Technical University of Munich, Institute of Biochemical Engineering, Boltzmannstr. 15, 85748, Garching, Germany.
Gene Bridges GmbH, Im Neuenheimer Feld 584, 69120, Heidelberg, Germany.
Microb Cell Fact. 2020 Jan 28;19(1):14. doi: 10.1186/s12934-020-1283-x.
Today there is an increasing demand for high yielding robust and cost efficient biotechnological production processes. Although cells in these processes originate from isogenic cultures, heterogeneity induced by intrinsic and extrinsic influences is omnipresent. To increase understanding of this mechanistically poorly understood phenomenon, advanced tools that provide insights into single cell physiology are needed.
Two Escherichia coli triple reporter strains have been designed based on the industrially relevant production host E. coli BL21(DE3) and a modified version thereof, E. coli T7E2. The strains carry three different fluorescence proteins chromosomally integrated. Single cell growth is followed with EmeraldGFP (EmGFP)-expression together with the ribosomal promoter rrnB. General stress response of single cells is monitored by expression of sigma factor rpoS with mStrawberry, whereas expression of the nar-operon together with TagRFP657 gives information about oxygen limitation of single cells. First, the strains were characterized in batch operated stirred-tank bioreactors in comparison to wildtype E. coli BL21(DE3). Afterwards, applicability of the triple reporter strains for investigation of population heterogeneity in bioprocesses was demonstrated in continuous processes in stirred-tank bioreactors at different growth rates and in response to glucose and oxygen perturbation simulating gradients on industrial scale. Population and single cell level physiology was monitored evaluating general physiology and flow cytometry analysis of fluorescence distributions of the triple reporter strains. Although both triple reporter strains reflected physiological changes that were expected based on the expression characteristics of the marker proteins, the triple reporter strain based on E. coli T7E2 showed higher sensitivity in response to environmental changes. For both strains, noise in gene expression was observed during transition from phases of non-growth to growth. Apparently, under some process conditions, e.g. the stationary phase in batch cultures, the fluorescence response of EmGFP and mStrawberry is preserved, whereas TagRFP657 showed a distinct response.
Single cell growth, general stress response and oxygen limitation of single cells could be followed using the two triple reporter strains developed in this study. They represent valuable tools to study population heterogeneity in bioprocesses significantly increasing the level of information compared to the use of single reporter strains.
如今，人们对高产、稳健且具有成本效益的生物技术生产工艺的需求日益增长。尽管这些工艺中的细胞源自于同基因培养物，但由内在和外在因素引起的异质性是普遍存在的。为了更深入地了解这一在机制上理解甚少的现象，需要先进的工具来深入了解单细胞生理学。
基于工业相关生产宿主大肠杆菌 BL21(DE3)及其改良版本大肠杆菌 T7E2，设计了两种三报告基因大肠杆菌菌株。这些菌株在染色体上携带三种不同的荧光蛋白。通过 EmeraldGFP（EmGFP）表达与核糖体启动子 rrnB 来跟踪单细胞生长。使用 mStrawberry 监测单个细胞的一般应激反应，通过表达 sigma 因子 rpoS 来监测单个细胞的一般应激反应，而表达 nar 操纵子与 TagRFP657 一起则提供了关于单个细胞氧限制的信息。首先，在分批搅拌罐生物反应器中对这些菌株进行了特征描述，并与野生型大肠杆菌 BL21(DE3)进行了比较。然后，在不同的生长速率下，在搅拌罐生物反应器中的连续过程中，以及在模拟工业规模梯度的葡萄糖和氧扰动的情况下，证明了三报告基因菌株在生物过程中种群异质性研究中的适用性。通过评估三报告基因菌株的一般生理学和荧光分布的流式细胞术分析，监测种群和单细胞水平的生理学。尽管两种三报告基因菌株都反映了基于标记蛋白表达特征预期的生理变化，但基于大肠杆菌 T7E2 的三报告基因菌株对环境变化的反应更为敏感。对于这两种菌株，在从非生长到生长的转变过程中，都观察到了基因表达的噪声。显然，在某些工艺条件下，例如分批培养的静止期，EmGFP 和 mStrawberry 的荧光响应得到了保留，而 TagRFP657 则表现出明显的响应。
使用本研究中开发的两种三报告基因菌株，可以跟踪单细胞生长、一般应激反应和单个细胞的氧限制。与使用单报告基因菌株相比，它们代表了研究生物过程中种群异质性的有价值的工具，大大增加了信息水平。
