3 Results & Discussion
3.1 FACS analysis of chemostat cultivations
with S. cerevisiae,C.U17
We cultivated the recombinant S. cerevisiae strain, C.U17, in
duplicated chemostats and performed time-resolved analysis of the
population with respect to particle size measured by FSC-A (used as a
proxy for Morphology), (Figure 1A). We observed the differentiation of
three main subpopulations over time with respect to FSC-A (Figure 2; See
Supplementary materials Figure S1 and S6 for replicates). We named the
three subpopulations Population 1 (small particles),Population 2 (medium particles) and Population 3 (large
particles), respectively. Population 1 corresponded to 23 % of
the total population after 270 hours of glucose-limited growth,Population 2 corresponded to 46 % and Population 3corresponded to 31 % (Figure 3A).
3.2 Morphology of FACS sorted
populations
We FACS sorted each of the three subpopulations based on FSC-A and
inspected the subpopulations by microscopy (Figure 1A, B). We observed
that Population 1 primarily contained single cells where about a
fourth presented small round buds, whereas Population 2 consisted
of multi-budded cells with a more ellipsoidal cell shape (Figure 3B).Population 3 contained branches of elongated cells with multiple
buds (pseudohyphae).
Morphological changes towards a more filamentous and pseudohyphal growth
are known effects of chemostat growth and also a known adaptive response
of cells in a nutrient poor environment as a strategy to forage for
nutrients (Ceccato-Antonini and Sudbery 2004; Hope et al. 2017; Rai et
al. 2019). This suggests that Population 3 has emerged as a
result of the glucose-limited conditions.
Flocculation and wall growth have been observed in prolonged chemostats
where cells stick to the surface of the culture vessels (Dykhuizen &
Hartl, 1983). However, wall growth has not been observed in this study.
Morphological changes exhibited by cell subpopulations comprising large
single-budded and multi-budded cells have previously been observed in
industrial scale chemostat cultivations with S. cerevisiae and
were related to hypoxia (Aon, Tecson, & Loladze, 2018). In the current
study, no sign of oxygen limitation can be found during the chemostat
cultures (Figure 4). Thus, we link the formation of the subpopulations
to the selective pressure of the constant glucose-limited environment.
We confirm this link by glucose-pulse experiments. A uniform particle
size and morphology with no significant changes over time could be
detected in cultures exposed to continuous glucose pulsing
(Supplementary materials Figure S11, S12, S13).
3.3 Maximum growth rate of FACS sorted
subpopulations
Each of the FACS sorted individual populations had maximum growth rates
equal to or higher than the initial cell clone (Figure 5A).Population 1 had a significant higher maximum growth rate on
glucose compared to the other populations, which can be explained by a
lower metabolic burden of the heterologous insulin production (this is
further discussed in section 3.4.2).
3.4 Reinitiated chemostat cultures of FACS
subpopulations
Each of the FACS sorted subpopulations were propagated in batch cultures
and stored as glycerol stocks (Figure 1A, B).
We cultured each of the propagated populations in new chemostat cultures
in order to characterize the populations individually, with respect to
particle size (FSC-A), heterologous insulin production and intracellular
proteome (Figure 1C). We also compared the subpopulations to theend sample cells from which they were sorted with respect to
heterologous insulin production. Biomass concentrations measured as cell
dry weight for each cultivation can be found in Supplementary materials
Figure S17.
3.4.1 Particle size (FSC-A)
We observed that the particle size of some of the cells inPopulation 2 and Population 3 after propagation in batch
cultures had changed towards particle sizes typical of Population
1 (Supplementary materials Figure S2, S3, S4, S7, S8, S9). However, the
particle size increased again over time in the reinitiated chemostats.
3.4.2 Heterologous insulin
production
The production of the recombinant insulin decreased over time when theinitial cell clone was cultivated under prolonged glucose-limited
conditions (Figure 5B). We have previously coupled this decline to
changes in the intracellular proteome of the strain observed in
measurements at the culture level (Wright et al., 2020). The end
sample cells , continued to produce insulin at the same low level when
cultured de novo in chemostats, while the subpopulations showed
divergent phenotypes with respect to heterologous insulin production
(Figure 5B). In the beginning of the reinitiated cultures, the sum of
the insulin yields of Population 1-3 was ~1.8
mg/gDW (adjusting for the ratios between the three subpopulations at the
end of the cultivations with the Initial cell clone ). This yield
approximates the insulin yield observed by bulk measurements in the end
of the cultivations with the initial cell clone . This indicates
that the insulin yield of the three populations were conserved during
the batch propagation of the FACS sorted cells (estimated to more than
40 generation).
Only small amounts of insulin could be detected in cultures withPopulation 1 . The production of a heterologous protein is a
burden for the cells (Peebo & Neubauer, 2018) and cells with reduced
productivity will have a growth advantage compared to cells, which are
not able to adapt (Rugbjerg & Olsson, 2020). We observe this advantage
as a higher maximum growth rate for Population 1 under glucose
rich conditions (Figure 5A). Therefore, we suggest that Population
1 has mainly arisen due to the burden of the heterologous protein
production and to a lesser extent due to the glucose-limited conditions.
The determined maximum growth rate for Population 1 is slightly
lower than the growth rate of the wildtype CEN.PK113-7D strain (0.37
h-1 ± 0.01), which has previously been used as
reference strain for comparison with the C.U17 strain (Seresht et al.,
2013).
The bulk measurements of insulin in the initial cell clonereached a new steady state after around 150 hours of glucose-limited
growth (Figure 5B). Moreover, a steady state between the three
morphological phenotypes seemed to occur after around 200 hours of
chemostat cultivation (Figure 2B). Cultivations with the end
sample cells for another 250 hours under glucose-limited conditions
confirm the steady state with respect to insulin production (Figure 5B).
This indicates that none of the three populations had a significant
higher fitness under chemostat conditions compared to the other. Based
on the maximum growth rate experiments, we would have expected thatPopulation 1 should take over the entire population in the
chemostat and that Population 2 and Population 3 gradually
would be washed out. However, the increase in fitness of
chemostat-adapted cells is often specific to the low nutrient-limited
environment and many organisms adapted to chemostat conditions show
reduced growth capabilities in nutrient rich environments (Gresham &
Hong, 2015; Hong & Gresham, 2014; Wenger et al., 2011). Thus, a higher
maximum growth rate under glucose rich conditions will not be an
adequate measure of fitness under glucose-limited conditions.
Yeast cells release a variety of different metabolites and prefer the
uptake of extracellular metabolites over self-synthesis (Campbell et
al., 2015). Population heterogeneity can emerge as a consequence of
metabolic cooperation between cells and a population as a whole can
benefit from division of labor between individuals (Ackermann, 2015;
Campbell, Vowinckel, & Ralser, 2016). We speculate that the three
populations in the chemostat cooperate in metabolism and exchange
metabolites and that this may explain the observed steady state between
the subpopulations. However, this suggestion needs to be further
investigated.
3.4.3 Intracellular
proteome
The FACS sorted cells were further characterized with respect to their
intracellular proteome during chemostat growth. We compared the
intracellular proteome in the beginning of chemostat cultures of the
three populations with our previously reported proteomics dataset from
the beginning of chemostat cultures with the initial cell clone(Wright et al., 2020), (Table 1). Moreover, we compared the
proteome of the three individual populations in the beginning of the
chemostat cultures and again in the end of the cultivations.
In the beginning of the chemostat cultures, Population 2 differed
from Population 1 and the initial cell clone with respect
to proteins involved in the central carbon metabolism (Table 1-3). The
levels of these proteins were significantly lower in Population 2(See Figure 6 for example of glycolytic proteins). Protein synthesis is
an energetically expensive process. Cells, which can economize the
protein synthesis, e.g., by reducing the production of overexpressed
proteins in a nutrient-limited environment, will have an advantage over
cells, which cannot adapt. Reduced capacity of the central carbon
metabolism including the glycolysis and TCA cycle is a well-known
adaptive response to chemostat conditions (Franchini & Egli, 2006;
Jansen et al., 2005; Mashego, Jansen, Vinke, van Gulik, & Heijnen,
2005). This suggests that the establishment of Population 2 is a
response to the glucose-limited conditions and confers a fitness
advantage in the chemostat.
6 % of the measured proteome differed between Population 1 and
the initial cell clone (log2 fold-change > 0.5 or
log2 fold-change < -0.5, q-value <0.05), (Table 1).
No differentially expressed GO terms could be found related to these
proteins (q-value <0.05). However, levels of the proteins
expressed from the selective markers, URA3 and HIS3 were significantly
lower in cultivations with Population 1 compared to cultivations
with the initial cell clone and Population 2 (Figure 5C).
This indicates that either a reduced plasmid copy number or a down
regulation of the plasmid genes caused the low insulin production inPopulation 1 . A decline in plasmid copy number over cultivation
time has previously been measured in the bulk of chemostat cultivations
with the initial cell clone in the time frame investigated in
this study (Seresht et al., 2013) and may be related to Population
1 growing over time. In general, several proteins involved in the
biosynthesis of uridine are significantly decreased in the beginning of
cultivations with Population 1 compared to Population 2(q-value<0.05) (see Supplementary materials Figure S16 for
levels of the specific proteins). This is not the case for the
biosynthesis pathway of histidine (see Supplementary materials Figure
S15 for levels of the specific proteins).
After 271 hours of glucose-limited growth, only 16 proteins differed
with more than 0.5 log2 fold-change between Population 1 andPopulation2 (q-value < 0.05), (Table 1, see
Supplementary materials Table S3 for the specific proteins). This
indicates that two adaptive mechanisms occurred over time inPopulation 1 . In the original chemostat cultures, the fitness of
the cells was increased towards the burden of the heterologous insulin
production by a decrease of the insulin productivity. In the reinitiated
chemostat cultures, the fitness was increased towards the
glucose-limited conditions by a decrease in the overcapacity of
especially enzymes linked to the central carbon metabolism. We have
previously shown that the adaptation to glucose-limited conditions is
enhanced by the production of a heterologous product (Wright et al.,
2020). This may explain the delayed adaptation in Population 1
with respect to the reduction of enzymatic overcapacity compared toPopulation 2 . During the first 100 hours of the reinitiated
chemostats with Population 2 there seems to be a further
reduction in the glycolytic capacity of the cells (Figure 6).
No significantly expressed proteins could be found betweenPopulation 2 and Population 3 in the beginning of
chemostat cultivations, whereas 50 proteins differed betweenPopulation 1 and Population 3 ( log2 fold-change
> 0.5 or log2 fold-change < -0.5, q-value
<0.05). Population 3 consisted of cells with
multiple-buds that span a larger variety of cells with respect to
particle size. Therefore, a larger variation was observed between
replicates with this population (Supplementary materials Figure S14).
This variation explains the lower level of significantly changing
proteins between Population 3 and the other subpopulations.
3.4.4 Cell synchrony
We have previously shown that the initial cell clone synchronized
growth during the first 100 hours of chemostat growth (Wright et al.,
2020). The time point where the synchronized growth stopped correlated
with the time point where the heterologous insulin production started to
decrease (Figure 4; Figure 5B). This also correlated well with the time
point where the population heterogeneity with respect to particle size
(FSC-A) arose in the culture (Figure 2B). Neither the end sample
cells, nor Population 3 showed synchronized growth in the
reinitiated cultures. We explain this by the larger fraction of
population heterogeneity in the reinitiated chemostats already from the
beginning of the cultures (Supplementary materials Figure S7-S10). This
heterogeneity was conserved during the initial batch propagation.Population 1 showed synchronized growth for 150 hours, whereasPopulation 2 showed synchronized growth for 50 hours. The stop of
synchronized growth of Population 2 correlated with an increase
in population heterogeneity in the reinitiated cultures with respect to
particle size (FSC-A), (Supplementary materials Figure S8).
3.5 Three apparent phenotypes underlie the adaptive response
observed at the bulk
level
We have previously shown that the recombinant S. cerevisiaestrain (C.U17) adapts in a reproducible manner at the average population
level for five replicated cultures with respect to changes in
heterologous insulin production and intracellular proteome under
prolonged glucose-limited conditions (Wright et al., 2020). Diversity of
adaptation varies as a function of the distribution of fitness effects
between beneficial outcomes (Gresham et al., 2008). Thus, if an adaptive
path confers a much greater selective advantage compared to other
selective outcomes, that path will be highly reproducible. Due to the
reproducibility of the phenotypic outcome of chemostat cultivations with
the initial cell clone, we expected the adaptation to be driven
by the selection of a single clone with a large relative selective
advantage. In the present study, however, we demonstrate that the
adaptation is not caused by the selection towards a single beneficial
phenotype. Instead, the isogenic strain differentiated into
subpopulations and reproducibly established three main subpopulations
after 271 hours of glucose-limited growth (Figure 3A). Our results
indicate that three apparent phenotypes underlie the adaptive response
observed at the bulk level (Figure 7). We speculate that this phenotypic
heterogeneity is a result of different mechanisms to increase fitness.Population 1 has increased fitness by downregulating heterologous
insulin production most likely by decreasing the plasmid copy number
whereas Population 2 has adapted towards the glucose-limited
conditions. Population 3 seems to be a response to both the
burden of the heterologous insulin production and the glucose-limited
conditions having a phenotype with reduced insulin productivity and
pseudo-hyphal growth.
Adaptation and evolution in chemostats have been highly investigated at
the average population level. However, only few studies investigate
adaptation in terms of isogenic cells differentiating into phenotypic
subpopulations (Kundu et al., 2020). Our results highlight the
importance of considering population heterogeneity when studying
adaptation as bulk adaptive outcomes observed at the culture level can
be a mixed response composed of different phenotypes or subpopulations.