In vivo evolutionary engineering for ethanol-tolerance of Saccharomyces cerevisiae haploid cells triggers diploidization.

Microbial ethanol production is an important alternative energy resource to replace fossil fuels, but at high level, this product is highly toxic, which hampers its efficient production. Towards increasing ethanol-tolerance of Saccharomyces cerevisiae, the so far best industrial ethanol-producer, we evaluated an in vivo evolutionary engineering strategy based on batch selection under both constant (5%, v v-1) and gradually increasing (5-11.4%, v v-1) ethanol concentrations. Selection under increasing ethanol levels yielded evolved clones that could tolerate up to 12% (v v-1) ethanol and had cross-resistance to other stresses. Quite surprisingly, diploidization of the yeast population took place already at 7% (v v-1) ethanol level during evolutionary engineering, and this event was abolished by the loss of MKT1, a gene previously identified as being implicated in ethanol tolerance (Swinnen et al., Genome Res., 22, 975-984, 2012). Transcriptomic analysis confirmed diploidization of the evolved clones with strong down-regulation in mating process, and in several haploid-specific genes. We selected two clones exhibiting the highest viability on 12% ethanol, and found productivity and titer of ethanol significantly higher than those of the reference strain under aerated fed-batch cultivation conditions. This higher fermentation performance could be related with a higher abundance of glycolytic and ribosomal proteins and with a relatively lower respiratory capacity of the evolved strain, as revealed by a comparative transcriptomic and proteomic analysis between the evolved and the reference strains. Altogether, these results emphasize the efficiency of the in vivo evolutionary engineering strategy for improving ethanol tolerance, and the link between ethanol tolerance and diploidization.

Mechanisms other than activation of the iron regulon account for the hyper-resistance to cobalt of a Saccharomyces cerevisiae strain obtained by evolutionary engineering.

Cobalt is an important metal ion with magnetic properties that is widely used for several industrial applications. Overexposure to cobalt ions can be highly toxic for the organisms because they usually overwhelm the endogenous physiological system that maintains their homeostasis causing (geno)toxic effects. To gain insight into the mechanism of cobalt toxicity, we characterized at the molecular and genetic levels a cobalt resistant CI25E Saccharomyces cerevisiae strain previously isolated by an in vivo evolutionary engineering strategy, and which was able to grow on 5 to 10 mM CoCl2. This evolved strain showed cross-resistance to other metal ions including iron, manganese, nickel and zinc, but not to copper. Moreover, the cobalt resistant trait was semi-dominant, and linked to more than one gene, as indicated by the absence of 2(+):2(-) segregation of the cobalt resistance. Genome wide transcriptional profiling revealed a constitutive activation of the iron regulon that could be accounted for by a constitutive nuclear localization of the transcriptional activator Aft1. However, the presence of Aft1 in the nucleus was not a prerequisite for hyper-resistance to cobalt, since a mutant defective in nuclear monothiol glutaredoxin encoding GRX3 and GRX4 that also leads to nuclear localization of Aft1 was cobalt hypersensitive. In addition, the loss of AFT1 only partially abolished the cobalt resistance in the evolved strain, and the deletion of COT1 encoding the major vacuolar transporter of cobalt had only a minor effect on this trait. Paradoxically to the activation of iron regulon, the evolved strain was hypersensitive to the iron chelator BPS, and this hypersensitivity was abrogated by cobalt ions. Taken together, this work suggested that cobalt resistance is not merely dependent upon activation of AFT1, but it likely implicates other mechanisms including intracellular reallocation of iron into important compartments whose function is dependent on this metal and adaptation of some cellular proteins to use Co(2+) in place of Fe(2+) for their catalytic activities.

Exposure to high static or pulsed magnetic fields does not affect cellular processes in the yeast Saccharomyces cerevisiae.

We report results of a study of the effects of strong static (up to 16 T for 8 h) and pulsed (up to 55 T single-shot and 4 x 20 T repeated shots) magnetic fields on Saccharomyces cerevisiae cultures in the exponential phase of growth. In contrast to previous reports restricted to only a limited number of cellular parameters, we have examined a wide variety of cellular processes: genome-scale gene expression, proteome profile, cell viability, morphology, and growth, metabolic and fermentation activity after magnetic field exposure. None of these cellular activities were impaired in response to static or pulsed magnetic field exposure. Our results confirm and extend previous reports on the absence of magnetic field effects on yeast and support the hypothesis that magnetic fields have no impact on the transcriptional machinery and on the integrity of unicellular biological systems.

Isolation of two cell populations from yeast during high-level alcoholic fermentation that resemble quiescent and nonquiescent cells from the stationary phase on glucose.

High-level production of bioethanol (140 g L(-1) in 45 h) in aerated fed-batch cultures of Saccharomyces cerevisiae was shown to be linked to the length of a production phase uncoupled to the growth. The induction of this phase was characterized by metabolic and morphologic changes reminiscent of those occurring in the stationary phase of growth on glucose. Global transcriptomic analysis of ethanol-stressed yeast cells in the uncoupling phase harboured features similar to those from stationary-phase cells on glucose. Two distinct cellular populations were isolated by Percoll density-gradient centrifugation in this uncoupling phase. The lower fraction was enriched by yeast cells that were mostly uniform in size and opalescent, containing a large amount of glycogen and trehalose, and exhibiting high respiratory activity. In contrast, the upper fraction was characterized by cells heterogeneous in size, with one to several small buds, which did not contain storage carbohydrates and which exhibited a poor respiratory competence while retaining a high relative glycolytic activity. These results are discussed in terms of a possible induction of a state similar to the quiescence state previously observed from yeast stationary-phase cultures, in response to ethanol toxicity, whose acquisition may be critical for performing high-level alcoholic fermentation.

Isolation of cobalt hyper-resistant mutants of Saccharomyces cerevisiae by in vivo evolutionary engineering approach.

Cobalt is an important element with magnetic properties used in various industrial applications, but is also needed for biological activity. Very little is known about the cellular response of living systems to cobalt stress. Towards investigating this mechanism, we isolated individual Saccharomyces cerevisiae cells resistant to high cobalt concentrations up to 8 mmoll(-1), by employing four different 'in vivo' evolutionary engineering strategies: selection under constant or gradually increasing stress levels, and selection under continuous or pulse exposure to cobalt stress. Selection under continuous exposure to gradually increasing cobalt stress levels yielded the most resistant cell population to cobalt. However, the resistance was highly heterogeneous within the mutant populations ranging from 3- to 3700-fold survival rate of isolated individuals to 8 mmoll(-1) CoCl2 in the most resistant population. Moreover, cobalt-resistant individual colonies were associated with 2-4-times lower intracellular cobalt contents as compared to wild-type, and with cross-resistance to metals such as nickel, zinc, manganese, but not to copper and chromium ions. Contrary to mutants evolved under continuous exposure to cobalt, those isolated by pulse exposure strategy also exhibited resistance to heat shock and hydrogen peroxide stress. Taken together, this study reinforced the fact that evolutionary engineering is useful in selecting strains with very specific phenotypes, and further illustrated the importance of the strategy chosen to isolate the best evolved strain.

Physiological behaviour of Saccharomyces cerevisiae in aerated fed-batch fermentation for high level production of bioethanol.

Saccharomyces cerevisiae was able to produce 20% (v/v) of ethanol in 45 h in a fully aerated fed-batch process recently developed in our laboratory. A notable feature of this process was a production phase uncoupled to growth, the extent of which was critical for high-level ethanol production. As the level of production was found to be highly variable, we investigated on this high variability by means of a detailed physiological analysis of yeast cells in two fed-batch fermentations showing the most extreme behaviour. We found a massive leakage of intracellular metabolites into the growth medium which correlated with the drop of cell viability. The loss of viability was also found to be proportional to the reduction of plasma membrane phospholipids. Finally, the fed-batch processes with the longest uncoupling phase were characterized by induction of storage carbohydrates at the onset of this phase, whereas this metabolic event was not seen in processes with a short uncoupling phase. Taken together, our results suggested that reproducible high-level bioethanol production in aerated fed-batch processes may be linked to the ability of yeast cells to impede ethanol toxicity by triggering a metabolic remodelling reminiscent to that of cells entering a quiescent GO/G1 state.