Inclusion Body Production in Fed-Batch and Continuous Cultivation.

Various fermentation strategies in industrial biotechnology are applied to produce recombinant target proteins using Escherichia coli. These proteins are often expressed as inclusion bodies (IBs), resulting in a high purity, high stability, and high product titers. In state-of-the-art fed-batch processes, product formation takes place in a short period of time. Sterilization, cleaning, and biomass growth are time consuming steps and reduce the space-time yield. Thus, the interest in establishing continuous cultivations, facilitating higher space-time yields, has been increased in recent years. In this protocol, we provide information and a guide to set-up the production of recombinant proteins in fed-batch, as well as in chemostat continuous cultivations using E. coli.

Recombinant Protein L: Production, Purification and Characterization of a Universal Binding Ligand.

Protein L (PpL) is a universal binding ligand that can be used for the detection and purification of antibodies and antibody fragments. Due to the unique interaction with immunoglobulin light chains, it differs from other affinity ligands, like protein A or G. However, due to its current higher market price, PpL is still scarce in applications. In this study, we investigated the recombinant production and purification of PpL and characterized the product in detail. We present a comprehensive roadmap for the production of the versatile protein PpL in E. coli.

Cascaded processing enables continuous upstream processing with E. coli BL21(DE3).

In many industrial sectors continuous processing is already the golden standard to maximize productivity. However, when working with living cells, subpopulation formation causes instabilities in long-term cultivations. In cascaded continuous cultivation, biomass formation and recombinant protein expression can be spatially separated. This cultivation mode was found to facilitate stable protein expression using microbial hosts, however mechanistic knowledge of this cultivation strategy is scarce. In this contribution we present a method workflow to reduce workload and accelerate the establishment of stable continuous processes with E. coli BL21(DE3) exclusively based on bioengineering methods.

Repetitive Fed-Batch: A Promising Process Mode for Biomanufacturing With E. coli.

Recombinant protein production with Escherichia coli is usually carried out in fed-batch mode in industry. As set-up and cleaning of equipment are time- and cost-intensive, it would be economically and environmentally favorable to reduce the number of these procedures. Switching from fed-batch to continuous biomanufacturing with microbials is not yet applied as these cultivations still suffer from time-dependent variations in productivity. Repetitive fed-batch process technology facilitates critical equipment usage, reduces the environmental fingerprint and potentially increases the overall space-time yield. Surprisingly, studies on repetitive fed-batch processes for recombinant protein production can be found for yeasts only. Knowledge on repetitive fed-batch cultivation technology for recombinant protein production in E. coli is not available until now. In this study, a mixed feed approach, enabling repetitive fed-batch technology for recombinant protein production in E. coli, was developed. Effects of the cultivation mode on the space-time yield for a single-cycle fed-batch, a two-cycle repetitive fed-batch, a three-cycle repetitive fed batch and a chemostat cultivation were investigated. For that purpose, we used two different E. coli strains, expressing a model protein in the cytoplasm or in the periplasm, respectively. Our results demonstrate that a repetitive fed-batch for E. coli leads to a higher space-time yield compared to a single-cycle fed-batch and can potentially outperform continuous biomanufacturing. For the first time, we were able to show that repetitive fed-batch technology is highly suitable for recombinant protein production in E. coli using our mixed feeding approach, as it potentially (i) improves product throughput by using critical equipment to its full capacity and (ii) allows implementation of a more economic process by reducing cleaning and set-up times.

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

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 ß-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.