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.