Design of Experiments

To study the effect of temperature and qS on growth, productivity, lysis and leakiness during SpA production, a full-factorial screening DoE was performed. Since growth of the X-press strain is repressed by Gp2 expression during induction, we chose to apply a constant substrate feed rate in our experiments. In the DoE, this is reflected in the first factor as the specific glucose uptake rate with respect to biomass at start of induction (qS,0 ). It was set to 0.13, 0.25 and 0.50 g/g/h, respectively. Temperature during induction, the second factor, was 25, 30 or 35°C, respectively. The different parameter settings are hereafter referred to as “temperature [°C]/glucose uptake rate [g/g/h]”, e.g. 30/0.25. Thus, the five factor combinations for the SpA cultivations were: 25/0.13, 25/0.5, 30/0.25/, 35/0.13, 35/0.5. AtqS,0 = 0.5 g/g/h, the physiological capabilities of the X-press strain were far exceeded (manifested in glucose accumulation and lysis, see Supporting Information 2) and linear regression did not result in significant model coefficients, thus we decided to omit the data of these experiments from further analysis in this study. In addition to the SpA cultivations, the growth arrest experiment with the plasmid free X-press strain was conducted at conditions 30/0.13. Furthermore, the process conditions leading to the highest productivity during SpA cultivations were repeated in both strains containing the plasmid with the VHH sequence.