Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan IT-20126, Italy.
Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg SE-405 30, Sweden.
Microbiology (Reading). 2012 Sep;158(Pt 9):2325-2335. doi: 10.1099/mic.0.058024-0. Epub 2012 Jul 12.
In living organisms, copper (Cu) contributes to essential functions but at high concentrations it may elicit toxic effects. Cu-tolerant yeast strains are of relevance for both biotechnological applications and studying physiological and molecular mechanisms involved in stress resistance. One way to obtain tolerant strains is to exploit experimental methods that rely on the principles of natural evolution (evolutionary engineering) and allow for the development of complex phenotypic traits. However, in most cases, the molecular and physiological basis of the phenotypic changes produced have not yet been unravelled. We investigated the determinants of Cu resistance in a Saccharomyces cerevisiae strain that was evolved to tolerate up to 2.5 g CuSO(4) l(-1) in the culture medium. We found that the content of intracellular Cu and the expression levels of several genes encoding proteins involved in Cu metabolism and oxidative stress response were similar in the Cu-tolerant (evolved) and the Cu-sensitive (non-evolved) strain. The major difference detected in the two strains was the copy number of the gene CUP1, which encodes a metallothionein. In evolved cells, a sevenfold amplification of CUP1 was observed, accounting for its strongly and steadily increased expression. Our results implicate CUP1 in protection of the evolved S. cerevisiae cells against Cu toxicity. In these cells, robustness towards Cu is stably inheritable and can be reproducibly selected by controlling environmental conditions. This finding corroborates the effectiveness of laboratory evolution of whole cells as a tool to develop microbial strains for biotechnological applications.
在生物体内，铜（Cu）对一些基本功能至关重要，但在高浓度时，它可能会产生毒性作用。耐铜酵母菌株在生物技术应用和研究与应激抗性相关的生理和分子机制方面都具有重要意义。获得耐受菌株的一种方法是利用基于自然进化原理的实验方法（进化工程），从而允许复杂表型特征的发展。然而，在大多数情况下，产生的表型变化的分子和生理基础尚未被揭示。我们研究了在一种酿酒酵母菌株中铜抗性的决定因素，该菌株经过进化可以耐受培养基中高达 2.5 g CuSO(4) l(-1)的 Cu。我们发现，耐铜（进化）和铜敏感（非进化）菌株的细胞内 Cu 含量和编码与 Cu 代谢和氧化应激反应相关的几种蛋白的基因表达水平相似。在两种菌株中检测到的主要差异是编码金属硫蛋白的基因 CUP1 的拷贝数。在进化细胞中，CUP1 被扩增了七倍，导致其表达水平强烈而稳定地增加。我们的结果表明 CUP1 参与了保护进化后的酿酒酵母细胞免受 Cu 毒性的作用。在这些细胞中，对 Cu 的稳健性是稳定可遗传的，可以通过控制环境条件来重复选择。这一发现证实了整个细胞的实验室进化作为开发生物技术应用微生物菌株的工具的有效性。
