RESUMO
Many microorganisms are capable of riboflavin oversynthesis and accumulation in a medium, suggesting that they efficiently excrete riboflavin. The mechanisms of riboflavin efflux in microorganisms remain elusive. Candida famata are representatives of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2) in response to iron limitation. The riboflavin overproducers of this yeast species have been obtained by classical mutagenesis and metabolic engineering. Overproduced riboflavin accumulates in the cultural medium rather than in the cells suggesting existence of the special mechanisms involved in riboflavin excretion. The appropriate protein and gene have not been identified in yeasts till recently. At the same time, the gene BCRP (breast cancer resistance protein) has been identified in mammal mammary glands. Several homologs of the mammal BCRP gene encoding putative riboflavin efflux protein (excretase) were identified in the flavinogenic yeasts Debaryomyces hansenii and C. famata. Here we evaluate the yeast homologs of BCRP with respect to improvement of a riboflavin production by C. famata. The closest homologs from D. hansenii or C. famata were expressed under the control of TEF1 promoter of these yeasts in the wild-type and riboflavin-overproducing strains of C. famata. Resulted transformants overexpressed the corresponding genes (designated as DhRFE and CfRFE) and produced 1.4- to 6-fold more riboflavin as compared to the corresponding parental strains. They also were characterized by overexpression of RIB1 and RIB6 genes which encode the first and the last structural enzymes of riboflavin synthesis and exhibited elevated specific activity of GTP cyclohydrolase II. Thus, overexpression of yeast homolog of mammal gene BCRP may be useful to increase the riboflavin yield in a riboflavin production process using a recombinant overproducing C. famata strain or other flavinogenic microorganisms.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Candida/crescimento & desenvolvimento , Engenharia Metabólica/métodos , Riboflavina/biossíntese , Candida/genética , Candida/metabolismo , Clonagem Molecular , Meios de Cultura/química , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Fator 1 de Elongação de Peptídeos/genética , Regiões Promotoras Genéticas , Regulação para CimaRESUMO
Candida famata is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2 ) in response to iron limitation. Overproduced riboflavin accumulates in the cultural medium rather than in the cells suggesting existence of the special mechanisms involved in riboflavin excretion. The corresponding protein and gene have not been identified in yeasts. At the same time, the corresponding gene BCRP has been identified in mammal mammary glands. Several homologs of the mammal BCRP gene encoding putative riboflavin efflux protein (excretase) were identified in Debaryomyces hansenii. The closest homolog was expressed under the control of D. hansenii TEF1 promoter in the riboflavin overproducing strain of C. famata. Resulted transformants overexpressed the corresponding gene and produced 1.4- to 1.8-fold more riboflavin as compared with the parental strain. They also were characterized by overexpression of RIB1 and RIB6 genes of riboflavin synthesis and exhibited elevated specific activity of GTP-cyclohydrolase II. Membrane localization of the riboflavin excretase was confirmed by fluorescent microscopy.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Candida/genética , Proteínas Fúngicas/genética , Mamíferos/genética , Riboflavina/metabolismo , Animais , Candida/classificação , Clonagem Molecular , DNA Fúngico/genética , Riboflavina/biossínteseRESUMO
A set of 185 strains of Candida albicans from patients with vulvovaginal candidiasis (VVC) and from non-VVC clinical sources in southwest China was analysed. Strains were subjected to genotyping using CAI microsatellite typing and amplification of an intron-containing region of the 25S rRNA gene. Microsatellite genotypes of strains from non-VVC sources showed high polymorphism, whereas those of VVC were dominated by few, closely similar genotypes. However, among non-VVC strains, two genotypes were particularly prevalent in patients with lung cancer. 25S rDNA genotype A was dominant in VVC sources (86.7%), whereas genotypes A, B, and C were rather evenly distributed among non-VVC sources; known genotypes D and E were not found. In an experimental mouse model, isolates from lung cancer and AIDS patients proved to have higher virulence than VVC strains. Among 156 mice infected with C. albicans, 19 developed non-invasive urothelial carcinoma. No correlation could be established between parameters of virulence, source of infection, and incidence of carcinoma. C. albicans strains from VVC were less susceptible to itraconazole than the strains from non-VVC sources, whereas there was small difference in antifungal susceptibility between different 25S rDNA genotypes of C. albicans tested against amphotericin B, itraconazole, fluconazole, and flucytosine.
Assuntos
Candida albicans/patogenicidade , Genótipo , Repetições de Microssatélites , Polimorfismo Genético , Síndrome da Imunodeficiência Adquirida/microbiologia , Animais , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candidíase/microbiologia , Candidíase Vulvovaginal/microbiologia , DNA Fúngico/genética , Feminino , Humanos , Itraconazol/farmacologia , Itraconazol/uso terapêutico , Neoplasias Pulmonares/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Técnicas de Tipagem Micológica , Neoplasias/microbiologia , Reação em Cadeia da Polimerase , RNA Ribossômico/genética , VirulênciaRESUMO
Tumor cells often exhibit specific metabolic defects due to the aberrations in oncogene-dependent regulatory and/or signaling pathways that distinguish them from normal cells. Among others, many malignant cells are deficient in biosynthesis of certain amino acids and concomitantly exhibit elevated sensitivity to deprivation of these amino acids. Although the underlying causes of such metabolic changes are still not fully understood, this feature of malignant cells is exploited in metabolic enzymotherapies based on single amino acid, e.g., arginine, deprivation. To achieve efficient arginine depletion in vivo, two recombinant enzymes, bacterial arginine deiminase and human arginase I have been evaluated and are undergoing further development. This review is aimed to summarize the current knowledge on the application of arginine-degrading enzymes as anticancer agents and as bioanalytical tools for arginine assays. The problems that have to be solved to optimize this therapy for clinical application are discussed.
Assuntos
Arginase/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Arginase/genética , Arginase/uso terapêutico , Arginina/metabolismo , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Hidrolases/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêuticoRESUMO
BACKGROUND: The methylotrophic yeast, Hansenula polymorpha is an industrially important microorganism, and belongs to the best studied yeast species with well-developed tools for molecular research. The complete genome sequence of the strain NCYC495 of H. polymorpha is publicly available. Some of the well-studied strains of H. polymorpha are known to ferment glucose, cellobiose and xylose to ethanol at elevated temperature (45 - 50°C) with ethanol yield from xylose significantly lower than that from glucose and cellobiose. Increased yield of ethanol from xylose was demonstrated following directed metabolic changes but, still the final ethanol concentration achieved is well below what is considered feasible for economic recovery by distillation. RESULTS: In this work, we describe the construction of strains of H. polymorpha with increased ethanol production from xylose using an ethanol-non-utilizing strain (2EthOH-) as the host. The transformants derived from 2EthOH- overexpressing modified xylose reductase (XYL1m) and native xylitol dehydrogenase (XYL2) were isolated. These transformants produced 1.5-fold more ethanol from xylose than the original host strain. The additional overexpression of XYL3 gene coding for xylulokinase, resulted in further 2.3-fold improvement in ethanol production with no measurable xylitol formed during xylose fermentation. The best ethanol producing strain obtained by metabolic engineering approaches was subjected to selection for resistance to the known inhibitor of glycolysis, the anticancer drug 3-bromopyruvate. The best mutant selected had an ethanol yield of 0.3 g/g xylose and produced up to 9.8 g of ethanol/l during xylose alcoholic fermentation at 45°C without correction for ethanol evaporation. CONCLUSIONS: Our results indicate that xylose conversion to ethanol at elevated temperature can be significantly improved in H. polymorpha by combining methods of metabolic engineering and classical selection.
Assuntos
Adaptação Fisiológica , Etanol/metabolismo , Fermentação , Engenharia Metabólica/métodos , Metano/metabolismo , Pichia/metabolismo , Temperatura , Xilose/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Aldeído Redutase/metabolismo , Antineoplásicos/farmacologia , D-Xilulose Redutase/metabolismo , Fermentação/efeitos dos fármacos , Pichia/efeitos dos fármacos , Pichia/enzimologia , Pichia/isolamento & purificação , Plasmídeos/metabolismo , Piruvatos/farmacologia , Transformação Genética/efeitos dos fármacos , Xilitol/metabolismoRESUMO
BACKGROUND: The production of ethyl alcohol by fermentation represents the largest scale application of Saccharomyces cerevisiae in industrial biotechnology. Increased worldwide demand for fuel bioethanol is anticipated over the next decade and will exceed 200 billion liters from further expansions. Our working hypothesis was that the drop in ATP level in S. cerevisiae cells during alcoholic fermentation should lead to an increase in ethanol production (yield and productivity) with a greater amount of the utilized glucose converted to ethanol. Our approach to achieve this goal is to decrease the intracellular ATP level via increasing the unspecific alkaline phosphatase activity. RESULTS: Intact and truncated versions of the S. cerevisiae PHO8 gene coding for vacuolar or cytosolic forms of alkaline phosphatase were fused with the alcohol dehydrogenase gene (ADH1) promoter. The constructed expression cassettes used for transformation vectors also contained the dominant selective marker kanMX4 and S. cerevisiae δ-sequence to facilitate multicopy integration to the genome. Laboratory and industrial ethanol producing strains BY4742 and AS400 overexpressing vacuolar form of alkaline phosphatase were characterized by a slightly lowered intracellular ATP level and biomass accumulation and by an increase in ethanol productivity (13% and 7%) when compared to the parental strains. The strains expressing truncated cytosolic form of alkaline phosphatase showed a prolonged lag-phase, reduced biomass accumulation and a strong defect in ethanol production. CONCLUSION: Overexpression of vacuolar alkaline phosphatase leads to an increased ethanol yield in S. cerevisiae.
Assuntos
Trifosfato de Adenosina/metabolismo , Fosfatase Alcalina/metabolismo , Etanol/metabolismo , Glucose/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Álcool Desidrogenase/genética , Fosfatase Alcalina/genética , Biomassa , Mutagênese Insercional , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
In yeast and filamentous fungi, sulfide can be condensed either with O-acetylhomoserine to generate homocysteine, the precursor of methionine, or with O-acetylserine to directly generate cysteine. The resulting homocysteine and cysteine can be interconverted through transsulfuration pathway. Here, we systematically analyzed the sulfur metabolic pathway of the thermotolerant methylotrophic yeast Hansenula polymorpha, which has attracted much attention as an industrial yeast strain for various biotechnological applications. Quite interestingly, the detailed sulfur metabolic pathway of H. polymorpha, which was reconstructed based on combined analyses of the genome sequences and validation by systematic gene deletion experiments, revealed the absence of de novo synthesis of homocysteine from inorganic sulfur in this yeast. Thus, the direct biosynthesis of cysteine from sulfide is the only pathway of synthesizing sulfur amino acids from inorganic sulfur in H. polymorpha, despite the presence of both directions of transsulfuration pathway Moreover, only cysteine, but no other sulfur amino acid, was able to repress the expression of a subset of sulfur genes, suggesting its central and exclusive role in the control of H. polymorpha sulfur metabolism. 35S-Cys was more efficiently incorporated into intracellular sulfur compounds such as glutathione than 35S-Met in H. polymorpha, further supporting the cysteine-centered sulfur pathway. This is the first report on the novel features of H. polymorpha sulfur metabolic pathway, which are noticeably distinct from those of other yeast and filamentous fungal species.
Assuntos
Cisteína/metabolismo , Pichia/metabolismo , Enxofre/metabolismo , Sequência de Aminoácidos , Aminoácidos Sulfúricos/metabolismo , Carbono/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Glutationa/biossíntese , Redes e Vias Metabólicas , Dados de Sequência Molecular , Mutação , Pichia/genética , Reprodutibilidade dos Testes , Alinhamento de Sequência , Serina/análogos & derivados , Serina/metabolismoRESUMO
Arginine deiminase is a promising anticancer drug active against melanoma, hepatocarcinoma and other tumors. Recombinant strains of Escherichia coli that express arginine deiminase from pathogenic bacteria Mycoplasma have been developed. However, production costs of heterologous arginine deiminase are high due to use of an expensive inducer and extraction buffer, as well as using diluted culture for enzyme induction. We report on a new advanced protocol for Mycoplasma hominis arginine deiminase expression, extraction and renaturation. The main improvements include manipulation with dense suspensions of E. coli, use of lactose instead of isopropyl ß-D-1-thiogalactopyranoside as an inducer and a cheaper but not less efficient buffer for solubilization of arginine deiminase inclusion bodies. In addition, supplementation of the storage culture medium with glucose and substrate (arginine) significantly stabilized the recombinant arginine deiminase producer. Homogenous preparations of recombinant arginine deiminase were obtained using anion-exchange and hydrophobic chromatography. The purified enzyme retained a specific activity of 30-34 U/mg for 12 months when stored at 4°C in 20 mM sodium phosphate buffer pH 7.2 containing 1 M NaCl.
Assuntos
Clonagem Molecular , Escherichia coli/enzimologia , Hidrolases/isolamento & purificação , Hidrolases/metabolismo , Mycoplasma hominis/enzimologia , Antineoplásicos/farmacologia , Biotecnologia , Meios de Cultura , Escherichia coli/genética , Hidrolases/economia , Hidrolases/genética , Corpos de Inclusão , Lactose/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The ability of baker's yeast Saccharomyces cerevisiae and of the thermotolerant methylotrophic yeast Hansenula polymorpha to produce ethanol during alcoholic fermentation of glucose was compared between wild-type strains and recombinant strains possessing an elevated level of intracellular glutathione (GSH) due to overexpression of the first gene of GSH biosynthesis, gamma-glutamylcysteine synthetase, or of the central regulatory gene of sulfur metabolism, MET4. The analyzed strains of H. polymorpha with an elevated pool of intracellular GSH were found to accumulate almost twice as much ethanol as the wild-type strain during glucose fermentation, in contrast to GSH1-overexpressing S. cerevisiae strains, which also possessed an elevated pool of GSH. The ethanol tolerance of the GSH-overproducing strains was also determined. For this, the wild-type strain and transformants with an elevated GSH pool were compared for their viability upon exposure to exogenous ethanol. Unexpectedly, both S. cerevisiae and H. polymorpha transformants with a high GSH pool proved more sensitive to exogenous ethanol than the corresponding wild-type strains.
Assuntos
Etanol/metabolismo , Fermentação , Glutationa/biossíntese , Pichia/metabolismo , Saccharomyces cerevisiae/metabolismo , Glucose/metabolismo , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Pichia/genética , Saccharomyces cerevisiae/genética , Xilose/metabolismoRESUMO
Candida guilliermondii is an opportunistic emerging fungal agent of candidiasis often associated with oncology patients. This yeast also remains an interesting biotechnological model for the industrial production of value-added metabolites. The recent whole-genome sequencing of the C. guilliermondii ATCC 6260 reference strain provides an interesting resource for elucidating new molecular events supporting pathogenicity, antifungal resistance and for exploring the potential of yeast metabolic engineering. In the present study, we designed an efficient transformation system for C. guilliermondii wild-type strains using both nourseothricin- and hygromycin B-resistant markers. To demonstrate the potential of these drug-resistant cassettes, we carried out the disruption and the complementation of the C. guilliermondii FCY1 gene (which encodes cytosine deaminase) known to be associated with flucytosine sensitivity in yeast. These two new dominant selectable markers represent powerful tools to study the function of a large pallet of genes in this yeast of clinical and biotechnological interest.
Assuntos
Candida/genética , Genética Microbiana/métodos , Mutagênese Insercional/métodos , Seleção Genética , Transformação Genética , Antifúngicos/farmacologia , Farmacorresistência Fúngica , Higromicina B/farmacologia , Estreptotricinas/farmacologiaRESUMO
BACKGROUND: Tripeptide glutathione (gamma-glutamyl-L-cysteinyl-glycine) is the most abundant non-protein thiol that protects cells from metabolic and oxidative stresses and is widely used as medicine, food additives and in cosmetic industry. The methylotrophic yeast Hansenula polymorpha is regarded as a rich source of glutathione due to the role of this thiol in detoxifications of key intermediates of methanol metabolism. Cellular and extracellular glutathione production of H. polymorpha DL-1 in the wild type and recombinant strains which overexpress genes of glutathione biosynthesis (GSH2) and its precursor cysteine (MET4) was studied. RESULTS: Glutathione producing capacity of H. polymorpha DL-1 depending on parameters of cultivation (dissolved oxygen tension, pH, stirrer speed), carbon substrate (glucose, methanol) and type of overexpressed genes of glutathione and its precursor biosynthesis during batch and fed-batch fermentations were studied. Under optimized conditions of glucose fed-batch cultivation, the glutathione productivity of the engineered strains was increased from ~900 up to ~ 2300 mg of Total Intracellular Glutathione (TIG) or GSH+GSSGin, per liter of culture medium. Meantime, methanol fed-batch cultivation of one of the recombinant strains allowed achieving the extracellular glutathione productivity up to 250 mg of Total Extracellular Glutathione (TEG) or GSH+GSSGex, per liter of the culture medium. CONCLUSIONS: H. polymorpha is an competitive glutathione producer as compared to other known yeast and bacteria strains (Saccharomyces cerevisiae, Candida utilis, Escherichia coli, Lactococcus lactis etc.) with good perspectives for further improvement especially for production of extracellular form of glutathione.
Assuntos
Engenharia Genética/métodos , Glutationa/biossíntese , Pichia/metabolismo , Aerobiose , Biomassa , Southern Blotting , Meios de Cultura , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Concentração de Íons de Hidrogênio , Metanol , Pichia/enzimologia , Pichia/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Single amino acid Arg (arginine) deprivation is currently considered as a therapeutic approach to treat certain types of tumours; the molecular mechanisms that underlie tumour cell sensitivity or resistance to Arg restriction are still little understood. Here, we address the question of whether endogenous levels of key Arg metabolic enzymes [catabolic: arginases, ARG1 (arginase type 1) and ARG2 (arginase type 2), and anabolic: OTC (ornithine transcarbamylase) and ASS (argininosuccinate synthetase)] affect cellular responses to arginine deprivation in vitro. Human epithelial cancer cells of different organs of origin exhibiting variable sensitivity to Arg deprivation provided the experimental models. Neither the basal expression status of the analysed enzymes, nor their changes upon arginine withdrawal correlated with cancer cell sensitivity to arginine deprivation. However, the ability to utilize exogenous Arg precursors (ornithine and citrulline) for growth in Arg-deficient medium strongly correlated with expression of the corresponding enzymes, OTC and ASS. We also observed that OTC expression was below the level of detection in all the types of tumour cells analysed, suggesting that in vitro, at least for them, Arg is an essential amino acid.
Assuntos
Arginase/genética , Arginina/deficiência , Neoplasias/enzimologia , Arginase/metabolismo , Arginina/metabolismo , Argininossuccinato Sintase/genética , Argininossuccinato Sintase/metabolismo , Citrulina/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Neoplasias/metabolismo , Ornitina/metabolismo , Ornitina Carbamoiltransferase/genética , Ornitina Carbamoiltransferase/metabolismo , Células Tumorais CultivadasRESUMO
Pichia guilliermondii is a representative of yeast species that overproduce riboflavin (vitamin B2) in response to iron deprivation. P. guilliermondii YFH1 gene coding for frataxin homologue, eukaryotic mitochondrial protein involved in iron trafficking and storage, was identified and deleted. Constructed P. guilliermondii Δyfh1 mutant grew very poorly in a sucrose-containing synthetic medium supplemented with sulfate or sulfite as a sole sulfur source. Addition of sodium sulfide, glutathione, cysteine, methionine, N-acetyl-L-cysteine partially restored growth rate of the mutant suggesting that it is impaired in sulfate assimilation. Cellular iron content in Δyfh1 mutant was ~3-3.5 times higher as compared to the parental strain. It produced 50-70 times more riboflavin in iron sufficient synthetic media relative to the parental wildtype strain. Biomass yield of the mutant in the synthetic glutathione containing medium supplemented with glycerol as a sole carbon source was 1.4- and 2.6-fold increased as compared to sucrose and succinate containing media, respectively. Oxygen uptake of the Δyfh1 mutant on sucrose, glycerol or succinate, when compared to the parental strain, was decreased 5.5-, 1.7- and 1.5-fold, respectively. Substitution of sucrose or glycerol in the synthetic iron sufficient medium with succinate completely abolished riboflavin overproduction by the mutants. Deletion of the YFH1 gene caused hypersensitivity to hydrogen peroxide and exogenously added riboflavin and led to alterations in superoxide dismutase activities. Thus, deletion of the gene coding for yeast frataxin homologue has pleiotropic effect on metabolism in P. guilliermondii.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Ferro/metabolismo , Organismos Geneticamente Modificados/metabolismo , Pichia/metabolismo , Riboflavina/farmacologia , Proteínas Fúngicas/genética , Deleção de Genes , Glicerol/metabolismo , Peróxido de Hidrogênio/farmacologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/genética , Proteínas de Ligação ao Ferro/genética , Mitocôndrias/metabolismo , Organismos Geneticamente Modificados/genética , Pichia/genética , Riboflavina/biossíntese , Riboflavina/genética , Ácido Succínico/metabolismo , Sacarose/metabolismo , Compostos de Enxofre/metabolismo , Ésteres do Ácido Sulfúrico/metabolismo , Superóxido Dismutase/análise , FrataxinaRESUMO
In yeast cell, glucose induces various changes of cellular metabolism on genetic and metabolic levels. One of such changes is autophagic degradation of dispensable peroxisomes (pexophagy) which occurs in vacuoles. We have found that in Saccharomyces cerevisiae, defect of G-protein-coupled receptor Gpr1 and G-protein Gpa2, both the components of cAMP-signaling pathway, strongly suppressed glucose-induced degradation of matrix peroxisomal protein thiolase. We conclude that proteins Gpr1 and Gpa2 are involved in glucose sensing and signal transduction during pexophagy process in yeast.
Assuntos
AMP Cíclico/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Glucose/farmacologia , Peroxissomos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/efeitos dos fármacos , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Mutação , Peroxissomos/efeitos dos fármacos , Receptores Acoplados a Proteínas G/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. However, the mechanisms of such regulation are not known. We found that mutations causing riboflavin overproduction and iron hyperaccumulation (rib80, rib81 and hit1), as well as cobalt excess or iron deficiency all provoke oxidative stress in the Pichia guilliermondii yeast. Iron content in the cells, production both of riboflavin and malondialdehyde by P. guilliermondii wild type and hit1 mutant strains depend on a type of carbon source used in cultivation media. The data suggest that the regulation of riboflavin biosynthesis and iron assimilation in P. guilliermondii are linked with cellular oxidative state.
Assuntos
Ferro/metabolismo , Mutação , Estresse Oxidativo , Pichia/metabolismo , Riboflavina/biossíntese , Cobalto/metabolismo , Regulação Fúngica da Expressão Gênica , Malondialdeído/metabolismo , Microscopia Eletrônica de Transmissão , Pichia/genética , Pichia/ultraestruturaRESUMO
Intracellular cadmium (Cd(2+)) ion accumulation and the ability to produce specific Cd(2+) ion chelators was studied in the methylotrophic yeast Hansenula polymorpha. Only one type of Cd(2+) intracellular chelators, glutathione (GSH), was identified, which suggests that sequestration of this heavy metal in H. polymorpha occurs similarly to that found in Saccharomyces cerevisiae, but different to Schizosaccharomys pombe and Candida glabrata which both synthesize phytochelatins. Cd(2+) ion uptake in the H. polymorpha wild-type strains appeared to be an energy dependent process. It was found that Deltagsh2 mutants, impaired in the first step of GSH biosynthesis, are characterized by increase in net Cd(2+) ion uptake by the cells, whereas Deltagsh1/Deltamet1 and Deltaggt1 mutants impaired in sulfate assimilation and GSH catabolism, respectively, lost the ability to accumulate Cd(2+) intracellularly. Apparently H. polymorpha, similarly to S. cerevisiae, forms a Cd-GSH complex in the cytoplasm, which in turn regulates Cd(2+) uptake. Genes GSH1/MET1 and GGT1 are involved in maturation and metabolism of cellular Cd-GSH complex, respectively. Transport of [(3)H]N-ethylmaleimide-S-glutathione ([(3)H]NEM-SG) conjugate into crude membrane vesicules, purified from the wild-type cells of H. polymorpha appeared to be MgATP dependent, uncoupler insensitive and vanadate sensitive. We suggest that MgATP dependent transporter involved in Cd-GSH uptake in H. polymorpha, is similar to S. cerevisiae Ycf1-mediated vacuolar transporter responsible for accumulation of organic GS-conjugates and Cd-GSH complex.
Assuntos
Cádmio/metabolismo , Quelantes/metabolismo , Glutationa/metabolismo , Pichia/metabolismo , Trifosfato de Adenosina/metabolismo , Glutationa/análogos & derivados , Maleimidas/metabolismo , Pichia/efeitos dos fármacos , Pichia/genética , Azida Sódica/farmacologiaRESUMO
GGT1 gene of the methylotrophic yeast Hansenula polymorpha appears to be a structural and functional homologue of Saccharomyces cerevisiae CIS2/ECM38 gene encoding gamma-glutamyltranspeptidase (gammaGT). This is confirmed by the absence of the corresponding activity of gammaGT in the mutant with disrupted GGT1 gene. It was shown that gammaGT of both H. polymorpha and S. cerevisiae are involved in detoxification of electrophilic xenobiotics, as the corresponding mutants appeared to be defective in the disappearance of the fluorescent vacuolar complex of GSH with xenobiotic bimane and the further diffuse distribution of this complex in the cytosol. We hypothesize that metabolism of electrophilic xenobiotics in the yeasts H. polymorpha and S. cerevisiae occurs through a gammaGT-dependent mercapturic acid pathway of GSH-xenobiotic detoxification, similar to that known for mammalian cells, with cysteine-xenobiotics and/or N-acetylcysteine-xenobiotics as the end products.
Assuntos
Proteínas Fúngicas/metabolismo , Pichia/metabolismo , Saccharomyces cerevisiae/metabolismo , Xenobióticos/metabolismo , gama-Glutamiltransferase/metabolismo , Sequência de Aminoácidos , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Proteínas Fúngicas/genética , Glutationa/metabolismo , Dados de Sequência Molecular , Pichia/genética , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , gama-Glutamiltransferase/genéticaRESUMO
It is known for many years that iron represses synthesis of riboflavin (RF) and most of RF-synthesizing enzymes in several yeast species, known as flavinogenic yeasts. However, the mechanism of such repression is not known. We have found that iron represses transcription of RIB1 and RIB7 genes coding for the first and the last enzymes of RF biosynthesis in the model flavinogenic organism Pichia guilliermondii. To decipher molecular mechanisms of iron-dependent repression, isolation and study of the regulatory mutants defective in corresponding regulation is desirable. However, no suitable methods for isolation of such mutants were previously available. We have produced a single-point transition mutation in the RIB1 gene. The corresponding rib1-86 mutant exhibits leaky phenotype and is unable to grow in iron-sufficient minimal medium without exogenous RF. However, it can grow in minimal iron-deficient medium without RF, or in iron-sufficient medium upon introduction of the previously-isolated regulatory mutation rib81, which leads to increase in RF production. Using the rib1-86 mutant as parental strain, a collection of mutants able to grow in iron-sufficient medium without exogenous RF has been isolated. The mutants appeared to be defective in regulation of RF biosynthesis and iron homeostasis and were divided into six new complementation groups. Study of one corresponding mutant, red6, showed derepression of RIB1 mRNA synthesis in iron-sufficient medium.