Impact of process parameters onYX/S
It has long been known that heterologous expression in plasmid-basedE. coli systems has a grave impact on cell physiology, widely
known as metabolic burden (Mairhofer, Scharl, Marisch,
Cserjan-Puschmann, & Striedner, 2013). This burden is often associated
with a decrease in growth rate and ultimately cell lysis (Bentley,
Mirjalili, Andersen, Davis, & Kompala, 1990; Bienick et al., 2014). We
assessed the impact of the selected process parameters on growth by
measuring YX/S . To investigate the induced growth
repression in the X-press strain without metabolic burden from
recombinant product formation, we performed a cultivation without an
exogenous plasmid and solely inducing Gp2 expression by addition of
L-arabinose. After induction, YX/S was reduced by
half from 0.48 in the uninduced state to levels between 0.24 and 0.27,
remaining almost constant throughout the cultivation (Figure 1A). We
assumed that any additional reduction of YX/S is
caused by the metabolic burden of heterologous gene expression. During
production of SpA in cultivation 25/0.13, the reduction ofYX/S was similar to the “basal” reduction by
Gp2 expression, thus the metabolic load of SpA expression had little
effect on growth. An additional reduction was observed at higher
temperature and qS,0 . In both cultivations
30/0.25 and 35/0.13, YX/S decreased throughout
the cultivation to values between 0.03 and 0.1. Hence, the metabolic
load of recombinant product expression still affected growth of the
X-press strain, but it was largely mitigated by induced growth
repression. Contrarily, in the reference strain BL21(DE3),YX/S varied greatly between different cultivation
conditions during SpA production (Figure 1B). It decreased at higher
induction temperature, so that at 25°C, biomass yield of BL21(DE3) was
least affected, while at 35°C, growth was fully arrested. This behavior
might stem from an increase in target gene transcript levels competing
with host mRNA at elevated temperatures (C. S. Shin, Hong, Bae, & Lee,
1997; Vind, Sorensen, Rasmussen, & Pedersen, 1993).