With the growing interest in continuous cultivation of Escherichia coli, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, it is shown that induced decoupling of growth and heterologous gene expression in the E. coli X‐press strain (derived from BL21(DE3)) facilitates extracellular recombinant protein production. The effect of the process parameters temperature and specific glucose consumption rate (qS) on growth, productivity, lysis and leakiness, is investigated, to find the parameter space allowing extracellular protein production. Two model proteins are used, Protein A (SpA) and a heavy‐chain single‐domain antibody (VHH), and performance is compared to the industrial standard strain BL21(DE3). It is shown that inducible growth repression in the X‐press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and qS are used to control productivity and leakiness. In the X‐press strain, extracellular SpA and VHH titer reach up to 349 and 19.6 mg g−1, respectively, comprising up to 90% of the total soluble product, while keeping cell lysis at a minimum. The findings demonstrate that the X‐press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli.

While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a His6-tagged fusion protein and characterized for function and structure. The enzyme exhibits dual cofactor specificity for NADPH and NADH and prefers D-xylose over other pentoses and investigated hexoses. A homology model based on a XR from Candida tenuis was generated and the architecture of the cofactor binding site was investigated in detail. Despite the outstanding thermophilicity of its host the enzyme is, however, not thermostable.

Raman spectroscopy is a nondestructive characterization method offering chemical-specific information. However, the cross-section of inelastically (Raman) scattered light is very low compared to elastically (Rayleigh) scattered light, resulting in weak signal intensities in Raman spectroscopy. Despite providing crucial information in off-line measurements, it usually is not sensitive enough for efficient, in-line process control in conjunction with low particle concentrations. To overcome this limitation, two custom-made 1.4404 stainless-steel prototype add-ons were developed for in-line Raman probes that enable ultrasound particle manipulation and thus concentration of particles in suspensions in the focus of the Raman excitation laser. Depending on size and density differences between particles and the carrier medium, particles are typically caught in the nodal planes of a quasi-standing wave field formed in an acoustic resonator in front of the sensor. Two arrangements were realized with regard to the propagation direction of the ultrasonic wave relative to the propagation direction of the laser. The parallel arrangement improved the limit of detection (LOD) by a factor of ≈30. In addition to increased sensitivity, the perpendicular arrangement offers increased selectivity: modifying the frequency of the ultrasonic wave field allows the liquid or solid phase to be moved into the focus of the Raman laser. The combination of in-line Raman spectroscopy with ultrasound particle manipulation holds promise to push the limits of conventional Raman spectroscopy, hence broadening its field of application to areas where previously Raman spectroscopy has not had sufficient sensitivity for accurate, in-line detection.

Recombinant proteins in Escherichia coli are usually expressed inside the cell. With the growing interest in continuous cultivation, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, we present the X-press strain, a novel E. coli production host for growth decoupled, extracellular recombinant protein production. We investigated the effect of the process parameters temperature and specific glucose uptake rate (qS) on the strain's growth, productivity, lysis and leakiness, to find the parameter space allowing extracellular protein production. Two model proteins were used, Protein A and a VHH single-domain antibody, and performance was compared to the industrial standard strain BL21(DE3). We show that inducible growth repression in the X-press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and qS were used to control productivity and leakiness. In the X-press strain, extracellular Protein A and VHH titer reached up to 349 mg/g and 19.6 mg/g, respectively, comprising up to 90% of total soluble product, while keeping cell lysis at a minimum. Our findings demonstrate that the X-press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli.

Background Escherichia coli is of central interest to biotechnological research and a widely used organism for producing proteins at both lab and industrial scales. However, many proteins remain difficult to produce efficiently in E. coli. This is particularly true for proteins that require post translational modifications such as disulfide bonds. Results In this study we develop a novel approach for quantitatively investigating the ability of E. coli to produce disulfide bonds in its own proteome. We summarise the existing knowledge of the E. coli disulfide proteome and use this information to investigate the demand on this organism’s quantitative oxidative folding apparatus under different growth conditions. Furthermore, we built an ordinary differential equation-based model describing the cells oxidative folding capabilities. We use the model to infer the kinetic parameters required by the cell to achieve the observed oxidative folding requirements. We found that the cellular requirement for disulfide bonded proteins changes significantly between growth conditions. Fast growing cells require most of their oxidative folding capabilities to keep up their proteome while cells growing in chemostats appear limited by their disulfide bond isomerisation capacities. Conclusion This study establishes a novel approach for investigating the oxidative folding capacities of an organism. We show the capabilities and limitations of E. coli for producing disulfide bonds under different growth conditions and predict under what conditions excess capability is available for recombinant protein production.

Background Escherichia coli is of central interest to biotechnological research and a widely used organism for producing proteins at both lab and industrial scales. However, many proteins remain difficult to produce efficiently in E. coli. This is particularly true for proteins that require post translational modifications such as disulfide bonds. Results In this study we develop a novel approach for quantitatively investigating the ability of E. coli to produce disulfide bonds in its own proteome. We summarise the existing knowledge of the E. coli disulfide proteome and use this information to investigate the demand on this organism’s quantitative oxidative folding apparatus under different growth conditions. Furthermore, we built an ordinary differential equation-based model describing the cells oxidative folding capabilities. We use the model to infer the kinetic parameters required by the cell to achieve the observed oxidative folding requirements. We find that the cellular requirement for disulfide bonded proteins changes significantly between growth conditions. Fast growing cells require most of their oxidative folding capabilities to keep up their proteome while cells growing in chemostats appear limited by their disulfide bond isomerisation capacities. Conclusion This study establishes a novel approach for investigating the oxidative folding capacities of an organism. We show the capabilities and limitations of E. coli for producing disulfide bonds under different growth conditions and predict under what conditions excess capability is available for recombinant protein production.

BackgroundRecombinant proteins in Escherichia coli are expressed inside the cell. With the growing interest in continuous cultivation, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, we present a novel E. coli production host for growth decoupled recombinant protein production that can leak up to 90% of recombinant protein to the extracellular space. We investigated the effects of the process parameters temperature and specific glucose uptake rate on physiology, productivity, lysis and leakiness. Two model proteins were used, Protein A and a VHH single-domain antibody, and performance was compared to the industrial standard strain BL21(DE3). ResultsWe show that inducible growth repression in the novel E. coli strain enGenes-X-press, the effect of the metabolic burden on host physiology can be greatly reduced compared to BL21(DE3). Furthermore, in both strains, increasing temperature and specific substrate enhanced productivity and leakiness.Using the enGenes-X-press strain, extracellular Protein A and VHH titer reached up to 349 mg/g and 19.6 mg/g, respectively, comprising between 80 and 90% of total soluble product, while keeping cell lysis to a minimum. BL21(DE3) leaked 198 mg/g and 3.9 mg/g of Protein A and VHH to the medium, accounting for only 56% and 34% of total soluble product, respectively. ConclusionsWe confined the parameter space in which outer membrane leakiness can be controlled, while maintaining cell viability. Moreover, our findings demonstrate that the enGenes-X-press strain constitutes a superior host for extracellular production of recombinant protein.