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

doi:10.1016/j.jbiotec.2009.06.024

Journal of Biotechnology

Volume 143, Issue 2, 20 August 2009, Pages 130-138

https://doi.org/10.1016/j.jbiotec.2009.06.024 Get rights and content

Abstract

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 mmol l⁻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 mmol l⁻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.

Introduction

Cobalt is an important magnetic element that has a widespread use in several industrial applications such as the production and the refining of alloys, jet engines, gas turbines, electrochemical materials and permanent magnets (Stadler and Schweyen, 2002). Additionally, cobalt is used in varnishes, paints, catalysts, inks, pigments, ceramics, and surgical implants (Beyersmann and Hartwig, 1992, Kazantzis, 1981). Biologically, cobalt is used as a cofactor of vitamin B₁₂ and other enzymes in yeast, animals, bacteria, archaea and plants (Kobayashi and Shimizu, 1999). Cobalt can be toxic for living systems when present at high concentrations, but the exact mechanism of this toxicity is still poorly understood. Transcriptomic and toxicogenomic studies have been carried out in Escherichia coli and human lung cells to identify potential signature to cobalt exposure. While in bacteria, excess cobalt causes inactivation of some Fe–S proteins and activates iron uptake as a potential compensatory mechanism (Ranquet et al., 2007), in mammalian cells, acute exposure to cobalt induces upregulation of cobalt carriers and stress-responsive genes (Malard et al., 2007). Some yeast studies have shown that cobalt ions are transported into the vacuole and/or mitochondria by a cobalt transporter encoded by COT1, and overexpression of COT1 confers increased tolerance to cobalt and to rhodium (Conklin et al., 1992). Another gene, COT2, was also found to be implicated in cobalt resistance as well as to other divalent cations including Zn²⁺, Mn²⁺; and Ni²⁺. It was found that COT2 is identical to GRR1 encoding a F-box protein component of the SCF ubiquitin-ligase complex, also implicated in glucose-catabolite repression and expression of high-affinity glucose transport, morphogenesis, and sulfite detoxification (Flick and Johnston, 1991, Vallier et al., 1994).

To further investigate the mechanism of cobalt resistance, and hence to unravel potential targets of cobalt in yeast, we sought to apply our recent selection procedure for multi-stress resistant phenotype of Saccharomyces cerevisiae (Çakar et al., 2005). While classical selection is based on screening in plates of chemically or UV-mutagenized cells for a given phenotype, e.g. growth on certain levels of toxic metals, evolutionary engineering exploits evolutionary principles to enhance microbial properties in a biotechnological context, provided that the desired phenotype is amenable to direct or indirect selection. This methodology is expected to gain relevance both as a complementary strategy to elucidate the molecular basis of desired phenotypes, as well as in metabolic engineering strain performance (reviewed in Sauer, 2001). Many examples of evolutionary engineering have been reported in the literature, such as isolation of ethanol and acetate stress resistant strains (Çakar et al., 2005, Aarnio et al., 1991, Brown and Oliver, 1982), improved strains for xylose utilization (Sonderegger and Sauer, 2003, Sonderegger et al., 2004) and increased pyruvate production (van Maris et al., 2004).

In this report, we have assessed the potential of four different evolutionary engineering strategies to isolate S. cerevisiae cells resistant to high-toxic-levels of cobalt. Cobalt resistant yeast populations were obtained upon pulse and continuous selection at gradually increasing cobalt stress levels. The degree of resistance of mutants to high cobalt concentration as monitored by survival rate was significantly different for continuous and pulse selection strategies. Overall, selection under continuous exposure to gradually increasing cobalt stress yielded the most resistant mutants to cobalt stress. Furthermore, for a given selection procedure, the resistant population expressed high heterogeneity in cobalt-resistance when assessed at the levels of single cells.

Section snippets

Strain, media and growth conditions

The S. cerevisiae wild-type strain CEN.PK 113-7D (MATa, MAL2-8^c, SUC2) was used as the initial wild-type strain in this work (P. Kötter, Johann Wolfgang Goethe-University, Frankfurt, Germany) (van Dijken et al., 2000). Unless otherwise stated, yeast cultivations were performed in yeast minimal medium (YMM), containing 6.7 g l⁻¹ yeast nitrogen base without amino acids (Difco) and 2.0% (w/v) glucose as the sole carbon source, in test tubes under aerobic conditions at 30 °C and 200 rpm. Cell growth

Determination of cobalt concentration that causes 50% of growth reduction

Before starting the evolutionary engineering experiments, a yeast culture of CEN.PK 113-7D strain was EMS-mutagenized in conditions resulting in 10% survival following the procedure described previously (Lawrence, 1991). The minimum inhibitory concentration (MIC) for cobalt was then measured for both wild-type and the mutagenized strain, termed S101. The method was to cultivate the two types of cell population in the presence of CoCl₂ varying from 0 to 8 mmol l⁻¹ and measure final OD₆₀₀ after 72 h

Conclusions

In this study, four different strategies of in vivo evolutionary engineering have been employed to generate cobalt-resistant yeast cells. The two main conclusions that could be made from this evolutionary engineering approach were, on the one hand, that each strategy led to significant differences in resistance to cobalt among the final evolved yeast populations, and on the other hand, the cobalt-resistance of single cells within the evolved population was highly heterogeneous, suggesting that

Acknowledgements

We thank Aslı Baysal, Ülkü Yılmaz, Hüseyin Tayran, Çeşminaz Karabulut and Esma Nur Özkan for technical assistance. This work was supported in part by the Turkish State Planning Organization DPT (Advanced Technologies and ITU-ARINANOTEK 2008K120710), TUBITAK (105T314, to Z.P. Çakar), TUBITAK-EGIDE (PIA-BOSPHORUS 107T284, to Z.P. Çakar and J.M. François) and by NSF-MRSEC and ARO-DURINT projects at the University of Washington, Seattle (M. Sarikaya and C. Tamerler).

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¹: These authors contributed equally to this paper.

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

Abstract

Introduction

Section snippets

Strain, media and growth conditions

Determination of cobalt concentration that causes 50% of growth reduction

Conclusions

Acknowledgements

Toxicol. Appl. Pharmacol.

FEMS Microbiol. Rev.

FEMS Yeast Res.

FEMS Yeast Res.

J. Biol. Chem.

J. Biol. Chem.

Enzyme Microb. Technol.

Isolation of acetic acid-tolerant baker's yeast variants in a turbidostat

Appl. Biochem. Biotechnol.

Isolation of ethanol-tolerant mutants of yeast by continuous selection

Eur. J. Appl. Microbiol. Biotechnol.

COT1, a gene involved in cobalt accumulation in Saccharomyces cerevisiae

Mol. Cell Biol.