有价值的工具，可以帮助理解环境条件对微生物细胞种群异质性的影响，从而了解工业过程条件下细胞的反应。它可以用于识别更稳健的亚群，并开发出用于菌株改进和过程设计的新策略，以实现更有效的生物加工。
