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.