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1.
Proc Natl Acad Sci U S A ; 119(30): e2108245119, 2022 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-35858410

RESUMEN

Heme is an oxygen carrier and a cofactor of both industrial enzymes and food additives. The intracellular level of free heme is low, which limits the synthesis of heme proteins. Therefore, increasing heme synthesis allows an increased production of heme proteins. Using the genome-scale metabolic model (GEM) Yeast8 for the yeast Saccharomyces cerevisiae, we identified fluxes potentially important to heme synthesis. With this model, in silico simulations highlighted 84 gene targets for balancing biomass and increasing heme production. Of those identified, 76 genes were individually deleted or overexpressed in experiments. Empirically, 40 genes individually increased heme production (up to threefold). Heme was increased by modifying target genes, which not only included the genes involved in heme biosynthesis, but also those involved in glycolysis, pyruvate, Fe-S clusters, glycine, and succinyl-coenzyme A (CoA) metabolism. Next, we developed an algorithmic method for predicting an optimal combination of these genes by using the enzyme-constrained extension of the Yeast8 model, ecYeast8. The computationally identified combination for enhanced heme production was evaluated using the heme ligand-binding biosensor (Heme-LBB). The positive targets were combined using CRISPR-Cas9 in the yeast strain (IMX581-HEM15-HEM14-HEM3-Δshm1-HEM2-Δhmx1-FET4-Δgcv2-HEM1-Δgcv1-HEM13), which produces 70-fold-higher levels of intracellular heme.


Asunto(s)
Hemo , Ingeniería Metabólica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Simulación por Computador , Hemo/biosíntesis , Hemo/genética , Hemoproteínas/biosíntesis , Hemoproteínas/genética , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
Metab Eng ; 72: 311-324, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35508267

RESUMEN

High-level production of recombinant proteins in industrial microorganisms is often limited by the formation of misfolded proteins or protein aggregates, which consequently induce cellular stress responses. We hypothesized that in a yeast Alzheimer's disease (AD) model overexpression of amyloid-ß peptides (Aß42), one of the main peptides relevant for AD pathologies, induces similar phenotypes of cellular stress. Using this humanized AD model, we previously identified suppressors of Aß42 cytotoxicity. Here we hypothesize that these suppressors could be used as metabolic engineering targets to alleviate cellular stress and improve recombinant protein production in the yeast Saccharomyces cerevisiae. Forty-six candidate genes were individually deleted and twenty were individually overexpressed. The positive targets that increased recombinant α-amylase production were further combined leading to an 18.7-fold increased recombinant protein production. These target genes are involved in multiple cellular networks including RNA processing, transcription, ER-mitochondrial complex, and protein unfolding. By using transcriptomics and proteomics analyses, combined with reverse metabolic engineering, we showed that reduced oxidative stress, increased membrane lipid biosynthesis and repressed arginine and sulfur amino acid biosynthesis are significant pathways for increased recombinant protein production. Our findings provide new insights towards developing synthetic yeast cell factories for biosynthesis of valuable proteins.


Asunto(s)
Enfermedad de Alzheimer , Proteínas de Saccharomyces cerevisiae , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Humanos , Estrés Oxidativo/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
Metab Eng ; 66: 259-267, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33984513

RESUMEN

With the increasing demand for blood transfusions, the production of human hemoglobin (Hb) from sustainable sources is increasingly studied. Microbial production is an attractive option, as it may provide a cheap, safe, and reliable source of this protein. To increase the production of human hemoglobin by the yeast Saccharomyces cerevisiae, the degradation of Hb was reduced through several approaches. The deletion of the genes HMX1 (encoding heme oxygenase), VPS10 (encoding receptor for vacuolar proteases), PEP4 (encoding vacuolar proteinase A), ROX1 (encoding heme-dependent repressor of hypoxic genes) and the overexpression of the HEM3 (encoding porphobilinogen deaminase) and the AHSP (encoding human alpha-hemoglobin-stabilizing protein) genes - these changes reduced heme and Hb degradation and improved heme and Hb production. The reduced hemoglobin degradation was validated by a bilirubin biosensor. During glucose fermentation, the engineered strains produced 18% of intracellular Hb relative to the total yeast protein, which is the highest production of human hemoglobin reported in yeast. This increased hemoglobin production was accompanied with an increased oxygen consumption rate and an increased glycerol yield, which (we speculate) is the yeast's response to rebalance its NADH levels under conditions of oxygen limitation and increased protein-production.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Sanguíneas , Fermentación , Proteínas Fúngicas , Hemo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , Chaperonas Moleculares , Peroxidasas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Methods ; 176: 82-90, 2020 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-31059831

RESUMEN

The yeast Candida glabrata, an opportunistic human fungal pathogen, is the second most prevalent cause of candidiasis worldwide, with an infection incidence that has been increasing in the past decades. The completion of the C. glabrata reference genome made fundamental contributions to the understanding of the molecular basis of its pathogenic phenotypes. However, knowledge of genome-wide genetic variations among C. glabrata strains is limited. In this study, we present a population genomic study of C. glabrata based on whole genome re-sequencing of 47 clinical strains to an average coverage of ∼63×. Abundant genetic variations were identified in these strains, including single nucleotide polymorphisms (SNPs), small insertion/deletions (indels) and copy number variations (CNVs). The observed patterns of variations revealed clear population structure of these strains. Using population genetic tests, we detected fast evolution of several genes involved in C. glabrata adherence ability, such as EPA9 and EPA10. We also located genome structural variations, including aneuploidies and large fragment CNVs, in regions that are functionally related to virulence. Subtelometric regions were hotspots of CNVs, which may contribute to variation in expression of adhesin genes that are important for virulence. We further conducted a genome-wide association study that identified two SNPs in the 5'UTR region of CST6 that were associated with fluconazole susceptibility. These observations provide convincing evidence for the highly dynamic nature of the C. glabrata genome with potential adaptive evolution to clinical environments, and offer valuable resources for investigating the mechanisms underlying drug resistance and virulence in this fungal pathogen. (249 words).


Asunto(s)
Candida glabrata/genética , Genes Fúngicos/genética , RNA-Seq/métodos , Candidiasis/tratamiento farmacológico , Candidiasis/microbiología , Variaciones en el Número de Copia de ADN , Farmacorresistencia Fúngica/genética , Evolución Molecular , Fluconazol/farmacología , Fluconazol/uso terapéutico , Variación Estructural del Genoma , Humanos , Infecciones Oportunistas/tratamiento farmacológico , Infecciones Oportunistas/microbiología , Polimorfismo de Nucleótido Simple
5.
J Ind Microbiol Biotechnol ; 46(2): 133-145, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30488364

RESUMEN

The use of thermotolerant yeast strains is an important attribute for a cost-effective high temperature biofermentation processes. However, the availability of thermotolerant yeast strains remains a major challenge. Isolation of temperature resistant strains from extreme environments or the improvements of current strains are two major strategies known to date. We hypothesised that bacteria are potential "hurdles" in the life cycle of yeasts, which could influence the evolution of extreme phenotypes, such as thermotolerance. We subjected a wild-type yeast, Lachancea thermotolerans to six species of bacteria sequentially for several generations. After coevolution, we observed that three replicate lines of yeasts grown in the presence of bacteria grew up to 37 °C whereas the controls run in parallel without bacteria could only grow poorly at 35 °C retaining the ancestral mesophilic trait. In addition to improvement of thermotolerance, our results show that the fermentative ability was also elevated, making the strains more ideal for the alcoholic fermentation process because the overall productivity and ethanol titers per unit volume of substrate consumed during the fermentation process was increased. Our unique method is attractive for the development of thermotolerant strains or to augment the available strain development approaches for high temperature industrial biofermentation.


Asunto(s)
Fermentación , Saccharomycetales/fisiología , Termotolerancia , Bacterias/crecimiento & desarrollo , Etanol , Reordenamiento Génico , Calor , Cariotipificación , Estrés Oxidativo , Saccharomycetales/aislamiento & purificación , Estrés Fisiológico
6.
Appl Microbiol Biotechnol ; 100(7): 3219-31, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26743658

RESUMEN

Dekkera bruxellensis is a non-conventional Crabtree-positive yeast with a good ethanol production capability. Compared to Saccharomyces cerevisiae, its tolerance to acidic pH and its utilization of alternative carbon sources make it a promising organism for producing biofuel. In this study, we developed an auxotrophic transformation system and an expression vector, which enabled the manipulation of D. bruxellensis, thereby improving its fermentative performance. Its gene ADH3, coding for alcohol dehydrogenase, was cloned and overexpressed under the control of the strong and constitutive promoter TEF1. Our recombinant D. bruxellensis strain displayed 1.4 and 1.7 times faster specific glucose consumption rate during aerobic and anaerobic glucose fermentations, respectively; it yielded 1.2 times and 1.5 times more ethanol than did the parental strain under aerobic and anaerobic conditions, respectively. The overexpression of ADH3 in D. bruxellensis also reduced the inhibition of fermentation by anaerobiosis, the "Custer effect". Thus, the fermentative capacity of D. bruxellensis could be further improved by metabolic engineering.


Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Dekkera/genética , Etanol/metabolismo , Proteínas Fúngicas/metabolismo , Glucosa/metabolismo , Aerobiosis , Alcohol Deshidrogenasa/genética , Anaerobiosis , Biocombustibles , Clonación Molecular , Dekkera/enzimología , Fermentación , Proteínas Fúngicas/genética , Expresión Génica , Ingeniería Genética , Plásmidos/química , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
Yeast ; 31(9): 323-32, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24932634

RESUMEN

Recently, the non-conventional yeast Dekkera bruxellensis has been gaining more and more attention in the food industry and academic research. This yeast species is a distant relative of Saccharomyces cerevisiae and is especially known for two important characteristics: on the one hand, it is considered to be one of the main spoilage organisms in the wine and bioethanol industry; on the other hand, it is 'indispensable' as a contributor to the flavour profile of Belgium lambic and gueuze beers. Additionally, it adds to the characteristic aromatic properties of some red wines. Recently this yeast has also become a model for the study of yeast evolution. In this review we focus on the recently developed molecular and genetic tools, such as complete genome sequencing and transformation, to study and manipulate this yeast. We also focus on the areas that are particularly well explored in this yeast, such as the synthesis of off-flavours, yeast detection methods, carbon metabolism and evolutionary history.


Asunto(s)
Cerveza/microbiología , Dekkera/crecimiento & desarrollo , Dekkera/metabolismo , Vino/microbiología , Cerveza/análisis , Bélgica , Dekkera/genética , Fermentación , Genética Microbiana , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Biología Molecular , Vino/análisis
8.
FEMS Yeast Res ; 14(4): 529-35, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24528571

RESUMEN

The yeast pathogen Candida glabrata is the second most frequent cause of Candida infections. However, from the phylogenetic point of view, C. glabrata is much closer to Saccharomyces cerevisiae than to Candida albicans. Apparently, this yeast has relatively recently changed its life style and become a successful opportunistic pathogen. Recently, several C. glabrata sister species, among them clinical and environmental isolates, have had their genomes characterized. Also, hundreds of C. glabrata clinical isolates have been characterized for their genomes. These isolates display enormous genomic plasticity. The number and size of chromosomes vary drastically, as well as intra- and interchromosomal segmental duplications occur frequently. The observed genome alterations could affect phenotypic properties and thus help to adapt to the highly variable and harsh habitats this yeast finds in different human patients and their tissues. Further genome sequencing of pathogenic isolates will provide a valuable tool to understand the mechanisms behind genome dynamics and help to elucidate the genes contributing to the virulence potential.


Asunto(s)
Adaptación Biológica , Candida glabrata/genética , Genoma Fúngico , Variación Estructural del Genoma , Orden Génico , Reordenamiento Génico
9.
Antonie Van Leeuwenhoek ; 104(1): 111-22, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23670790

RESUMEN

We analyzed 192 strains of the pathogenic yeast Candida glabrata from patients, mainly suffering from systemic infection, at Danish hospitals during 1985-1999. Our analysis showed that these strains were closely related but exhibited large karyotype polymorphism. Nine strains contained small chromosomes, which were smaller than 0.5 Mb. Regarding the year, patient and hospital, these C. glabrata strains had independent origin and the analyzed small chromosomes were structurally not related to each other (i.e. they contained different sets of genes). We suggest that at least two mechanisms could participate in their origin: (i) through a segmental duplication which covered the centromeric region, or (ii) by a translocation event moving a larger chromosome arm to another chromosome that leaves the centromere part with the shorter arm. The first type of small chromosomes carrying duplicated genes exhibited mitotic instability, while the second type, which contained the corresponding genes in only one copy in the genome, was mitotically stable. Apparently, in patients C. glabrata chromosomes are frequently reshuffled resulting in new genetic configurations, including appearance of small chromosomes, and some of these resulting "mutant" strains can have increased fitness in a certain patient "environment".


Asunto(s)
Candida glabrata/ultraestructura , Cromosomas Fúngicos/ultraestructura , Antifúngicos/farmacología , Secuencia de Bases , Candida glabrata/efectos de los fármacos , Candida glabrata/genética , Candida glabrata/aislamiento & purificación , Candidiasis/microbiología , Infección Hospitalaria/microbiología , ADN de Hongos/genética , ADN Ribosómico , Dinamarca , Farmacorresistencia Fúngica/genética , Evolución Molecular , Fluconazol/farmacología , Fungemia/microbiología , Duplicación de Gen , Genes Fúngicos , Inestabilidad Genómica , Haploidia , Humanos , Cariotipificación , Datos de Secuencia Molecular , Filogenia , Selección Genética , Especificidad de la Especie , Translocación Genética
10.
Methods Mol Biol ; 2477: 313-330, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35524125

RESUMEN

After its discovery RNA interference (RNAi) has become a powerful tool to study gene functions in different organisms. RNAi has been applied at genome-wide scale and can be nowadays performed using high-throughput automated systems (robotics). The simplest RNAi process requires the expression of two genes (Dicer and Argonaute) to function. To initiate the silencing, constructs generating either double-strand RNA or antisense RNA are required. Recently, RNAi was reconstituted by expressing Saccharomyces castellii genes in the human pathogenic yeast Candida glabrata and was used to identify new genes related to the virulence of this pathogen.In this chapter, we describe a method to make the C. glabrata pathogenic yeast competent for RNAi and to use RNA silencing as a tool for low- or high-resolution phenotypic screening in this species.


Asunto(s)
Proteínas Argonautas , Candida glabrata , Interferencia de ARN , Proteínas Argonautas/genética , Candida glabrata/genética , ARN Bicatenario
11.
Data Brief ; 42: 108322, 2022 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-35677454

RESUMEN

The production of recombinant proteins at high levels often induces stress-related phenotypes by protein misfolding or aggregation. These are similar to those of the yeast Alzheimer's disease (AD) model in which amyloid-ß peptides (Aß42) were accumulated [1], [2]. We have previously identified suppressors of Aß42 cytotoxicity via the genome-wide synthetic genetic array (SGA) [3] and here we use them as metabolic engineering targets to evaluate their potentiality on recombinant protein production in yeast Saccharomyces cerevisiae. In order to investigate the mechanisms linking the genetic modifications to the improved recombinant protein production, we perform systems biology approaches (transcriptomics and proteomics) on the resulting strain and intermediate strains. The RNAseq data are preprocessed by the nf-core/RNAseq pipeline and analyzed using the Platform for Integrative Analysis of Omics (PIANO) package [4]. The quantitative proteome is analyzed on an Orbitrap Fusion Lumos mass spectrometer interfaced with an Easy-nLC1200 liquid chromatography (LC) system. LC-MS data files are processed by Proteome Discoverer version 2.4 with Mascot 2.5.1 as a database search engine. The original data presented in this work can be found in the research paper titled "Suppressors of Amyloid-ß Toxicity Improve Recombinant Protein Production in yeast by Reducing Oxidative Stress and Tuning Cellular Metabolism", by Chen et al. [5].

13.
J Ind Microbiol Biotechnol ; 37(2): 213-8, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19967446

RESUMEN

Hansenula polymorpha is a naturally xylose-fermenting yeast; however, both its ethanol yield from xylose and ethanol resistance have to be improved before this organism can be used for industrial high-temperature simultaneous saccharification and fermentation of lignocellulosic materials. In the current research, we checked if the expression of the Saccharomyces cerevisiae MPR1 gene encoding N-acetyltransferase can increase the ethanol tolerance of H. polymorpha. The S. cerevisiae MPR1 gene was cloned in the H. polymorpha expression vector under the control of the H. polymorpha strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). H. polymorpha recombinant strains harboring 1-3 copies of the S. cerevisiae MPR1 gene showed enhanced tolerance to L: -azetidine-2-carboxylic acid and ethanol. The obtained results suggest that the expression of the S. cerevisiae MPR1 gene in H. polymorpha can be a useful approach in the construction of H. polymorpha strains with improved ethanol resistance.


Asunto(s)
Acetiltransferasas/genética , Ácido Azetidinocarboxílico/farmacología , Etanol/farmacología , Pichia/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Acetiltransferasas/metabolismo , Ácido Azetidinocarboxílico/metabolismo , Clonación Molecular , Farmacorresistencia Fúngica/genética , Etanol/metabolismo , Genes Fúngicos , Ingeniería Genética , Datos de Secuencia Molecular , Pichia/efectos de los fármacos , Pichia/metabolismo , Recombinación Genética , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Metab Eng ; 11(3): 163-7, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19558965

RESUMEN

Recombinant strains of the flavinogenic yeast Candida famata able to overproduce flavin mononucleotide (FMN) that contain FMN1 gene encoding riboflavin (RF) kinase driven by the strong constitutive promoter TEF1 (translation elongation factor 1alpha) were constructed. Transformation of these strains with the additional plasmid containing the FMN1 gene under the TEF1 promoter resulted in the 200-fold increase in the riboflavin kinase activity and 100-fold increase in FMN production as compared to the wild-type strain (last feature was found only in iron-deficient medium). Overexpression of the FMN1 gene in the mutant that has deregulated riboflavin biosynthesis pathway and high level of riboflavin production in iron-sufficient medium led to the 30-fold increase in the riboflavin kinase activity and 400-fold increase in FMN production of the resulted transformants. The obtained C. famata recombinant strains can be used for the further construction of improved FMN overproducers.


Asunto(s)
Candida/metabolismo , Mononucleótido de Flavina/biosíntesis , Proteínas Fúngicas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Candida/genética , Clonación Molecular , Mononucleótido de Flavina/genética , Proteínas Fúngicas/genética , Dosificación de Gen/genética , Hierro/metabolismo , Regiones Promotoras Genéticas , Riboflavina/metabolismo
15.
Biotechnol Bioeng ; 104(5): 911-9, 2009 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-19575437

RESUMEN

The methylotrophic yeast Hansenula polymorpha has the potential to be used in the process of simultaneous saccharification and fermentation (SSF) of xylan derived xylose at elevated temperatures. To improve parameters of high-temperature resistance and high-temperature fermentation of H. polymorpha, strains carrying deletion of acid trehalase gene (ATH1) and overexpressing genes coding for heat-shock proteins Hsp16p and Hsp104p were constructed. Results indicate that the corresponding recombinant strains have up to 12-fold increased tolerance to heat-shock treatment. The deletion of ATH1 gene and constitutive expression of HSP16 and HSP104 resulted in up to 5.8-fold improvement of ethanol production from xylose at 50 degrees C. Although the maximum ethanol concentration achieved from xylose was 0.9 g L(-1), our model H. polymorpha strains with elevated thermotolerance can be further modified by metabolic engineering to construct improved high-temperature ethanol producers from this pentose.


Asunto(s)
Ingeniería Genética , Calor , Pichia/fisiología , Pichia/efectos de la radiación , Estrés Fisiológico , Etanol/metabolismo , Proteínas Fúngicas/genética , Eliminación de Gen , Dosificación de Gen , Proteínas de Choque Térmico/genética , Metano/análogos & derivados , Metano/metabolismo , Pichia/genética , Trehalasa/genética , Xilosa/metabolismo
16.
Antibiotics (Basel) ; 9(1)2019 Dec 30.
Artículo en Inglés | MEDLINE | ID: mdl-31905828

RESUMEN

The opportunistic human fungal pathogen Candida albicans relies on cell morphological transitions to develop biofilm and invade the host. In the current study, we developed new regulatory molecules, which inhibit the morphological transition of C. albicans from yeast-form cells to cells forming hyphae. These compounds, benzyl α-l-fucopyranoside and benzyl ß-d-xylopyranoside, inhibit the hyphae formation and adhesion of C. albicans to a polystyrene surface, resulting in a reduced biofilm formation. The addition of cAMP to cells treated with α-l-fucopyranoside restored the yeast-hyphae switch and the biofilm level to that of the untreated control. In the ß-d-xylopyranoside treated cells, the biofilm level was only partially restored by the addition of cAMP, and these cells remained mainly as yeast-form cells.

17.
Methods Mol Biol ; 1923: 243-264, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30737744

RESUMEN

Human hemoglobin is an essential protein, whose main function as an oxygen carrier is indispensable for life. Hemoglobin is a cofactor-containing protein with heme as prosthetic group. Same as in humans, heme is synthesized in many organisms in a complex pathway involving two cellular compartments (mitochondria and cytosol), which is tightly regulated. Red blood cells (erythrocytes) are specialized and adapted for production and transport of the hemoglobin molecules. In addition to oxygen binding, hemoglobin can participate in a variety of chemical reactions by its iron and heme and may become toxic when released from erythrocytes. Hemoglobin is a major target for the development of blood substitutes/oxygen carriers, and therefore its microbial production is attractive, as it may provide a cheap and reliable source of human hemoglobin. Significant efforts have been dedicated to this task for the last three decades. Moreover since the first generation of cell-free blood substitutes based on unmodified hemoglobin failed human trials, mutant forms became of great interest.In this chapter we summarize the existing knowledge about human hemoglobin, challenges of its microbial production, and its improvement, with a particular focus upon yeast as production host.


Asunto(s)
Hemoglobinas/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Hemo/metabolismo , Humanos , Ingeniería Metabólica/métodos , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética
18.
RSC Adv ; 9(19): 10983-10989, 2019 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-35515281

RESUMEN

The vaginal microbiome of healthy women is a diverse and dynamic system of various microorganisms. Any sudden change in microbe composition can increase the vaginal pH and thus lead to vaginal infections, conditions that affect a large percentage of women each year. The most common fungal strains involved in infections belong to the yeast species Candida albicans. The main virulence factor of C. albicans is the ability to transform from planktonic yeast-form cells into a filamentous form (hyphae or pseudohyphae), with the subsequent formation of biofilm. The hyphal form, constituted by filamentous cells, has the ability to invade tissue and induce inflammation. Our hypothesis is that certain polyhydroxylated carboxylic acids, that may serve as an alternative carbohydrate source and at the same time lower the pH, function as an indicator of a nutrient-rich environment for C. albicans, which favors planktonic cells over hyphae, and thus diminish the formation of biofilm. We have shown that the biofilm formation in C. albicans and other Candida species can be significantly reduced by the addition of glucono-δ-lactone (GDL).

19.
Front Microbiol ; 10: 1679, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31396189

RESUMEN

The yeast Candida glabrata is a major opportunistic pathogen causing mucosal and systemic infections in humans. Systemic infections caused by this yeast have high mortality rates and are difficult to treat due to this yeast's intrinsic and frequently adapting antifungal resistance. To understand and treat C. glabrata infections, it is essential to investigate the molecular basis of C. glabrata virulence and resistance. We established an RNA interference (RNAi) system in C. glabrata by expressing the Dicer and Argonaute genes from Saccharomyces castellii (a budding yeast with natural RNAi). Our experiments with reporter genes and putative virulence genes showed that the introduction of RNAi resulted in 30 and 70% gene-knockdown for the construct-types antisense and hairpin, respectively. The resulting C. glabrata RNAi strain was used for the screening of a gene library for new virulence-related genes. Phenotypic profiling with a high-resolution quantification of growth identified genes involved in the maintenance of cell integrity, antifungal drugs, and ROS resistance. The genes identified by this approach are promising targets for the treatment of C. glabrata infections.

20.
PLoS One ; 11(8): e0161741, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27560164

RESUMEN

The wine and beer yeast Dekkera bruxellensis thrives in environments that are harsh and limiting, especially in concentrations with low oxygen and high ethanol. Its different strains' chromosomes greatly vary in number (karyotype). This study isolates two novel centromeric loci (CEN1 and CEN2), which support both the yeast's autonomous replication and the stable maintenance of plasmids. In the sequenced genome of the D. bruxellensis strain CBS 2499, CEN1 and CEN2 are each present in one copy. They differ from the known "point" CEN elements, and their biological activity is retained within ~900-1300 bp DNA segments. CEN1 and CEN2 have features of both "point" and "regional" centromeres: They contain conserved DNA elements, ARSs, short repeats, one tRNA gene, and transposon-like elements within less than 1 kb. Our discovery of a miniature inverted-repeat transposable element (MITE) next to CEN2 is the first report of such transposons in yeast. The transformants carrying circular plasmids with cloned CEN1 and CEN2 undergo a phenotypic switch: They form fluffy colonies and produce three times more biofilm. The introduction of extra copies of CEN1 and CEN2 promotes both genome rearrangements and ploidy shifts, with these effects mediated by homologous recombination (between circular plasmid and genome centromere copy) or by chromosome breakage when integrated. Also, the proximity of the MITE-like transposon to CEN2 could translocate CEN2 within the genome or cause chromosomal breaks, so promoting genome dynamics. With extra copies of CEN1 and CEN2, the yeast's enhanced capacities to rearrange its genome and to change its gene expression could increase its abilities for exploiting new and demanding niches.


Asunto(s)
Centrómero/genética , Dekkera/genética , Genes Fúngicos , Sitios Genéticos , Inestabilidad Genómica , Cerveza/microbiología , Biopelículas , Secuencia Conservada , Dekkera/fisiología , Recombinación Homóloga , Ploidias , Vino/microbiología
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