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.