Green fluorescent protein (GFP) leakage from microbial biosensors provides useful information for the evaluation of the scale‐down effect

Abstract

Mixing deficiencies can be potentially detected by the use of a dedicated whole cell microbial biosensor. In this work, a csiE promoter induced under carbon-limited conditions was involved in the elaboration of such biosensor. The cisE biosensor exhibited interesting response after up and down-shift of the dilution rate in chemostat mode. Glucose limitation was accompanied by green fluorescent protein (GFP) leakage to the extracellular medium. In order to test the responsiveness of microbial biosensors to substrate fluctuations in large-scale, a scale-down reactor (SDR) experiment was performed. The glucose fluctuations were characterized at the single cell level and tend to decrease the induction of GFP. Simulations run on the basis of a stochastic hydrodynamic model have shown the variability and the frequencies at which biosensors are exposed to glucose gradient in the SDR. GFP leakage was observed to a great extent in the case of a culture operated in well-mixed fed-batch mode, by comparison with those operated in SDR. GFP leakage seems to be correlated to a higher membrane permeability, confirming previous studies highlighting a better cell viability in cultures operated in a fluctuating environment. Our results suggest that GFP leakage could be used in parallel to the normal GFP biosensor function in order to assess microbial viability in process conditions.

A low-cost, multiplexable, automated flow cytometry procedure for the characterization of microbial stress dynamics in bioreactors

“…These glucose pulses tended to decrease the segregation between the R1 and R2 subpopulations, with a progressive disappearance of the latter subpopulation. This result is consistent with our previous results [23] as well as with other published works [25,29,30], suggesting that substrate limitation induces an increase in cell membrane permeability in order to increase substrate uptake. The value of the automated FC relies upon its generation of on-line, physiologically related variables that enable direct comparisons of these parameters between each other and with process variables.…”

“…This phenomenon was unexpected since GFP would remain in steady-state if the activation of the fis promoter is effectively proportional to the growth rate, suggesting that additional physiological mechanisms are involved. In our previous studies, we have shown that proteins are secreted to the extracellular medium when microbial cells are exposed to prolonged substrate limitation [23,24]. This phenomenon has been reported by several authors and is generally termed protein leakage [25,26].…”

Application of a Genetically Encoded Biosensor for Live Cell Imaging of L-Valine Production in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains

“…This is further supported by the high correlation of sensor output (eYFP) and the signal of the integrated plasmid marker (E2-Crimson) shown in Figure 3A, B. Similar correlations were recently described for studies based on a GFP-sensor enabling the detection of carbon-limited conditions in E. coli as well as for a reporter system in yeast, in which the expression of gfp was set under control of a ribosomal protein promoter [37], [38].…”

Microbial heterogeneity affects bioprocess robustness: Dynamic single‐cell analysis contributes to understanding of microbial populations

“…Whatever the application is, the scaleup procedures induce a loss of mixing capacity and trigger a stress response at the level of the microbial population. Several authors have reported that this stress response can be beneficial and lead to substantial bioprocess improvement, the origin of which origin remains unclear [26,32,106,107]. In this context, it is important to identify the respective part taken by the individual path and the coordinated heterogeneity hypotheses that have been put forward to explain the appearance of phenotypic heterogeneity in bioprocesses.…”