Monitoring of corynebacterium glutamicum cultivations by means of a genetically encoded fluorescence sensor

Abstract

pH (pHi) measurement has gotten a strong interest in biotechnology and molecular biology over the last decades. Internal pH homeostasis is essential for all cellular functions and directly linked to membrane integrity and functionality of energy generation processes. Therefore, the internal pH in relation to the external pH is a valuable parameter to assess the physiological state and the viability of bacteria. In this work, Corynebacterium glutamicum was used with the plasmid containing genetically encoded pHsensitive green fluorescent protein pHluorin. The sensor expression is connected to an expression system that is induced under the addition of IPTG. This inducer activates the tacpromotor and allows conditional sensor production. The goals of the work were the following: To validate the production of the pHluorin protein and to check the sensor production dependency on biomass concentration To tune fluorescence sensor expression levels To investigate the influence of additional stress factors on the production of pHluorin sensor To monitor the protein production and to correlate the internal pH to process parameters and predict the performance of the process The noninvasive sensor was calibrated by expressing it under IPTG induction at different extracellular pH values and adding membranedisturbing chemicals (Nigericin and CTAB). The calibration allows the calculation of pHi under experimental conditions. Also, the calibration showed no dependency on biomass concentration, meaning that no extra sample manipulations like dilution are necessary during bioreactor cultivation. As the next step, IPTG induction levels were tuned in order to find the appropriate inductor concentration, at which sensor production proved adequate to obtain a sufficiently strong fluorescent signal while minimizing the metabolic load on the strain, showing that low IPTG levels are already enough to observe an adequate signal. Corynebacterium glutamicum is widely used in industry for its high glutamate production, which gives this bacteria industrial importance and plays a principal role in the progress of the amino acid fermentation. Glutamate is produced under the addition of membrane stress conditions. In this study, Tween 40 and Penicillin G were applied to investigate how the cells can handle additional stress conditions and how they influence the production of the pHluorin sensor. The physiological response of the C.glutamicum cells under the production of the fluorescence pHluorin sensor was monitored. Internal pH homeostasis measurement showed the existence of a correlation of pHi with other process variables, like the specific substrate uptake rate (qs), respiration rates (qO2 and qCO2) and its relation to the carbon dioxide evaluation rate (CER). This novelty shows that sensor production can be used to monitor process performance.