Laboratory evolution of copper tolerant yeast strains

Abstract

Background: Yeast strains endowed with robustness towards copper and/or enriched in intracellular Cu might find application in biotechnology processes, among others in the production of functional foods. Moreover, they can contribute to the study of human diseases related to impairments of copper metabolism. In this study, we investigated the molecular and physiological factors that confer copper tolerance to strains of baker's yeasts.

Results: We characterized the effects elicited in natural strains of Candida humilis and Saccharomyces cerevisiae by the exposure to copper in the culture broth. We observed that, whereas the growth of Saccharomyces cells was inhibited already at low Cu concentration, C. humilis was naturally robust and tolerated up to 1 g · L-1 CuSO4 in the medium. This resistant strain accumulated over 7 mg of Cu per gram of biomass and escaped severe oxidative stress thanks to high constitutive levels of superoxide dismutase and catalase. Both yeasts were then "evolved" to obtain hyper-resistant cells able to proliferate in high copper medium. While in S. cerevisiae the evolution of robustness towards Cu was paralleled by the increase of antioxidative enzymes, these same activities decreased in evolved hyper-resistant Candida cells. We also characterized in some detail changes in the profile of copper binding proteins, that appeared to be modified by evolution but, again, in a different way in the two yeasts.

Conclusions: Following evolution, both Candida and Saccharomyces cells were able to proliferate up to 2.5 g · L-1 CuSO4 and to accumulate high amounts of intracellular copper. The comparison of yeasts differing in their robustness, allowed highlighting physiological and molecular determinants of natural and acquired copper tolerance. We observed that different mechanisms contribute to confer metal tolerance: the control of copper uptake, changes in the levels of enzymes involved in oxidative stress response and changes in the copper-binding proteome. However, copper elicits different physiological and molecular reactions in yeasts with different backgrounds.

Figure 1

Assay of copper tolerance in yeast strains. Five μl of 1:10 serial dilutions were plated on minimal medium without or with 0.5 g · L^-1 CuSO₄ (a) or YPD medium containing 0-2.5 g · L^-1 CuSO₄ (b). Plates were incubated two days at 30°C. AL5 is the C. humilis strain; BL7, EL1 and GL6 are different S. cerevisiae strains.

Figure 2

Growth of yeast cells in YPD + 2.5 g · L^-1 CuSO₄. Evolved (black cirles), non-evolved (black triangles) and de-adapted (black squares) cells of C. humilis AL5 (a), S. cerevisiae BL7 (b), S. cerevisiae EL1 (c), S. cerevisiae GL6 (d). The values reported are averages of three replicates. Calculated standard deviations are ≤ 0.6, making error bars not appreciable.

Figure 3

Intracellular copper measured during growth in YPD + 2.5 g · L^-1 CuSO₄. C. humilis AL5 (a); S. cerevisiae BL7 (b); S. cerevisiae EL1 (c) and S. cerevisiae GL6 (d). White bars: evolved cells; black bars: de-adapted cells; grey bars: non-evolved cells. The amount of Cu is reported as mg · g^-1 biomass. Values are the average of three replicates. Note the change of scale in (a).

Figure 4

Cytofluorimetric analysis. Evolved and non-evolved C. humilis (a) and S. cerevisiae (b) cells. Cells were collected during exponential growth in YPD + 2.5 g ^. L^-1 CuSO₄ and stained with propidium iodide. The x- and y-axes of the histogram display the log fluorescence intensity (PI) and the number of collected cells (events) per sample, respectively. Propidium positive cells are on the right side of the distribution whereas viable cells are on the left side. Fluorescence distributions are representative of three replicates obtained in independent experiments. This analysis was carried out with S. cerevisiae cells despite their extreme poor growth in copper medium thanks to the very small number of cells required.

Figure 5

Fluorimetric analysis of superoxide anion (OH^-•) production. Evolved and non-evolved C. humilis (a) and S. cerevisiae (b) cells exponentially growing in YPD (white bars) and in YPD + 2.5 g ^. L^-1 CuSO₄ (grey bars). OH^-• formation is expressed as fluorescence intensity of ethidium in arbitrary units. Data presented are the mean of at least three independent analyses.

Figure 6

SDS-PAGE of copper-binding proteins. (a) S. cerevisiae. Lane 1: proteins from non-evolved cells grown in YPD; lane 2: proteins from evolved cells grown in YPD; lane 3: proteins from evolved cells grown in YPD + CuSO₄ 2.5 g · L^-1. (b) C. humilis. Lane 1: proteins from non-evolved cells grown in YPD; lane 2: proteins from evolved cells grown in YPD; lane 3: proteins from non-evolved cells grown in YPD + CuSO₄ 2.5 g · L^-1; lane 4: proteins from evolved cells grown in YPD + CuSO₄ 2.5 g · L^-1.