Chair of Biochemical Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstr. 15, 85748, Garching, Germany.
Microb Cell Fact. 2023 Aug 13;22(1):153. doi: 10.1186/s12934-023-02165-4.
The omnipresence of population heterogeneity in industrial bioprocesses originates from prevailing dynamic bioprocess conditions, which promote differences in the expression of cellular characteristics. Despite the awareness, the concrete consequences of this phenomenon remain poorly understood.
Therefore, for the first time, a L-phenylalanine overproducing Escherichia coli quadruple reporter strain was established for monitoring of general stress response, growth behavior, oxygen limitation and product formation of single cells based on mTagBFP2, mEmerald, CyOFP1, and mCardinal2 expression measured by flow cytometry. This strain was applied for the fed-batch production of L-phenylalanine from glycerol and ammonia in a stirred-tank bioreactor at homogeneous conditions compared to the same process in a novel two-compartment bioreactor. This two-compartment bioreactor consists of a stirred-tank bioreactor with an initial volume of 0.9 L (homogeneous zone) with a coiled flow inverter with a fixed working volume of 0.45 L as a bypass (limitation zone) operated at a mean hydraulic residence time of 102 s. The product formation was similar in both bioreactor setups with maximum L-phenylalanine concentrations of 21.1 ± 0.6 g L demonstrating the consistency of this study's microbial L-phenylalanine production. However, cell growth was vulnerable to repetitive exposure to the dynamically changing conditions in the two-compartment bioreactor with maximum biomass yields reduced by 21%. The functionality of reporter molecules was approved in the stirred-tank bioreactor cultivation, in which expressed fluorescence levels of all four markers were in accordance with respective process state variables. Additional evaluation of the distributions on single-cell level revealed the presence of population heterogeneity in both bioprocesses. Especially for the marker of the general stress response and the product formation, the corresponding histograms were characterized by bimodal shapes and broad distributions. These phenomena were pronounced particularly at the beginning and the end of the fed-batch process.
The here shown findings confirm multiple reporter strains to be a noninvasive tool for monitoring cellular characteristics and identifying potential subpopulations in bioprocesses. In combination with experiments in scale-down setups, these can be utilized for a better physiological understanding of bioprocesses and support future scale-up procedures.
工业生物过程中普遍存在的种群异质性源于普遍存在的动态生物过程条件，这些条件促进了细胞特征表达的差异。尽管已经意识到这一现象，但对其具体后果仍知之甚少。
因此，首次建立了一种 L-苯丙氨酸高产大肠杆菌四重报告菌株，用于基于流式细胞术测量的 mTagBFP2、mEmerald、CyOFP1 和 mCardinal2 表达，监测单细胞的一般应激反应、生长行为、氧限制和产物形成。该菌株在搅拌罐生物反应器中应用于甘油和氨的 L-苯丙氨酸分批补料生产，与相同工艺在新型两室生物反应器中的生产相比，条件更为均匀。该两室生物反应器由一个初始体积为 0.9 L 的搅拌罐生物反应器组成（均相区），带有一个固定工作体积为 0.45 L 的盘管流逆变器作为旁路（限制区），水力停留时间平均值为 102 s。两种生物反应器设置中的产物形成相似，最大 L-苯丙氨酸浓度为 21.1±0.6 g/L，证明了本研究微生物 L-苯丙氨酸生产的一致性。然而，细胞生长容易受到两室生物反应器中动态变化条件的反复暴露的影响，最大生物量产率降低了 21%。在搅拌罐生物反应器培养中，报告分子的功能得到了验证，所有四种标记物的表达荧光水平均与相应的过程状态变量一致。对单细胞水平分布的进一步评估表明，两种生物过程中均存在种群异质性。特别是对于一般应激反应和产物形成的标记物，相应的直方图表现出双峰形状和广泛的分布。这些现象在补料分批过程的开始和结束时尤为明显。
这里显示的研究结果证实，多重报告菌株是监测细胞特征和识别生物过程中潜在亚群的非侵入性工具。与小型化实验相结合，这些工具可用于更好地了解生物过程的生理学，并支持未来的放大程序。
