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).