Abstract

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 andqS 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 .
Keywords
continuous manufacturing; leakiness; outer membrane integrity; periplasmic protein release; secretion
Introduction
Escherichia coli is a widely used expression host for recombinant protein production. Its advantages lie in short doubling times, growth on cheap media to high cell densities and straightforward cloning procedures (Kleiner-Grote, Risse, & Friehs, 2018; Rosano & Ceccarelli, 2014; Yoon, Kim, & Kim, 2010). However, the product is usually expressed inside the cell, which requires cell disruption in downstream processing, leading to release of unwanted host cell proteins and other contaminants, like lipids and DNA (Balasundaram, Harrison, & Bracewell, 2009). If the target protein is produced as insoluble inclusion bodies (IBs), additional IB processing is needed.
Especially with the growing interest in continuous manufacturing (C. Chen, Wong, & Goudar, 2018; Kateja, Agarwal, Hebbi, & Rathore, 2017), extracellular production is an important enabler for future bioprocessing with E. coli . Secretion of recombinant protein to the medium furthermore enhances solubility, stability and biological activity of the product (Mergulhão & Monteiro, 2007). This can be achieved either by one-step-secretion (directly from the cytoplasm to the extracellular space) via the T1SS or T3SS system, or by two-step-secretion: In the first step, the protein is directed through the inner membrane via the Sec- or Tat-pathway. In the second step, the outer membrane (OM) is made permeable, or “leaky”, to release the product to the medium (Kleiner-Grote et al., 2018). Numerous studies on how to increase leakiness during cultivation exist and several reviews cover this research in detail (Kleiner-Grote et al., 2018; Mergulhão, Summers, & Monteiro, 2005; Yoon et al., 2010).
One approach to increase leakiness is chemical permeabilization by addition of media supplements, like Triton-X, glycine or EDTA. However, those additives usually have detrimental effects on the viability of the cells and might harm the product (Kleiner-Grote et al., 2018). Another approach is the generation of leaky E. coli mutants. Many expression systems that show permanently high leakiness have been engineered to date. Their outer membrane structure is usually altered by mutations in cell envelope genes and signal peptides are optimized for higher translocation efficiency (Kleiner-Grote et al., 2018; Zhou, Lu, Wang, Selvaraj, & Zhang, 2018). However, detailed process information at bioreactor scale is often missing for these strains.
Another reported strategy is the enhancement of OM permeability via temperature or specific growth rate (µ ). Shokri, Sanden, and Larsson (2002) showed that growth rate dependent changes in the membrane composition have an effect on protein leakage. In their study, in continuous cultivation of E. coli W3110, leakiness had an optimum at a dilution rate of 0.3 h-1 and declined upon lowering or increasing µ . Similar results were obtained in fed-batch studies of W3110 (Voulgaris, Finka, Uden, & Hoare, 2015) and a K12 derivate (Bäcklund et al., 2008), in which an increase in µled to enhanced leakiness. Contrarily, Rinas and Hoffmann (2004) stated that µ had no significant effect on periplasmic protein release during heat induction of E. coli TG1 strains. In another fed-batch study using a C41(DE3) strain, it has even been stated thatµ and leakiness are inversely correlated (Wurm, Marschall, Sagmeister, Herwig, & Spadiut, 2017). Adverse reports can also be found about the influence of temperature on OM leakiness for differentE. coli strains. While Rodríguez-Carmona et al. (2012) found that leakage of a Fab was enhanced at lower temperatures, several other studies suggest that increased temperature drives periplasmic protein release (Rinas & Hoffmann, 2004; Wurm, Marschall, et al., 2017; Wurm, Slouka, Bosilj, Herwig, & Spadiut, 2017). Controlling leakiness via temperature and µ is an interesting approach, since it does not require alteration of the chemical environment and is easy to implement, however, the contrary results in the aforementioned studies illustrate that the mechanisms that temperature and µ exert on outer membrane leakiness might depend on a variety of factors, like the strain, product or promoter, and are not fully understood yet.
In this study, we investigated the influence of the process parameters cultivation temperature and specific glucose uptake rate (qS , linked to µ via the biomass yieldYX/S ) on OM leakiness of a novel E. coliexpression host. The X-press strain is a proprietary expression technology recently developed by enGenes Biotech GmbH (Mairhofer, Striedner, Grabherr, & Wilde, 2016; Stargardt, Feuchtenhofer, Cserjan-Puschmann, Striedner, & Mairhofer, 2020). It is derived from BL21(DE3) and carries a genomically integrated sequence coding for the bacteriophage-derived RNA polymerase inhibitor Gp2 under control of thearaB promoter. This protein from the T7 phage inhibits the host RNA polymerase, while the T7 RNA polymerase stays unaffected. Thus, upon induction with L-arabinose, host mRNA levels and cell proliferation are reduced, while IPTG-induced target protein expression is enhanced. This approach to decouple growth from recombinant protein production has already been shown to increase specific yield and product quality (Lemmerer et al., 2019; Stargardt et al., 2020). In previous experiments, the X-press strain showed high tendency to leak periplasmic protein to the medium (Stargardt et al., 2020). Therefore, in the present research, we further investigated its response to the process parameters temperature and qS , both known to affect leakiness of other E. colistrains, in fed-batch cultivations. We performed a screening Design of Experiments (DoE) to find the adequate parameter space for enhancing leakiness while maintaining high productivity and viability. We compared the X-press strain to the industrial standard strain BL21(DE3), using two industrially relevant model proteins: Protein A (SpA) fromStaphylococcus aureus and a VHH sdAb (VHH). The processes were analyzed with respect to YX/S , productivity, lysis and leakiness. With this holistic approach, we aimed at 1) characterization of a novel E. coli expression host for growth decoupled protein secretion and 2) finding the parameter space that allows tight control of leakiness and productivity for extracellular recombinant protein production.
Materials and Methods