Mechanistic platform knowledge of concomitant sugar uptake in Escherichia coli BL21(DE3) strains

Abstract

When producing recombinant proteins, the use of Escherichia coli strain BL21(DE3) in combination with the T7-based pET-expression system is often the method of choice. In a recent study we introduced a mechanistic model describing the correlation of the specific glucose uptake rate (qs,glu) and the corresponding maximum specific lactose uptake rate (qs,lac,max) for a pET-based E. coli BL21(DE3) strain producing a single chain variable fragment (scFv). We showed the effect of qs,lac,max on productivity and product location underlining its importance for recombinant protein production. In the present study we investigated the mechanistic qs,glu/qs,lac,max correlation for four pET-based E. coli BL21(DE3) strains producing different recombinant products and thereby proved the mechanistic model to be platform knowledge for E. coli BL21(DE3). However, we found that the model parameters strongly depended on the recombinant product. Driven by this observation we tested different dynamic bioprocess strategies to allow a faster investigation of this mechanistic correlation. In fact, we succeeded and propose an experimental strategy comprising only one batch cultivation, one fed-batch cultivation as well as one dynamic experiment, to reliably determine the mechanistic model for qs,glu/qs,lac,max and get trustworthy model parameters for pET-based E. coli BL21(DE3) strains which are the basis for bioprocess development.

Hybrid Modelling and Multi- Parametric Control of Bioprocesses

“…Also, expansion of MSCs on microcarrier was performed successfully in spinner flasks [37] and CSTRs [38]. However, the quantum system was mainly developed for use at a clinical scale, and thus might be oversized for research scale while cell harvest is challenging in microcarrier-based systems [39,40]. Also, both approaches are limited by their surface capacity.…”

“…For instance, numerous studies show a higher matrix deposition or upregulation of relevant genes in comparison to static culture conditions when bone precursor cells are exposed to fluid shear stress [38][39][40][41][42][43]. Although several tailor-made [10,11,44] and commercially available [45][46][47] perfusion systems were developed and successfully used, there is a lack of automated sensor-controlled systems that allow the cultivation of multiple independent replicates under different conditions.…”

“…Induction was always performed with a lactose pulse of 100 mL of a 300 g/L sterile lactose solution-resulting in a lactose concentration in the fermentation broth of approximately 30 g/L. Induction period always lasted 7 h. The q s control used here was performed using Equation (1) according to an exponential feed forward approach to keep q s constant [3,27,40,46]: …”

“…In addition, mixtures of glucose, glycerol, and lactose have shown promising results for diverse products gained via autoinduction systems [20,25]. Recent studies [3,40] showed that the dependence of the inducer lactose influences the maximum IB production even on a quite low level of the specific glucose uptake rate. Low feeding rates of glucose would therefore result in the maximum inducer uptake rate, as cyclic adenosine monophosphate (cAMP) levels increase at higher glucose addition and therefore decrease the affinity for the RNA polymerase, decreasing the expression of the genes coding for the lac operon [35].…”

“…Usage of such, in combination with lactose, may result in diauxic growth and catabolite repression, which are caused by the regulatory network that is induced by glucose [37][38][39]. Catabolite repression results in decreased lactose uptake rates when glucose is present in excess [27,39,40]. Glycerol, first noticed in biotechnology as a by-product in the biodiesel production [41], has shown quite promising results in terms of biomass/substrate yield in E. coli cultivations [22,25].…”