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