Utilization of whole cell Green Fluorescent Protein (GFP) reporter systems as tracers to estimate the bioreactor efficiency

Brognaux A., Delvigne F., Thonart P.

Unité de Bio-industries- Gembloux Agro Bio-Tech - Université de Liège

Objectives

Material and methods

Strategy and results

Conclusion

Potential application and key benefits

Escherichia coli is a microorganism widely used in the industry for the production of recombinant proteins. The performances obtained at the laboratory level are not reproducible at a large scale. Actually, the mixing operation is not efficient enough: gradients of glucose and oxygen appear when operating in fed-batch mode (addition of glucose during the culture). These gradients cause adverse impacts on the production of biomass and recombinant protein. The aim of this work is to use the microbial population as biosensor of the encountered stress inside heterogeneous industrial bioreactors to better scale-up and regulate these reactors

The coding sequence of the Green Fluorescent Protein (GFP) is introduced after a specific stress promoter in a plasmid. This one is inserted in Escherichia coli. When these cells are submitted to a given stress condition (depending on the function of the promoter),GFP synthesis is induced and accumulated into the cytoplasm

Fluorescence analysis by flow cytometry

The corresponding stressed cells becomes fluorescent!

Flow cytometry allows the green fluorescence analysis at the single cell level:obtained results are frequency histograms of fluorescence-intensity in the microbial population . So, it is possible to link the GFP content among the microbial population with the bioreactor heterogeneity (stress condition)

Three classes of fluorescence have been defined: GFP-, GFP+ and GFP++. When reactors are heterogeneous, there is a delay at the level of the passage from the non-induced (GFP- in the case of rpoS strain and GFP+ in the case of csiE strain) to the induced state (GFP+ and GFP++ in the case of the rpoS strain and GFP++ in the case of csiE train). This result can be explained by the repression of the stress promoter when cells are exposed to local glucose excess..

prpoS::gfp and csiE::gfp strains can be used as biosensors of the heterogeneity of glucose encountered inside industrial reactors.

These strains could be used to validate a fed-batch regulation (addition of glucose) at the industrial level.

This research is financed by a FRIA fellowship received from the FRS-FNRS.

Cultures with Escherichia coli prpoS::gfp and csiE:gfp strains have been carried out in several types of bioreactors that have different mixing efficiencies: an homogeneous reactor and two kinds of heterogeneous scale-down reactors that mimic industrial conditions.

The rpoS gene is a gene of the general stress response, mainly induced at the entrance to stationary phase (during a lack of glucose) and the csiE gene is specifically induced during a glucose limitation. The GFP gene has been introduced after the rpoS and csiE promoters. The fluorescence has been monitored at the single cell level by flow cytometry.

The cytograms opposite show a progessive increase of the GFP signal for the whole population with csiE::gfp strain in an homogeneous reactor. Evolution of the FSC-FL1 cytogram during the culture of the csiE strain in a reference reactor (correctly mixed and

homogeneous) after A: 0h, B: 5h, C:6h, D:7h, F:13h, G:24h

Evolution of the different GFP classes during the culture of the rpoS:gfp (right panel) and the csiE:gfp strain (left panel). From top to bottom, decrease of the global bioreactor mixing effeciency by considering a reference bioreactor, a SDR with tR = 38.2 s and a SDR with tR = 79.8 s