Mustafi Nurije, Grünberger Alexander, Mahr Regina, Helfrich Stefan, Nöh Katharina, Blombach Bastian, Kohlheyer Dietrich, Frunzke Julia
IBG-1: Biotechnology, Forschungszentrum Jülich, Jülich, Germany.
Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany.
PLoS One. 2014 Jan 17;9(1):e85731. doi: 10.1371/journal.pone.0085731. eCollection 2014.
The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ΔaceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ΔaceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains.
大多数与生物技术相关的代谢产物不会给产生这些代谢产物的细胞带来明显的表型。因此，微生物代谢产物生产的分析仍然主要依赖于整体技术，而这些技术可能会掩盖单细胞水平上的显著差异。在本研究中，我们应用了最近开发的Lrp生物传感器，用于监测基于丙酮酸脱氢酶复合物缺陷型（PDHC）菌株谷氨酸棒杆菌ΔaceE逐步工程改造的L-缬氨酸生产谷氨酸棒杆菌菌株单细胞中的氨基酸生产。在分批培养过程中对传感器输出（增强型黄色荧光蛋白荧光）进行在线监测，证明了该传感器适用于可视化不同的生产水平。接下来，我们使用微流控芯片装置对谷氨酸棒杆菌传感器菌株进行了活细胞成像研究。正如预期的那样，与基础菌株谷氨酸棒杆菌ΔaceE相比，高产生产者的微菌落中的传感器输出更高。在基本培养基中进行微流控培养显示，在生产阶段单细胞荧光呈现典型的高斯分布。值得注意的是，少量的复合营养物质完全改变了所有菌株观察到的表型模式，导致群体出现表型分化。一些细胞停止生长并开始产生L-缬氨酸，而另一些细胞继续生长或显示出向生产的延迟转变。根据培养条件，观察到相当一部分非荧光细胞，这表明代谢活性丧失。这些研究表明，基因编码的生物传感器是监测单细胞生产力和研究微生物生产菌株表型模式的有价值工具。
