Evolutionary Engineering of an Iron-Resistant Saccharomyces cerevisiae Mutant and Its Physiological and Molecular Characterization

Abstract

Iron plays an essential role in all organisms and is involved in the structure of many biomolecules. It also regulates the Fenton reaction where highly reactive hydroxyl radicals occur. Iron is also important for microbial biodiversity, health and nutrition. Excessive iron levels can cause oxidative damage in cells. Saccharomyces cerevisiae evolved mechanisms to regulate its iron levels. To study the iron stress resistance in S. cerevisiae, evolutionary engineering was employed. The evolved iron stress-resistant mutant “M8FE” was analysed physiologically, transcriptomically and by whole genome re-sequencing. M8FE showed cross-resistance to other transition metals: cobalt, chromium and nickel and seemed to cope with the iron stress by both avoidance and sequestration strategies. PHO84, encoding the high-affinity phosphate transporter, was the most down-regulated gene in the mutant, and may be crucial in iron-resistance. M8FE had upregulated many oxidative stress response, reserve carbohydrate metabolism and mitophagy genes, while ribosome biogenesis genes were downregulated. As a possible result of the induced oxidative stress response genes, lower intracellular oxidation levels were observed. M8FE also had high trehalose and glycerol production levels. Genome re-sequencing analyses revealed several mutations associated with diverse cellular and metabolic processes, like cell division, phosphate-mediated signalling, cell wall integrity and multidrug transporters.

Genomic, transcriptomic and physiological analyses of silver‐resistant Saccharomyces cerevisiae obtained by evolutionary engineering

“…The comparative transcriptomic and whole‐genome resequencing results of the silver‐resistant strain 2E revealed that several genes associated with cell wall biogenesis and integrity were either differentially expressed or mutated in 2E, compared with the reference strain (Tables 1 and 2). To test if there were any differences in cell wall integrity between 2E and the reference strain, we performed the lyticase sensitivity assay (Balaban et al, 2020; Kuranda et al, 2006) to both strains, in the presence and absence of silver (200‐μM AgNO 3 ) stress, using the cell wall β‐1,3‐glucan‐degrading enzyme lyticase. Our results revealed that the evolved strain 2E was significantly more resistant to lyticase than the reference strain (Figure 9), implying that the silver‐resistant evolved strain had a higher cell wall integrity or robustness than the reference strain.…”

“…Lyticase sensitivity assay was adapted from Kuranda, Leberre, Sokol, Palamarczyk, and François (2006) and performed exactly as described previously (Balaban et al, 2020), using the reference and evolved strain cultures grown under both control and 200-μM AgNO 3 stress conditions; 40-mM 2-mercaptoethanol (Merck, Hohenbrunn, Germany) and 2-U/mL lyticase (Sigma-Aldrich, St. Louis, MO, USA) were used during the experimental procedure. Cell lysis was monitored by absorbance (OD 600 ) measurements every 20 min.…”

“…Evolutionary engineering, also known as adaptive laboratory evolution, is a powerful inverse metabolic engineering strategy to improve genetically complex, microbial phenotypes (Cakar, Turanli‐Yildiz, Alkim, & Yilmaz, 2012). Using evolutionary engineering, we have previously obtained multiple‐stress resistant (Çakar, Seker, Tamerler, Sonderegger, & Sauer, 2005), cobalt‐resistant (Alkım, Benbadis, Yılmaz, Çakar, & Francois, 2013; Çakar et al, 2009), nickel‐resistant (Küçükgöze et al, 2013), ethanol‐tolerant (Turanli‐Yildiz et al, 2017), coniferyl aldehyde‐resistant (Hacısalihoğlu, Holyavkin, Topaloğlu, Kısakesen, & Çakar, 2019), caffeine‐resistant (Sürmeli et al, 2019) and iron‐resistant (Balaban et al, 2020) S. cerevisiae . Apart from stress resistance, we have also increased chronological life span of S. cerevisiae using an evolutionary engineering strategy (Arslan et al, 2018).…”

A Review on the Resistance and Accumulation of Heavy Metals by Different Microbial Strains

“…The fungal strains useful in iron resistance are Aspergillus niger and Aspergillus foetidus and some Penicillium species too. Fungal strains have good ability for bio leaching process by interfering functional groups of enzymes [105] (Table 12).…”

Physiological and Molecular Characterization of an Oxidative Stress-Resistant Saccharomyces cerevisiae Strain Obtained by Evolutionary Engineering

“…Evolutionary selection principles have been used for the improvement of biotechnologically important characteristics, including novel catabolic activities, improved enzyme properties, plasmid functions, and stress resistance ( Sauer, 2001 ; Çakar et al, 2005 , 2012 ). S. cerevisiae strains that are resistant against diverse stress types have been successfully obtained by evolutionary engineering, including nickel-resistant ( Küçükgöze et al, 2013 ), cobalt-resistant ( Çakar et al, 2009 ; Alkim et al, 2013 ), iron-resistant ( Balaban et al, 2020 ), coniferyl aldehyde-resistant ( Hacısalihoğlu et al, 2019 ), caffeine-resistant ( Surmeli et al, 2019 ), ethanol-tolerant ( Turanlı-Yıldız et al, 2017 ), chronologically long-lived ( Arslan et al, 2018a , b ), and silver-resistant ( Terzioğlu et al, 2020 ) S. cerevisiae .…”