Development and characterization of Escherichia coli triple reporter strains for investigation of population heterogeneity in bioprocesses

Abstract

Background: 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. Results: 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. Conclusions: 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.

Advances in automated real‐time flow cytometry for monitoring of bioreactor processes

“…Moreover, the integration of a possibility to sort during ART‐FCM could further extend bioreactor process monitoring, as it will allow to isolate events of interest for further analysis. Consequently, the demand for deeper investigation of population heterogeneities in bioreactor processes with reporter strains [ 84 ], can be fulfilled. However, to our knowledge, such a system does not exist yet.…”

“…For both tools standardized functions to load FC data as FCS files exist. For MATLAB the fcs_read and fcs_readfcs algorithms are popular [ 19 , 26 , 52 , 84 , 85 ]. For R, the Bioconductor platform exists, that hosts the largest collection of open source FCM software covering data analysis and visualization of FCM data [ 86 ].…”

Measurement Techniques to Resolve and Control Population Dynamics of Mixed-Culture Processes

“…experiments are typically restricted to a maximum of three fluorescent proteins to achieve acceptable spectral separation [101]. Nevertheless, theoretically, more complex communities involving more than three members could also be resolved using spectral unmixing algorithms [102].…”

Single-Cell Technologies to Study Phenotypic Heterogeneity and Bacterial Persisters

“…Others have developed whole-genome reporter libraries to serve as a resource to better resolve transcriptional distinctions between cells [13]. Additionally, cutting edge "multi-reporter" constructs have been effectively optimized in E. coli and P. aeruginosa to enable observation of multiple transcripts simultaneously while subverting traditional limitations of spectral overlap (Figure 2A) [20][21][22].…”