The Lazarus Escherichia coli Effect: Recovery of Productivity on Glycerol/Lactose Mixed Feed in Continuous Biomanufacturing

Abstract

Continuous cultivation with Escherichia coli has several benefits compared to classical fed-batch cultivation. The economic benefits would be a stable process, which leads to time independent quality of the product, and hence ease the downstream process. However, continuous biomanufacturing with E. coli is known to exhibit a drop of productivity after about 4-5 days of cultivation depending on dilution rate. These cultivations are generally performed on glucose, being the favorite carbon source for E. coli and used in combination with isopropyl β-D-1 thiogalactopyranoside (IPTG) for induction. In recent works, harsh induction with IPTG was changed to softer induction using lactose for T7-based plasmids, with the result of reducing the metabolic stress and tunability of productivity. These mixed feed systems based on glucose and lactose result in high amounts of correctly folded protein. In this study we used different mixed feed systems with glucose/lactose and glycerol/lactose to investigate productivity of E. coli based chemostats. We tested different strains producing three model proteins, with the final aim of a stable long-time protein expression. While glucose fed chemostats showed the well-known drop in productivity after a certain process time, glycerol fed cultivations recovered productivity after about 150 h of induction, which corresponds to around 30 generation times. We want to further highlight that the cellular response upon galactose utilization in E. coli BL21(DE3), might be causing fluctuating productivity, as galactose is referred to be a weak inducer. This "Lazarus" phenomenon has not been described in literature before and may enable a stabilization of continuous cultivation with E. coli using different carbon sources.

Keywords: E. coli; continuous biomanufacturing; mixed-feeding; productivity recovery; recombinant protein production.

FIGURE 1

Comparison between (A) fed batch, (B) glucose/lactose fed chemostat, and (C) glycerol/lactose chemostat for production of recombinant GFP using Bl21(DE3).

FIGURE 2

HMS174 expressing GFP (A) using a glucose/lactose mixed feed and (B) a glycerol/lactose mixed feed. In neither of the cultivations a recovery of productivity is observed. Based on consumption of galactose, the overall DCW is higher in both cultivations.

FIGURE 3

Chemostat cultures with Bl21(DE3) with glycerol/lactose mixed feed expressing (A) mCherry, (B) Blitzenblue. The recovery of the productivity is clearly visible at 200 h in both cultivations. Blitzenblue (B) exhibits only soluble protein in fed batch and continuous cultivation.

FIGURE 4

(A) C-balances of all three chemostat cultivations with glycerol/lactose feed. Blitzenblue does not reach a balance of 1 in the entire cultivation time and does not have adaption phase I. (B) Inclusion body (IB) fractions over the timespan of glycerol-lactose chemostat cultivations: GFP IB amount increased up until 100 h and followed a subsequent drop. A recovery after 125 h of the IB fraction could be observed, mCherry showed higher IB content in the beginning of the cultivation but only recovered in productivity of the soluble fraction whereas the IB formation stopped after 100 h. Blitzenblue showed no IB-formation.

FIGURE 5

(A) Bl21(DE3) expressing GFP: glycerol/lactose cultivation: specific substrate uptake rates are constant throughout the whole induction time, while titer of the recombinant protein and extracellular galactose concentrations show high fluctuations; (B) HMS174 expressing GFP: glycerol/lactose cultivation with no accumulation of galactose and no respective recovery of productivity.