RNA-seq reveals multifaceted gene expression response to Fab production in Escherichia coli fed-batch processes with particular focus on ribosome stalling.

BACKGROUND: Escherichia coli is a cost-effective expression system for production of antibody fragments like Fabs. Various yield improvement strategies have been applied, however, Fabs remain challenging to produce. This study aimed to characterize the gene expression response of commonly used E. coli strains BL21(DE3) and HMS174(DE3) to periplasmic Fab expression using RNA sequencing (RNA-seq). Two Fabs, Fabx and FTN2, fused to a post-translational translocation signal sequence, were produced in carbon-limited fed-batch cultivations. RESULTS: Production of Fabx impeded cell growth substantially stronger than FTN2 and yields of both Fabs differed considerably. The most noticeable, common changes in Fab-producing cells suggested by our RNA-seq data concern the cell envelope. The Cpx and Psp stress responses, both connected to inner membrane integrity, were activated, presumably by recombinant protein aggregation and impairment of the Sec translocon. The data additionally suggest changes in lipopolysaccharide synthesis, adjustment of membrane permeability, and peptidoglycan maturation and remodeling. Moreover, all Fab-producing strains showed depletion of Mg2+, indicated by activation of the PhoQP two-component signal transduction system during the early stage and sulfur and phosphate starvation during the later stage of the process. Furthermore, our data revealed ribosome stalling, caused by the Fabx amino acid sequence, as a contributor to low Fabx yields. Increased Fabx yields were obtained by a site-specific amino acid exchange replacing the stalling sequence. Contrary to expectations, cell growth was not impacted by presence or removal of the stalling sequence. Considering ribosome rescue is a conserved mechanism, the substantial differences observed in gene expression between BL21(DE3) and HMS174(DE3) in response to ribosome stalling on the recombinant mRNA were surprising. CONCLUSIONS: Through characterization of the gene expression response to Fab production under industrially relevant cultivation conditions, we identified potential cell engineering targets. Thereby, we hope to enable rational approaches to improve cell fitness and Fab yields. Furthermore, we highlight ribosome stalling caused by the amino acid sequence of the recombinant protein as a possible challenge during recombinant protein production.

Interaction of Periplasmic Fab Production and Intracellular Redox Balance in Escherichia coli Affects Product Yield.

Antibody fragments such as Fab's require the formation of disulfide bonds to achieve a proper folding state. During their recombinant, periplasmic expression in Escherichia coli, oxidative folding is mediated by the DsbA/DsbB system in concert with ubiquinone. Thereby, overexpression of Fab's is linked to the respiratory chain, which is not only immensely important for the cell's energy household but also known as a major source of reactive oxygen species. However, the effects of an increased oxidative folding demand and the consequently required electron flux via ubiquinone on the host cell have not been characterized so far. Here, we show that Fab expression in E. coli BL21(DE3) interfered with the intracellular redox balance, thereby negatively impacting host cell performance. Production of four different model Fab's in lab-scale fed-batch cultivations led to increased oxygen consumption rates and strong cell lysis. An RNA sequencing analysis revealed transcription activation of the oxidative stress-responsive soxS gene in the Fab-producing strains. We attributed this to the accumulation of intracellular superoxide, which was measured using flow cytometry. An exogenously supplemented ubiquinone analogue improved Fab yields up to 82%, indicating that partitioning of the quinone pool between aerobic respiration and oxidative folding limited ubiquinone availability and hence disulfide bond formation capacity. Combined, our results provide a more in-depth understanding of the profound effects that periplasmic Fab expression and in particular disulfide bond formation has on the host cell. Thereby, we show new possibilities to elaborate cell engineering and process strategies for improved host cell fitness and process outcome.

Microbioreactor Cultivations of Fab-Producing Escherichia coli Reveal Genome-Integrated Systems as Suitable for Prospective Studies on Direct Fab Expression Effects.

Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli (E. coli) and the high degree of similarity within this protein class, a generic platform technology is still not available. Indeed, feasible production of new Fab candidates remains challenging. In this study, a setup that enables direct characterization of host cell response to Fab expression by utilizing genome-integrated (GI) systems is established. Among the multitude of factors that influence Fab expression, the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest (GOI) are varied. The resulting 32 production clones are characterized in carbon-limited microbioreactor cultivations with yields of 0-7.4 mg Fab per gram of cell dry mass. Antigen-binding region variations have the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performs better than the BL21(DE3) strain. Translocation mechanism variations mainly influence leader peptide cleavage efficiency. Plasmid-free systems, with a single copy of the GOI integrated into the chromosome, reach Fab yields in the range of 80-300% of plasmid-based counterparts. Consequently, the GI Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions.