Adaptive evolution of baker's yeast in a dough-like environment enhances freeze and salinity tolerance

Abstract

We used adaptive evolution to improve freeze tolerance of industrial baker's yeast. Our hypothesis was that adaptation to low temperature is accompanied by enhanced resistance of yeast to freezing. Based on this hypothesis, yeast was propagated in a flour-free liquid dough model system, which contained sorbitol and NaCl, by successive batch refreshments maintained constantly at 12°C over at least 200 generations. Relative to the parental population, the maximal growth rate (µ(max)) under the restrictive conditions, increased gradually over the time course of the experiment. This increase was accompanied by enhanced freeze tolerance. However, these changes were not the consequence of genetic adaptation to low temperature, a fact that was confirmed by prolonged selection of yeast cells in YPD at 12°C. Instead, the experimental populations showed a progressive increase in NaCl tolerance. This phenotype was likely achieved at the expense of others traits, since evolved cells showed a ploidy reduction, a defect in the glucose derepression mechanism and a loss in their ability to utilize gluconeogenic carbon sources. We discuss the genetic flexibility of S. cerevisiae in terms of adaptation to the multiple constraints of the experimental design applied to drive adaptive evolution and the technologically advantageous phenotype of the evolved population.

Figure 1

Freeze tolerance of cells harvested from the evolved populations. Molasses‐grown yeast cells from the parental (●), 50‐ (), 100‐ () and 200‐generation (○) evolved populations were transferred to LD medium and total CO₂ production at 30°C was measured before (control, time 0) and after freezing and frozen storage at −20°C for 7, 14 or 21 days. The amount of CO₂ produced for 180 min at 30°C was recorded in a home‐made fermentometer. Frozen samples were thawed at 30°C for 30 min before measuring gassing power. Values are expressed as ml of CO₂ per sample. In all cases, values represent the means of at least three independent experiments. The error associated with the points was calculated by using the formula: , where n is the number of measurements. Additional details are given in the Experimental procedures section.

Figure 2

The evolved strains show increased freeze tolerance in flour‐based dough. Molasses‐plate‐grown cells of the parental CR and evolved CR19 and CR20 baker's yeast strains were used to prepare lean dough as described in the Experimental procedures section. Samples were quickly frozen at −80°C for 1 h and stored at −20°C for 21 days. Then, the frozen dough (grey bars) was left to thaw at 30°C for 30 min and CO₂ production was recorded in a home‐made fermentometer (Chittick apparatus). Unfrozen samples (black bars) were used as control. Values are expressed as ml of CO₂ produced after 180 min of dough fermentation and represent the means of at least three independent experiments. The error was calculated as described in Fig. 1.

Figure 3

Flow cytometric analysis is used to estimate approximate genome size. Logarithmic YPD‐grown cells of the industrial (CR and CR20) and laboratory (BY4743) S. cerevisiae strains were stained with propidium iodine and the DNA content of 30 000 individuals cells was measured by flow citometry. The horizontal axes in the graphs are measures of dye fluorescence and are proxies for genome size. Diploid cells of the BY4743 strain were used as control. The first peak indicates the unreplicated DNA content of the population, G1 phase (2n for the BY4743 strain), while the second peak indicates the replicated DNA content, G2 phase (4n for the BY4743 strain). A representative experiment is shown.

Figure 4

Phenotypic characterization of evolved clones and petite mutants. Cells of the parental CR and evolved CR19 and CR20 strains were assayed for growth on different culture media and/or conditions. (A) YPD at 30°C or 12°C. (B) YPD, LD or YPD containing 1 M NaCl at 30°C. (C) YP containing raffinose (YPRaf), maltose (YPMal) or ethanol (YPEtOH) as the sole carbon source at 30°C. In some cases, two petite yeast mutants of the CR strain, CRρ1 and CRρ2, were tested under the same conditions (panel B and C). YPD‐exponentially growing cultures (OD₆₀₀ = 1.0) were diluted (1–10⁻³) and aliquots were extended (10 µl 10⁻³, A) or spotted (2.5 µl 1–10⁻², B and C) on Petri dishes. Cells were inspected for growth after 2–4 (30°C) or 10 (12°C) days. Results of a representative experiment are shown.

Figure 5

Enzymatic and metabolic profile. Molasses‐grown cells of the parental CR (●) and evolved CR20 (○) strains were transferred to LD medium and incubated at 30°C. At the indicated times, samples of the cultures were taken for further analysis.
A. Residual glucose and maltose in the culture supernatant.
B. Production of glycerol and ethanol.
C. Invertase and maltase activity in cell‐free extracts. Experimental details are given in the Experimental procedures section.
In all cases, values represent the means of at least three independent experiments. The error associated with the points was calculated as described in Fig. 1.