Figure 1: Overview of the experimental setup. A) The monoclonal initial cell clone was used to inoculate a chemostat culture. After 271 hours of continuous cultivation a sample of adapted cells were taken (end sample cells) and stored as glycerol stock. The end sample cells were further separated by FACS sorting into three different subpopulations based on FSC-A. B) The FACS sorted subpopulations were inspected by microscopy, propagated in batch cultures and stored as glycerol stock. C) Each of the FACS sorted subpopulations were cultured in reinitiated chemostats and compared in terms of particle size (FSC-A), intracellular proteome and heterologous insulin production. In addition, the end sample cells were cultured in reinitiated chemostats and analyzed for heterologous insulin production and particle size (FSC-A).

Figure 2: Distribution of cells with FSC-A corresponding to Population 1 (log2(FSC-A) < 16.6), Population 2 (16.6 < log2(FSC-A) < 17.7) or Population 3 (log2(FSC-A) > 17.7) at different time points during a representative culture of the initial cell clone. A) Density plot of log2(FSC-A) at different cultivation time points. 100,000 cells were analyzed at each time point. B) Fraction of cells with a FSC-A corresponding to each of the three populations as function of cultivation time.

Figure 3: A) Cell count with respect to log2(FCS-A) measured by FACS after 271 hours of chemostat culture with the initial cell clone. The color code indicates the separation of cells into three subpopulations for FACS sorting. 100,000 cells were analyzed. B) Images of cell morphology of the three FACS sorted subpopulations.

Figure 4: Dissolved oxygen tension during chemostat cultivations with the Initial cells clone, the end sample cells and the sorted subpopulations population1, Population 2 and Population 3. The pO₂ electrode was defective for replicate 2 with the initial cell clone.

Figure 5: A) Maximum growth rate in batch cultures. Error bars indicate differences between three biological replicates, B) Insulin yield over time in chemostat cultivations. C) Levels of His3p and Ura3p over time in chemostat cultivations.

Figure 6: Levels of glycolytic enzymes measured during replicated chemostat cultivations with the initial cell clone, Population 1, Population 2 and Population 3. Only proteins, which differ significantly between Population 1 and Population 2 in the beginning of the cultivations (≤ 48 hours of chemostat growth) are pictured (q-value <0.05, log2 fold-change >0.5).

Figure 7: The adaptive outcome observed at the culture level is a mixed response of at least three apparent phenotypes. We speculate that Population 1 is mainly an outcome of the selective pressure of the glucose-limited conditions whereas Population 2 is a reaction towards the protein burden of the heterologous insulin production. Population 3 seems to be a product of both the selective pressure of the heterologous insulin burden and the glucose-limited conditions.