Evolution of copper tolerance in yeast cells

For all living organisms, copper (Cu) is an essential micronutrient taking part, with its redox chemistry, to several metabolic and regulatory cellular events. However, the same redox properties that make Cu essential are responsible for its toxicity. Indeed, Cu participates in reactions that generate Reactive Oxygen Species (ROS). ROS target main cellular macromolecules (proteins, lipids, DNA and RNA), leading to cellular dysfunctions and in the extreme case, to cell death. All living organisms evolved molecular mechanisms for Cu homeostasis. Indeed, uptake, transport and detoxification systems that actively prevent both Cu deficiency and poisoning are well conserved along the phylogenetic tree. Among eukaryotes, these mechanisms have been mainly investigated in the yeast Saccharomyces cerevisiae used as a model organism. Evolutionary engineering is a rational approach that uses the evolutionary principles to direct the selection of organisms with a desired set of phenotypes, allowing for the improvement of microbial properties. This approach can be exploited to obtain Cu-tolerant and Cu-accumulating yeast cells, with potential application in nutraceutics, as nutritional supplements, as well as in bioremediation, for the removal or recovery of metal ions. At the same time, evolutionary engineering is a valuable strategy to gain more insight into the molecular aspects of Cu tolerance in microbial cells. In the present work is described an evolutionary engineering strategy to improve Cu tolerance of natural yeasts. Strains of Saccharomyces cerevisiae and of Candida humilis originally endowed with different sensitivity and tolerance toward Cu have been exposed to increasing concentrations of Cu during cell cultivation in liquid medium. This treatment stably improved Cu tolerance of all strains. One evolved strain for each yeast species was then chosen to analyze in detail the physiological response to Cu. Compared with the original Cu-sensitive strains the two evolved strains showed improved cell viability and attenuated production of ROS. A reshaping of the profile of antioxidant enzymes and Cu-binding proteins was observed in both strains as a specific response to copper. Further investigations carried out on S. cerevisiae strains demonstrated a pivotal role of the CUP1 gene, encoding for a metallothionein. A 7-fold amplification of this gene was found associated with evolution of Cu tolerance. Finally, Cu tolerance in C. humilis cells was studied by proteomic analyses. Changes were observed in the levels of several proteins involved in the oxidative stress response (such as glycolytic enzymes), heat shock proteins, proteins involved in protein synthesis and energy production, proteins with a role in phospholipids synthesis. Cu exposure resulted in differential protein expression, in both non-evolved and Cu evolved cells. In general, changes in protein levels detected in evolved cells were smaller. On this basis, it was hypothesized that in the evolved cells copper tolerance relies only partly on the molecular mechanisms associated with the oxidative stress response. This work shows once again that evolutionary engineering is a powerful strategy to drive the gain of stable phenotypic traits. The evolved strains might found direct application in several biotechnological fields, and provide a kind of “molecular platform” for the investigation on the mechanisms of stress tolerance. The availability of data about the S. cerevisiae genome allowed a focused investigation on the molecular actors involved in Cu tolerance. In the case of C. humilis, the use of a proteomic approach allowed to compensate for the poor information available on the determinants of Cu tolerance.