Toxicity of Copper Oxide (CuO) Nanoparticles on Human Blood Lymphocytes

CuO nanoparticles (CuO-NPs) serve several important functions in human life, particularly in the fields of medicine, engineering, and technology. These nanoparticles have been utilized as catalysts, semiconductors, sensors, gaseous and solid ceramic pigments, and magnet rotatable devices. Further use for CuO-NPs has been employed in the pharmaceutical industry especially in the production of anti-microbial fabric treatments or prevention of infections caused by Escherichia coli and methicillin-resistant Staphylococcus aureus. Two key potential routes of exposure to CuO-NPs exist through inhalation and skin exposure. Toxicity of these nanoparticles has been reported in various studies; however, no study as of yet has investigated the complete cellular mechanisms involved in CuO-NPs toxicity on human cells. The aim of this study was to determine the cytotoxicity of CuO-NPs on human blood lymphocytes. Blood lymphocytes were obtained from healthy male subjects through the use of Ficoll polysaccharide subsequently by gradient centrifugation. The following parameters were assayed in blood lymphocytes after a 6-h incubation with different concentrations of CuO-NPs: cell viability, reactive oxygen species (ROS) formation, lipid peroxidation, cellular glutathione levels, and mitochondrial and lysosomal damage. Our results demonstrate that CuO-NPs, in particular, decreased cell viability in a concentration-dependent manner and the IC50 determined was 382 μM. CuO-NP cytotoxicity was associated with significant increase at intracellular ROS level and loss of mitochondrial membrane potential and lysosomal membrane leakiness. Hence, CuO-NPs are shown to effectively induce oxidative stress in addition to inflict damage on mitochondria and lysosomes in human blood lymphocytes.     

Uptake of Intact Copper Oxide Nanoparticles Causes Acute Toxicity in Cultured Glial Cells

Copper oxide nanoparticles (CuO-NPs) dispersions are known for their high cell toxic potential but contaminating copper ions in such dispersions are a major hurdle in the investigation of specific nanoparticle-mediated toxicity. In order to distinguish between the adverse effects exhibited by CuO-NPs and/or by contaminating ionic copper, the membrane-impermeable copper chelator bathocuproine disulfonate (BCS) was added in a low molar ratio (20% of the total copper applied) in order to chelate the copper ions that had been released extracellularly from the CuO-NPs before or during the incubation. Physicochemical characterization of synthesized CuO-NPs revealed that the presence of this low concentration of BCS did not alter the size or zeta potential of the CuO-NPs. Application of CuO-NPs to C6 glioma cells and primary astrocytes induced a concentration- and temperature-dependent copper accumulation which was accompanied by a severe loss in cell viability. The adverse consequences of the CuO-NP application were not affected by the presence of 20% BCS, while the copper accumulation and cell toxicity observed after application of ionic copper were significantly lowered in the presence of BCS. These results demonstrate that for the experimental conditions applied the adverse consequences of an exposure of cultured glial cells to dispersions of CuO-NPs are mediated by accumulated NPs and not caused by the uptake of contaminating copper ions.

     

Isolation of copper oxide (CuO) nanoparticles resistant Pseudomonas strains from soil and investigation on possible mechanism for resistance

The present study deals with isolation and characterization of copper oxide nanoparticles resistant Pseudomonas strains that were isolated from the soil collected from mining and refining sites of Sarcheshmeh copper mine in the Kerman Province of Iran. The three isolates were selected based on high level of copper oxide nanoparticles (CuO NPs) resistance. The isolates were authentically identified as Pseudomonas fluorescens CuO-1, Pseudomonas fluorescens CuO-2 and Pseudomonas sp. CuO-3 by morphological, biochemical and 16S rDNA gene sequencing analysis. The growth pattern of these isolates with all the studied CuO NPs concentrations was similar to that of control (without CuO NPs) indicating that CuO NPs would not affect the growth of isolated strains. A reduction in the amount of exopolysaccharides was observed after CuO NPs—P. fluorescens CuO-1 culture supernatant interaction. The Fourier transform infrared spectroscopy (FT-IR) peaks for the exopolysaccharides extracted from the bacterial culture supernatant and the interacted CuO NPs were almost similar. The exopolysaccharide capping of the CuO NPs was confirmed by FT-IR and X-ray diffraction analysis. The study of bacterial exopolysaccharides capped CuO NPs with E. coli PTCC 1338 and S. aureus PTCC 1113 showed less toxicity compared to uncoated CuO NPs. Our study suggests that the capping of nanoparticles by bacterially produced exopolysaccharides serve as the probable mechanism of tolerance.     

Resistance to cellobiose lipids and specific features of lipid composition in yeast

The significance of the fatty acid composition and ergosterol content in cells for resistance to cellobiose lipids has been investigated in the cells of mutant Saccharomyces cerevisiae strains that are unable to produce ergosterol or sphingomyelin and in the cells of microorganisms that produce cellobiose lipids. S. cerevisiae mutants were shown to be less sensitive to cellobiose lipids from Cryptococcus humicola than the wild-type strain, and the strains that produced cellobiose lipids were virtually insensitive to this compound as well. The sensitivity of Pseudozyma fusiformata yeast to its own cellobiose lipids was reduced under conditions that favored the production of these compounds. No correlation between the content of ergosterol and sensitivity to cellobiose lipids was observed in S. cerevisiae or in the strains that produced cellobiose lipids. The ratio between the levels of saturated and unsaturated fatty acids in the cells of the mutant strains was correlated to the sensitivity of the cells to cellobiose lipids.     

CARDIOLIPIN CONTENT OF WILD TYPE AND MUTANT YEASTS IN RELATION TO MITOCHONDRIAL FUNCTION AND DEVELOPMENT

The phospholipid composition of various strains of the yeast, Saccharomyces cerevisiae, and several of their derived mitochondrial mutants grown under conditions designed to induce variations in the complement of mitochondrial membranes has been examined. Wild type and petite (cytoplasmic respiratory deficient) yeasts were fractionated into various subcellular fractions, which were monitored by electron microscopy and analyzed for cytochrome oxidase (in wild type) and phospholipid composition. 90% or more of the phospholipid, cardiolipin was found in the mitochondrial membranes of wild type and petite yeast. Cardiolipin content differed markedly under various growth conditions. Stationary yeast grown in glucose had better developed mitochondria and more cardiolipin than repressed log phase yeast. Aerobic yeast contained more cardiolipin than anaerobic yeast. Respiration-deficient cytoplasmic mitochondrial mutants, both suppressive and neutral, contained less cardiolipin than corresponding wild types. A chromosomal mutant lacking respiratory function had normal cardiolipin content. Log phase cells grown in galactose and lactate, which do not readily repress the development of mitochondrial membranes, contained as much cardiolipin as stationary phase cells grown in glucose. Cytoplasmic mitochondrial mutants respond to changes in the glucose concentration of the growth medium by variations in their cardiolipin content in the same way as wild type yeast does under similar growth conditions. It is concluded that cardiolipin content of yeast is correlated with, and is a good indicator of, the state of development of mitochondrial membrane.     

Uptake and Toxicity of Copper Oxide Nanoparticles in C6 Glioma Cells

Copper oxide nanoparticles (CuO-NPs) are frequently used for many technical applications, but are also known for their cell toxic potential. In order to investigate a potential use of CuO-NPs as a therapeutic drug for glioma treatment, we have investigated the consequences of an application of CuO-NPs on the cellular copper content and cell viability of C6 glioma cells. CuO-NPs were synthesized by a wet-chemical method and were coated with dimercaptosuccinic acid and bovine serum albumin to improve colloidal stability in physiological media. Application of these protein-coated nanoparticles (pCuO-NPs) to C6 cells caused a strong time-, concentration- and temperature-dependent copper accumulation and severe cell death. The observed loss in cellular MTT-reduction capacity, the loss in cellular LDH activity and the increase in the number of propidium iodide-positive cells correlated well with the specific cellular copper content. C6 glioma cells were less vulnerable to pCuO-NPs compared to primary astrocytes and toxicity of pCuO-NPs to C6 cells was only observed for incubation conditions that increased specific cellular copper contents above 20 nmol copper per mg protein. Both cellular copper accumulation as well as the pCuO-NP-induced toxicity in C6 cells were prevented by application of copper chelators, but not by endocytosis inhibitors, suggesting that liberation of copper ions from the pCuO-NPs is the first step leading to the observed toxicity of pCuO-NP-treated glioma cells.     

Isolation of Suppressive Sensitive Mutants from Killer and Neutral Strains of SACCHAROMYCES CEREVISIAE

Dominant sensitive mutants were isolated from a killer and a neutral strain of Saccharomyces cerevisiae by treatment with nitrosomethylurethane. Genetic studies suggest that these sensitives arose by mutation of the wild-type cytoplasmic genetic determinants (k) or (n) to a mutant form (s). (s) determinants lack wild-type (k) and (n) activity but are retained in the cell and suppress the replication or activity of the wild-type determinants, converting killer and neutral cells to the sensitive phenotype. These mutants show an obvious similarity in behavior to suppressive petite mutants of yeast.     

The effect of altered sterol composition on cytochrome oxidase and S-adenosylmethionine: delta 24 sterol methyltransferase enzymes of yeast mitochondria

Arrhenius kinetics of two mitochondrial enzymes, cytochrome oxidase and S-adenosylmethionine: Δ 24 sterol methyltransferase were analyzed in wild-type and sterol mutant strains of yeast. Temperature effects on the enzymes isolated from the ergosterol producing wild-type and nystatin resistant mutants (major sterol Δ⁸(9), 22 ergostadiene-3-β-ol) were compared. Transition temperatures were lower in both mutant strains compared to wild-type. Lipid analysis shows a relationship between sterol content and the temperature dependent transition phases.     

Effect of hydrostatic pressure on a mutant of Saccharomyces cerevisiae deleted in the trehalose-6-phosphate synthase gene

Mutants Saccharomyces cerevisiae deleted on the trehalose-6-phosphate synthase gene (tps1) and their parental wild-type cells were submitted to hydrostatic pressure in the range of 0–200 MPa. Experimental evidence showed that viability for both strains decreased with increasing pressure and that tps1 mutants, unable to accumulate trehalose, were more sensitive to hydrostatic pressure than the wild-type cells. Additionally, both tps1 and wild-type cells in the stationary phase, when there is an accumulation of endogenous trehalose, were more resistant to pressure than proliferating cells. Under these conditions, mutant cells were also more sensitive to pressure treatment than the wild type. The present work also showed that mild pressure pretreatment did not induce hydrostatic pressure resistance (barotolerance) in yeast cells.     

Analysis of proteomic changes in colored mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin as its most prevalent xanthophyll derivative. Comparisons between the protein profiles of mutant lines of this yeast can provide insight into the carotenogenic pathway. Differently colored mutants (red, orange, pink, yellow, and white) were obtained from this yeast species, and their protein profiles were determined using two-dimensional polyacrylamide gel electrophoresis (2DE). Individual proteins differentially expressed were identified using mass spectrometry. The red mutants hyperproduced total carotenoids (mainly astaxanthin), while in white and orange mutants, mutagenesis affected the phytoene dehydrogenase activity as indicated by the accumulation of phytoene. Inactivation of astaxanthin synthase after the mutagenic treatment was evident in β-carotene accumulating mutants. Differences in the proteomic profiles of wild-type X. dendrorhous and its colored mutants were demonstrated using 2DE. Of the total number of spots detected in each gel (297–417), 128 proteins were present in all strains. The red mutant showed the greatest number of matches with respect to the wild type (305 spots), while the white and yellow mutants, which had reduced concentrations of total carotenoids, presented the highest correlation coefficient (0.6) between each other. A number of differentially expressed proteins were sequenced, indicating that tricarboxylic acid cycle and stress response proteins are closely related to the carotenogenic process.     

RBE of densely ionizing radiation for wild-type and radiosensitive mutants of yeast

Survival curves were obtained for haploid and diploid yeasts, Saccharmyces cerevisiae, of wild-type strains and radiosensitive mutants exposed to γ-rays and α-particles. A correlation between the values of the relative biological effectiveness (RBE) of high-LET radiation and cell-repair capacity was found. The difference in radiosensitivities of the wild-type diploid strain and homozygous rad mutants incapable of recovery was significantly higher after low-LET radiation than after high-LET radiation. Possible reasons for the observed radiation responses to low- and high-LET exposure of yeast cells with various genotype are discussed.     

Anaerobic respiration of superoxide dismutase-deficientSaccharomyces cerevisiae under oxidative stress

The ethanol productivity of superoxide dismutase (SOD)-deficient mutants ofSaccharomyces cerevisiae was examined under the oxidative stress by Paraquat. It was observed that MnSOD-deficient mutant ofS. cerevisiae had higher ethanol productivity than wild type or CuZnSOD-deficient yeast both in aerobic and in anaerobic culture condition. Pyruvate dehydrogenase activity decreased by 35% and alcohol dehydrogenase activity increased by 32% were observed in MnSOD-deficient yeast grown aerobically. When generating oxygen radicals by Paraquat, the ethanol productivity was increased by 40% in CuZnSOD-deficient or wild strain, resulting from increased activity of alcohol dehydrogenase and decreased activity of pyruvate dehydrogenase. However, the addition of ascorbic acid with Paraquat returned the enzyme activities at the level of control. These results imply that SOD-deficiency in yeast strains may cause the metabolic flux to shift into anaerobic ethanol fermentation in order to avoid their oxidative damages by Paraquat.     

Biochemical Studies of Paraquat-Tolerant Mutants of the Fern Ceratopteris richardii

Enzymes and metabolites associated with mitigation of paraquat toxicity were compared in two paraquat-tolerant mutants and a sensitive wild-type strain of the fern Ceratopteris richardii Brongn. In 21-day-old gametophytes, the specific activities of superoxide dismutase, catalase, peroxidase, glutathione reductase, dehydroascorbate reductase, and ascorbate peroxidase showed no differences that would explain mutant tolerance. Constitutive levels of ascorbate and glutathione also did not differ significantly in the three strains. An experiment testing the inducibility of paraquat tolerance revealed no change in the dose response of mutant or wild type gametophytes after exposure to sublethal concentrations of the herbicide. Uptake of paraquat by whole gametophytes was also equivalent in mutants and wild type. These data suggest that the physiological basis for tolerance in these mutants, unlike several other tolerant biotypes reported, does not lie in the oxygen radical scavenging system, in an inducible stress response, or in a block to whole-plant uptake.     

Mitochondrial control of sugar utilization in Saccharomyces cerevisiae.

When a number of wild-type strains of Saccharomyces cerevisiae—all capable of utilizing the three sugars galactose, maltose, and α-methyl-d-glucoside for growth—were converted by ethidium bromide (EtdBr) mutagenesis to stable cytoplasmic petite (rho^?) mutants, the latter lost the ability to grow on one or more of these sugars. The actual pattern of retention (or loss) or sugar utilization by these mutants depended on the wild-type strain, but was independent of the length of exposure to EtdBr during mutagenesis. This treatment varied from 0.5 to 24 h, by which time the majority of the mutants must have been of the mitochondrial (mt) DNA-deficient rho⁰ type. Furthermore, with one exception—involving the ability of one set of mutants to utilize α-methyl-glucoside—all rho^? mutants derived from the same wild type exhibited the same, discrete pattern of sugar utilization. Respiration-deficient mutants with defined lesions in their mtDNA (mit^? mutants) exhibited the same pattern of sugar utilization as did the petite mutants of the same strain. Diploid petite strains also exhibited discrete, but less stringent, patterns of sugar utilization. For any one genotype this pattern was identical whether the mutant was generated by crossing two haploid rho^? strains, themselves derived by EtdBr mutagenesis, or by EtdBr mutagenesis of the diploid obtained from a haploid wild-type × wild-type cross. In such mutant diploids the sugar-positive phenotype was usually dominant, but there were indications in some instances of modulation of this effect by virtue of nuclear gene interactions. Various respiration-deficient mutants incapable of utilizing α-methylglucoside also were unable to form α-glucosidase, but were able to do so after being rendered permeable by exposure to dimethyl sulfoxide. Arguments are advanced that respiring mitochondria generate an entity—probably not directly related to ATP production—required for the expression of nuclear genes or their products, some of which may be necessary for plasma membrane function.     

Dominant spore color mutants of Aspergillus nidulans defective in germination and sexual development.

The ascomycete Aspergillus nidulans produces green conidia (asexual spores). Recessive mutants which produce yellow conidia have been previously isolated from haploid strains and have been shown to be deficient in laccase (diphenol oxidase), an enzyme that requires copper for activity. Using a diploid parent strain, we isolated dominant yellow conidial mutants which, in the haploid state, produced even less laccase activity than a recessive mutant. Three isolates of such mutants behaved similarly and define a single complementation group (yB) on chromosome VIII distinct from the yA locus on chromosome I defined by recessive mutants. Unlike yA mutants, whose only discernable phenotype is their conidial color, yB mutants are pleiotropic: conidial germination was delayed relative to the wild type, and sexual development was blocked at an early stage. The three phenotypes of yB mutants were expressed on yeast extract-glucose medium containing 1.6 microM of added copper. When copper was added to above 5 microM, all three phenotypes were remediated, and near wild-type levels of laccase were produced. We conclude that yB mutants have a reduced availability of copper. The dominance of yB mutants could result, for example, from an alteration in transport or storage of copper. Using an immunological assay, we detected no laccase antigenic cross-reacting material in yB mutants grown on medium of low copper content. We conclude that either the synthesis or the stability of laccase is copper dependent.     

Pathways of ultraviolet mutability in Saccharomyces Cerevisiae. IV. The relation between canavanine toxicity and ultraviolet mutability to canavanine resistance.

Seven umr mutants of Saccharomyces cerevisiae which had reduced capacity for ultraviolet light (UV)-induced forward mutation from CAN1 to can1 were tested for sensitivity to L-canavanine relative to one wild-type UMR strain and one slightly UV-sensitive but phenotypically umr⁺ strain (mutant 306). Relative UV mutation resistance was estimated by dividing the UV fluence needed to yeild a particular induced mutation frequency by that needed to reach the same frequency in the genotypic wild-type strain. The umr5 and umr6 strains were especially sensitive to canavanine growth inhibition, while umr1 was no more sensitive than either wild type; umr2, umr3, umr4, a umr7, and α umr7 were equally sensitive to an intermediate degree. Incubation at 30°C of wildtype cells plated on canavanine-selective agar for increasingly longer times before UV irradiation resulted in decreasing UV mutation frequencies (reduced to 50% in 1.6 h). All umr strains tested in this way lost UV mutability faster than wild type, including mutant 306, umr1 (not sensitive to growth inhibition), and umr6 (very sensitive to growth inhibition). Cells were grown to stationary phase in YEDP growth medium and assayed for arginine and tryptophan transport into the cell. The umr6 strain, which had weak UV mutation resistance but high sensitivity to canavanine growth inhibition, transported arginine and tryptophan at essentially wild-type levels. The umr1 strain, however, which had moderate UV mutation resistance and normal canavanine toxicity, transported both amino acids at rates tenfold higher than wild type. The data suggest that increased canavanine toxicity does not necessarily lead to defective mutability at CAN1, and that mutational deficiency cannot result solely from increased canavanine toxicity. Although exposure to canavanine was shown to block mutation fixation and/or expression, it is suggested that the degree of growth inhibition is not strictly correlated with the degree of mutation resistance.     

Deletion of the dynein heavy-chain gene DYN1 leads to aberrant nuclear positioning and defective hyphal development in Candida albicans

Cytoplasmic dynein is a microtubule-associated minus-end-directed motor protein. CaDYN1 encodes the single dynein heavy-chain gene of Candida albicans. The open reading frames of both alleles of CaDYN1 were completely deleted via a PCR-based approach. Cadyn1 mutants are viable but grow more slowly than the wild type. In vivo time-lapse microscopy was used to compare growth of wild-type (SC5314) and dyn1 mutant strains during yeast growth and after hyphal induction. During yeast-like growth, Cadyn1 strains formed chains of cells. Chromosomal TUB1-GFP and HHF1-GFP alleles were used both in wild-type and mutant strains to monitor the orientation of mitotic spindles and nuclear positioning in C. albicans. In vivo fluorescence time-lapse analyses with HHF1-GFP over several generations indicated defects in dyn1 cells in the realignment of spindles with the mother-daughter axis of yeast cells compared to that of the wild type. Mitosis in the dyn1 mutant, in contrast to that of wild-type yeast cells, was very frequently completed in the mother cells. Nevertheless, daughter nuclei were faithfully transported into the daughter cells, resulting in only a small number of multinucleate cells. Cadyn1 mutant strains responded to hypha-inducing media containing l-proline or serum with initial germ tube formation. Elongation of the hyphal tubes eventually came to a halt, and these tubes showed a defect in the tipward localization of nuclei. Using a heterozygous DYN1/dyn1 strain in which the remaining copy was controlled by the regulatable MAL2 promoter, we could switch between wild-type and mutant phenotypes depending on the carbon source, indicating that the observed mutant phenotypes were solely due to deletion of DYN1.     

Desensitization of Feedback Inhibition of the Saccharomyces cerevisiae γ-Glutamyl Kinase Enhances Proline Accumulation and Freezing Tolerance

In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. The yeast Saccharomyces cerevisiae induces trehalose or glycerol synthesis but does not increase intracellular proline levels during various stresses. Using a proline-accumulating mutant, we previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. This mutant was recently shown to carry an allele of PRO1 which encodes the Asp154Asn mutant γ-glutamyl kinase (GK), the first enzyme of the proline biosynthetic pathway. Here, enzymatic analysis of recombinant proteins revealed that the GK activity of S. cerevisiae is subject to feedback inhibition by proline. The Asp154Asn mutant was less sensitive to feedback inhibition than wild-type GK, leading to proline accumulation. To improve the enzymatic properties of GK, PCR random mutagenesis in PRO1 was employed. The mutagenized plasmid library was introduced into an S. cerevisiae non-proline-utilizing strain, and proline-overproducing mutants were selected on minimal medium containing the toxic proline analogue azetidine-2-carboxylic acid. We successfully isolated several mutant GKs that, due to extreme desensitization to inhibition, enhanced the ability to synthesize proline better than the Asp154Asn mutant. The amino acid changes were localized at the region between positions 142 and 154, probably on the molecular surface, suggesting that this region is involved in allosteric regulation. Furthermore, we found that yeast cells expressing Ile150Thr and Asn142Asp/Ile166Val mutant GKs were more tolerant to freezing stress than cells expressing the Asp154Asn mutant.     

AP endonuclease deficiency results in extreme sensitivity to thymidine deprivation

Thymidine depletion is toxic to virtually all actively growing cells. The fundamental mechanism responsible for thymidineless death remains unknown. One event thought to be critical in causing the toxicity of thymidine depletion is a sharp rise in the ratio of dUTP to dTTP and subsequent incorporation of dUTP into DNA. Maneuvers to alter dUTP levels appear to alter the toxicity of thymidine depletion. However, loss of uracil-DNA-N-glycosylase activity does not appear to change the toxicity of thymidine deprivation significantly. This study proposes to define the role of uracil base excision repair (BER) in mediating thymidineless death. The toxicity of thymidine deprivation induced by the antifolate aminopterin was measured in a series of mutant Saccharomyces cerevisiae strains deficient in various steps in uracil-BER. Most mutants displayed modest changes in their sensitivity to aminopterin, with the exception of cells lacking the abasic endonuclease Apn1. apn1 mutants displayed a profound sensitivity to aminopterin that was relieved in an apn1 ung1 double mutant. Wild-type and apn1 mutants displayed similar levels of DNA damage and S-phase arrest during aminopterin treatment. A significant portion of cell killing occurred after removal of aminopterin in both wild-type and apn1 mutant cells. apn1 mutants showed a complete inability to re-initiate DNA replication following removal of aminopterin. These findings suggest recovery from arrest is a crucial step in determining the response to thymidine deprivation and that interruptions in uracil-BER increase the toxicity of thymidine deprivation by blocking re-initiation of replication rather than inciting global DNA damage. Inhibition of apurinic/apyrimidinic endonuclease may therefore be a reasonable approach to increase the efficacy of anticancer chemotherapies based on thymidine depletion.     

Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Strain by Using RNA-Guided Cas9 Nuclease

Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries.