Evolutionary engineering and molecular characterization of a caffeine-resistant Saccharomyces cerevisiae strain

Caffeine is a naturally occurring alkaloid, where its major consumption occurs with beverages such as coffee, soft drinks and tea. Despite a variety of reports on the effects of caffeine on diverse organisms including yeast, the complex molecular basis of caffeine resistance and response has yet to be understood. In this study, a caffeine-hyperresistant and genetically stable Saccharomyces cerevisiae mutant was obtained for the first time by evolutionary engineering, using batch selection in the presence of gradually increased caffeine stress levels and without any mutagenesis of the initial population prior to selection. The selected mutant could resist up to 50 mM caffeine, a level, to our knowledge, that has not been reported for S. cerevisiae so far. The mutant was also resistant to the cell wall-damaging agent lyticase, and it showed cross-resistance against various compounds such as rapamycin, antimycin, coniferyl aldehyde and cycloheximide. Comparative transcriptomic analysis results revealed that the genes involved in the energy conservation and production pathways, and pleiotropic drug resistance were overexpressed. Whole genome re-sequencing identified single nucleotide polymorphisms in only three genes of the caffeine-hyperresistant mutant; PDR1, PDR5 and RIM8, which may play a potential role in caffeine-hyperresistance. Graphic abstrac

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

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

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

International audienceOxidative stress is a major stress type observed in yeast bioprocesses, resulting in a decrease in yeast growth, viability, and productivity. Thus, robust yeast strains with increased resistance to oxidative stress are in highly demand by the industry. In addition, oxidative stress is also associated with aging and age-related complex conditions such as cancer and neurodegenerative diseases. Saccharomyces cerevisiae , as a model eukaryote, has been used to study these complex eukaryotic processes. However, the molecular mechanisms underlying oxidative stress responses and resistance are unclear. In this study, we have employed evolutionary engineering (also known as adaptive laboratory evolution – ALE) strategies to obtain an oxidative stress-resistant and genetically stable S. cerevisiae strain. Comparative physiological, transcriptomic, and genomic analyses of the evolved strain were then performed with respect to the reference strain. The results show that the oxidative stress-resistant evolved strain was also cross-resistant against other types of stressors, including heat, freeze-thaw, ethanol, cobalt, iron, and salt. It was also found to have higher levels of trehalose and glycogen production. Further, comparative transcriptomic analysis showed an upregulation of many genes associated with the stress response, transport, carbohydrate, lipid and cofactor metabolic processes, protein phosphorylation, cell wall organization, and biogenesis. Genes that were downregulated included those related to ribosome and RNA processing, nuclear transport, tRNA, and cell cycle. Whole genome re-sequencing analysis of the evolved strain identified mutations in genes related to the stress response, cell wall organization, carbohydrate metabolism/transport, which are in line with the physiological and transcriptomic results, and may give insight toward the complex molecular mechanisms of oxidative stress resistance

Genomic, transcriptomic, and metabolic characterization of 2-Phenylethanol-resistant Saccharomyces cerevisiae obtained by evolutionary engineering

International audience2-Phenylethanol is an aromatic compound commonly used in the food, cosmetic, and pharmaceutical industries. Due to increasing demand for natural products by consumers, the production of this flavor by microbial fermentation is gaining interest, as a sustainable alternative to chemical synthesis or expensive plant extraction, both processes relying on the use of fossil resources. However, the drawback of the fermentation process is the high toxicity of 2-phenylethanol to the producing microorganism. The aim of this study was to obtain a 2-phenylethanol-resistant Saccharomyces cerevisiae strain by in vivo evolutionary engineering and characterize the adapted yeast at the genomic, transcriptomic and metabolic levels. For this purpose, the tolerance to 2-phenylethanol was developed by gradually increasing the concentration of this flavor compound through successive batch cultivations, leading to an adapted strain that could tolerate 3.4 g/L of 2-phenylethanol, which was about 3-times better than the reference strain. Genome sequencing of the adapted strain identified point mutations in several genes, notably in HOG1 that encodes the Mitogen-Activated Kinase of the high-osmolarity signaling pathway. As this mutation is localized in the phosphorylation lip of this protein, it likely resulted in a hyperactive protein kinase. Transcriptomic analysis of the adapted strain supported this suggestion by revealing a large set of upregulated stress-responsive genes that could be explained in great part by HOG1 -dependent activation of the Msn2/Msn4 transcription factor. Another relevant mutation was found in PDE2 encoding the low affinity cAMP phosphodiesterase, the missense mutation of which may lead to hyperactivation of this enzyme and thereby enhance the stressful state of the 2-phenylethanol adapted strain. In addition, the mutation in CRH1 that encodes a chitin transglycosylase implicated in cell wall remodeling could account for the increased resistance of the adapted strain to the cell wall-degrading enzyme lyticase. Finally, the potent upregulation of ALD3 and ALD4 encoding NAD + -dependent aldehyde dehydrogenase together with the observed phenylacetate resistance of the evolved strain suggest a resistance mechanism involving conversion of 2-phenylethanol into phenylacetaldehyde and phenylacetate implicating these dehydrogenases

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

International audienceSilver is a non-essential metal used in medical applications as an antimicrobial agent, but it is also toxic for biological systems. To investigate the molecular basis of silver resistance in yeast, we employed evolutionary engineering using successive batch cultures at gradually increased silver stress levels up to 0.25-mM AgNO(3)in 29 populations and obtained highly silver-resistant and genetically stableSaccharomyces cerevisiaestrains. Cross-resistance analysis results indicated that the silver-resistant mutants also gained resistance against copper and oxidative stress. Growth physiological analysis results revealed that the highly silver-resistant evolved strain 2E was not significantly inhibited by silver stress, unlike the reference strain. Genomic and transcriptomic analysis results revealed that there were mutations and/or significant changes in the expression levels of the genes involved in cell wall integrity, cellular respiration, oxidative metabolism, copper homeostasis, endocytosis and vesicular transport activities. Particularly the missense mutation in theRLM1gene encoding a transcription factor involved in the maintenance of cell wall integrity and with 707 potential gene targets might have a key role in the high silver resistance of 2E, along with its improved cell wall integrity, as confirmed by the lyticase sensitivity assay results. In conclusion, the comparative physiological, transcriptomic and genomic analysis results of the silver-resistantS. cerevisiaestrain revealed potential key factors that will help understand the complex molecular mechanisms of silver resistance in yeast