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1.
Nucleic Acids Res ; 48(1): 421-431, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31724694

RESUMO

COMPlex ASsociating with SET1 (COMPASS) is a histone H3 Lys-4 methyltransferase that typically marks the promoter region of actively transcribed genes. COMPASS is a multi-subunit complex in which the catalytic unit, SET1, is required for H3K4 methylation. An important subunit known to regulate SET1 methyltransferase activity is the CxxC zinc finger protein 1 (Cfp1). Cfp1 binds to COMPASS and is critical to maintain high level of H3K4me3 in cells but the mechanisms underlying its stimulatory activity is poorly understood. In this study, we show that Cfp1 only modestly activates COMPASS methyltransferase activity in vitro. Binding of Cfp1 to COMPASS is in part mediated by a new type of monovalent zinc finger (ZnF). This ZnF interacts with the COMPASS's subunits RbBP5 and disruption of this interaction blunts its methyltransferase activity in cells and in vivo. Collectively, our studies reveal that a novel form of ZnF on Cfp1 enables its integration into COMPASS and contributes to epigenetic signaling.


Assuntos
Proteínas Fúngicas/química , Histona-Lisina N-Metiltransferase/química , Histonas/química , Fatores de Transcrição/química , Dedos de Zinco , Sequência de Aminoácidos , Sítios de Ligação , Chaetomium/genética , Chaetomium/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Epigênese Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Cinética , Metilação , Modelos Moleculares , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Zinco/metabolismo
2.
Mol Cell ; 77(1): 3-16.e4, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31607544

RESUMO

Tracing DNA repair factors by fluorescence microscopy provides valuable information about how DNA damage processing is orchestrated within cells. Most repair pathways involve single-stranded DNA (ssDNA), making replication protein A (RPA) a hallmark of DNA damage and replication stress. RPA foci emerging during S phase in response to tolerable loads of polymerase-blocking lesions are generally thought to indicate stalled replication intermediates. We now report that in budding yeast they predominantly form far away from sites of ongoing replication, and they do not overlap with any of the repair centers associated with collapsed replication forks or double-strand breaks. Instead, they represent sites of postreplicative DNA damage bypass involving translesion synthesis and homologous recombination. We propose that most RPA and recombination foci induced by polymerase-blocking lesions in the replication template are clusters of repair tracts arising from replication centers by polymerase re-priming and subsequent expansion of daughter-strand gaps over the course of S phase.


Assuntos
Replicação do DNA/genética , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/genética , Genoma/genética , Dano ao DNA/genética , Reparo do DNA/genética , Recombinação Homóloga/genética , Proteína de Replicação A/genética , Fase S/genética , Saccharomycetales/genética
3.
Microbiol Res ; 232: 126372, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31759230

RESUMO

The methylotrophic yeast, Ogataea thermomethanolica TBRC656, is an attractive host organism for heterologous protein production owing to the availability of protein expression vectors and a genome-editing tool. In this study, we focused on mating-type switching and gene expression in order to elucidate its sexual life cycle and establish genetic approaches applicable for the strain. A putative mating-type gene cluster was identified in TBRC656 that is syntenic to the cluster in Ogataea parapolymorpha DL-1 (previously named Hansenula polymorpha). Like DL-1, TBRC656 possesses two mating loci, namely MATa and MATα, and also shows flip-flop mating-type switching. Interestingly, unlike any other methylotrophic yeast, TBRC656 robustly switched mating type during late growth in rich medium (YPD). Under nutrient depletion, mating-type switching was observed within one hour. Transcription from both MATa and MATα mating loci was detected during growth in YPD, and possibly induced upon nitrogen depletion. Gene expression from MATα was detected as a single co-transcript from a three-gene array (α2-α1-a1S). Deletion of a putative a1S ORF at the MATα locus had no observed effect on mating-type switching but demonstrated significant effect on mating-type gene expression at both MATa and MATα loci.


Assuntos
Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos Tipo Acasalamento/genética , Saccharomycetales/genética , Saccharomycetales/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica/fisiologia , Genes Fúngicos Tipo Acasalamento/fisiologia , Haploidia , Proteínas de Homeodomínio/genética , Família Multigênica , Pichia/genética , Pichia/fisiologia , Proteínas Repressoras/genética , Reprodução/genética , Reprodução/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia
4.
Int J Food Microbiol ; 312: 108373, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31654841

RESUMO

The use of non-Saccharomyces yeast in conjunction with Saccharomyces cerevisiae in wine fermentation is a growing trend in the wine industry. Non-Saccharomyces, through their distinctive production of secondary metabolites, have the potential to positively contribute to wine sensory profile. To discover new candidate strains for development as starter cultures, indigenous non-Saccharomyces were isolated from un-inoculated fermenting Shiraz musts from a South Australian vineyard (McLaren Vale wine region) and characterised. Among the 77 isolates, 7 species belonging to 5 genera (Kazachstania, Aureobasidium, Meyerozyma, Wickerhamomyces and Torulaspora) were identified by sequencing the internal transcribed spacer regions of the 5.8S rRNA gene (ITS1-5.8S-ITS2 region). The indigenous isolates were evaluated for oenological properties, namely, ethanol tolerance, enzyme activity, and H2S production. To determine their potential industrial use as starter cultures, representative isolates of each species were assessed in a sterile chemically defined grape juice and Viognier grape juice to evaluate their contribution to fermentation kinetics and production of key metabolites, including volatile compounds.


Assuntos
Saccharomycetales/genética , Saccharomycetales/metabolismo , Vitis/metabolismo , Vinho/microbiologia , Leveduras/metabolismo , Austrália , DNA Intergênico/genética , Fazendas , Fermentação , RNA Ribossômico 5,8S/genética , Saccharomycetales/classificação , Saccharomycetales/isolamento & purificação , Austrália do Sul , Vinho/análise
5.
Clin Exp Dermatol ; 45(1): 36-40, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31220362

RESUMO

BACKGROUND: Palmoplantar pustulosis (PPP) is a distinct, chronic skin disorder characterized by intraepidermal pustules on the palms and soles. It is hypothesized that microorganisms on the skin might induce the symptoms of PPP via inflammatory cell activation. However, the microbiota has not been studied in detail because of the assumption that the pustules in PPP are sterile. AIM: To elucidate the role of microorganisms in pathogenesis of PPP. METHODS: PCR analysis was performed of microbial DNA fragments in the pustules of patients with PPP. The sequence of the D1/D2 LSU 26s rRNA gene and that of the 16S rRNA gene was used for fungal and bacterial DNA detection, respectively. RESULTS: In total, 71 samples were carefully collected from the pustules of patients with PPP. Fungal DNA bands were detected in 68 samples, and fungi including Malassezia spp. were identified in 30 of 71 samples (42.3%). Malassezia restricta was the most frequently encountered fungus (14/71; 19.7%). However, bacterial DNA was not detected by the methods used. Furthermore, identical fungal DNA was not detected in the outer lid of the pustules, suggesting that the fungi detected within the pustule did not derive from contamination via the skin surface. CONCLUSIONS: In the present study, we demonstrated for the first time that certain pustules in patients with PPP contain fungal DNA fragments, especially those of Malassezia spp. Our findings provide new insights on the role of skin microbiota in the pathogenesis of PPP.


Assuntos
DNA Bacteriano/isolamento & purificação , DNA Fúngico/isolamento & purificação , Malassezia/isolamento & purificação , Psoríase/microbiologia , Acremonium/genética , Acremonium/isolamento & purificação , Adulto , Idoso , Aspergillus/genética , Aspergillus/isolamento & purificação , Cladosporium/genética , Cladosporium/isolamento & purificação , Feminino , Humanos , Malassezia/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Saccharomycetales/genética
6.
World J Microbiol Biotechnol ; 35(11): 170, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31673816

RESUMO

Debaryomyces hansenii is a halotolerant yeast of importance in basic and applied research. Previous reports hinted about possible links between saline and oxidative stress responses in this yeast. The aim of this work was to study that hypothesis at different molecular levels, investigating after oxidative and saline stress: (i) transcription of seven genes related to oxidative and/or saline responses, (ii) activity of two main anti-oxidative enzymes, (iii) existence of common metabolic intermediates, and (iv) generation of damages to biomolecules as lipids and proteins. Our results showed how expression of genes related to oxidative stress was induced by exposure to NaCl and KCl, and, vice versa, transcription of some genes related to osmotic/salt stress responses was regulated by H2O2. Moreover, and contrary to S. cerevisiae, in D. hansenii HOG1 and MSN2 genes were modulated by stress at their transcriptional level. At the enzymatic level, saline stress also induced antioxidative enzymatic defenses as catalase and glutathione reductase. Furthermore, we demonstrated that both stresses are connected by the generation of intracellular ROS, and that hydrogen peroxide can affect the accumulation of in-cell sodium. On the other hand, no significant alterations in lipid oxidation or total glutathione content were observed upon exposure to both stresses tested. The results described in this work could help to understand the responses to both stressors, and to improve the biotechnological potential of D. hansenni.


Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Estresse Oxidativo/fisiologia , Saccharomycetales/fisiologia , Estresse Salino/fisiologia , Antioxidantes , Catalase/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Peróxido de Hidrogênio , Metabolismo dos Lipídeos , Osmorregulação/genética , Osmorregulação/fisiologia , Estresse Oxidativo/genética , Cloreto de Potássio/metabolismo , Proteômica , Saccharomycetales/genética , Estresse Salino/genética , Sódio/metabolismo , Cloreto de Sódio/metabolismo , Fatores de Transcrição/genética
7.
PLoS Genet ; 15(10): e1008466, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31652272

RESUMO

What makes a gene essential for cellular survival? In model organisms, such as budding yeast, systematic gene deletion studies have revealed that paralog genes are less likely to be essential than singleton genes and that this can partially be attributed to the ability of paralogs to buffer each other's loss. However, the essentiality of a gene is not a fixed property and can vary significantly across different genetic backgrounds. It is unclear to what extent paralogs contribute to this variation, as most studies have analyzed genes identified as essential in a single genetic background. Here, using gene essentiality profiles of 558 genetically heterogeneous tumor cell lines, we analyze the contribution of paralogy to variable essentiality. We find that, compared to singleton genes, paralogs are less frequently essential and that this is more evident when considering genes with multiple paralogs or with highly sequence-similar paralogs. In addition, we find that paralogs derived from whole genome duplication exhibit more variable essentiality than those derived from small-scale duplications. We provide evidence that in 13-17% of cases the variable essentiality of paralogs can be attributed to buffering relationships between paralog pairs, as evidenced by synthetic lethality. Paralog pairs derived from whole genome duplication and pairs that function in protein complexes are significantly more likely to display such synthetic lethal relationships. Overall we find that many of the observations made using a single strain of budding yeast can be extended to understand patterns of essentiality in genetically heterogeneous cancer cell lines.


Assuntos
Evolução Molecular , Modelos Genéticos , Neoplasias/genética , Linhagem Celular Tumoral , Deleção de Genes , Duplicação Gênica , Genes Essenciais , Humanos , Saccharomycetales/genética , Mutações Sintéticas Letais
8.
PLoS One ; 14(10): e0223374, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31581259

RESUMO

The spores of fungi come in a wide variety of forms and sizes, highly adapted to the route of dispersal and to survival under specific environmental conditions. The ascomycete Ashbya gossypii produces needle shaped spores with a length of 30 µm and a diameter of 1 µm. Formation of these spores relies on actin and actin regulatory proteins and is, therefore, distinct from the minor role that actin plays for spore formation in Saccharomyces cerevisiae. Using in vivo FRET-measurements of proteins labeled with fluorescent proteins, we investigate how the formin AgBnr2, a protein that promotes actin polymerization, integrates into the structure of the spindle pole body during sporulation. We also investigate the role of the A. gossypii homologs to the S. cerevisiae meiotic outer plaque proteins Spo74, Mpc54 and Ady4 for sporulation in A. gossypii. We found highest FRET of AgBnr2 with AgSpo74. Further experiments indicated that AgSpo74 is a main factor for targeting AgBnr2 to the spindle pole body. In agreement with these results, the Agspo74 deletion mutant produces no detectable spores, whereas deletion of Agmpc54 only has an effect on spore length and deletion of Agady4 has no detectable sporulation phenotype. Based on this study and in relation to previous results we suggest a model where AgBnr2 resides within an analogous structure to the meiotic outer plaque of S. cerevisiae. There it promotes formation of actin cables important for shaping the needle shaped spore structure.


Assuntos
Proteínas Fúngicas/metabolismo , Saccharomycetales/metabolismo , Corpos Polares do Fuso/metabolismo , Esporos Fúngicos/metabolismo , Proteínas Fúngicas/genética , Modelos Biológicos , Mutação , Fenótipo , Saccharomycetales/genética , Esporos Fúngicos/genética
9.
Proc Natl Acad Sci U S A ; 116(44): 22219-22228, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31611373

RESUMO

Horizontal acquisition of bacterial genes is presently recognized as an important contribution to the adaptation and evolution of eukaryotic genomes. However, the mechanisms underlying expression and consequent selection and fixation of the prokaryotic genes in the new eukaryotic setting are largely unknown. Here we show that genes composing the pathway for the synthesis of the essential vitamin B1 (thiamine) were lost in an ancestor of a yeast lineage, the Wickerhamiella/Starmerella (W/S) clade, known to harbor an unusually large number of genes of alien origin. The thiamine pathway was subsequently reassembled, at least twice, by multiple HGT events from different bacterial donors involving both single genes and entire operons. In the W/S-clade species Starmerella bombicola we obtained direct genetic evidence that all bacterial genes of the thiamine pathway are functional. The reconstructed pathway is composed by yeast and bacterial genes operating coordinately to scavenge thiamine derivatives from the environment. The adaptation of the newly acquired operons to the eukaryotic setting involved a repertoire of mechanisms until now only sparsely documented, namely longer intergenic regions, post-horizontal gene transfer (HGT) gene fusions fostering coordinated expression, gene relocation, and possibly recombination generating mosaic genes. The results provide additional evidence that HGT occurred recurrently in this yeast lineage and was crucial for the reestablishment of lost functions and that similar mechanisms are used across a broad range of eukaryotic microbes to promote adaptation of prokaryotic genes to their new environment.


Assuntos
Transferência Genética Horizontal , Genes Bacterianos , Óperon , Saccharomycetales/genética , Tiamina/genética , Bactérias/genética , Tiamina/metabolismo
10.
Curr Microbiol ; 76(12): 1537-1544, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31555854

RESUMO

One novel ascomycetous yeast strain TF5-16-2 was isolated from water samples of Tuofengling crater lake located in Da Hinggan Ling Mountain, in the Inner Mongolia province of China. Morphological, physiological characteristics, as well as phylogenetic analyses of D1/D2 domains of the large subunit rRNA (LSU), ITS region, small subunit rRNA (SSU), and elongation factor-1α (EF-1α) were performed and finally confirmed the phylogenetic placement of strain TF5-16-2 in the genus Wickerhamomyces. Sequences analysis revealed that strain TF5-16-2 differed from its most closely related phylogenetic neighbors 'Candida' silvicultrix CBS 6269T and Wickerhamomyces anomalus CBS 5759T by 8.0% (including 2.3% gaps), 8.5% (including 2.4% gaps) divergences in D1/D2 domains of LSU, and 11% (including 4.3% gaps) and 13% (including 4.4% gaps) divergences in ITS region, respectively. As the considerable sequence divergence and distinguishable physiological characteristics, strain TF5-16-2 was proposed as a new species of the genus Wickerhamomyces, with the name Wickerhamomyces kurtzmanii sp. nov. (holotype = CGMCC 2.5597, Mycobank number is MB829959).


Assuntos
Lagos/microbiologia , Saccharomycetales/isolamento & purificação , China , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Proteínas Fúngicas/genética , Técnicas de Tipagem Micológica , Fator 1 de Elongação de Peptídeos/genética , Filogenia , Saccharomycetales/classificação , Saccharomycetales/genética
11.
J Ind Microbiol Biotechnol ; 46(12): 1697-1706, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31512095

RESUMO

Free fatty acids are basic oleochemicals implemented in a range of applications including surfactants, lubricants, paints, plastics, and cosmetics. Microbial fatty acid biosynthesis has gained much attention as it provides a sustainable alternative for petrol- and plant oil-derived chemicals. The yeast Starmerella bombicola is a microbial cell factory that naturally employs its powerful lipid metabolism for the production of the biodetergents sophorolipids (> 300 g/L). However, in this study we exploit the lipidic potential of S. bombicola and convert it from the glycolipid production platform into a free fatty acid cell factory. We used several metabolic engineering strategies to promote extracellular fatty acid accumulation which include blocking competing pathways (sophorolipid biosynthesis and ß-oxidation) and preventing free fatty acid activation. The best producing mutant (Δcyp52m1Δfaa1Δmfe2) secreted 0.933 g/L (± 0.04) free fatty acids with a majority of C18:1 (43.8%) followed by C18:0 and C16:0 (40.0 and 13.2%, respectively). Interestingly, deletion of SbFaa1 in a strain still producing sophorolipids also resulted in 25% increased de novo sophorolipid synthesis (P = 0.0089) and when oil was supplemented to the same strain, a 50% increase in sophorolipid production was observed compared to the wild type (P = 0.03). We believe that our work is pivotal for the further development and exploration of S. bombicola as a platform for synthesis of environmentally friendly oleochemicals.


Assuntos
Ácidos Graxos/biossíntese , Glicolipídeos/metabolismo , Saccharomycetales/metabolismo , Glicolipídeos/química , Metabolismo dos Lipídeos , Engenharia Metabólica , Oxirredução , Saccharomycetales/genética
12.
Int J Mol Sci ; 20(18)2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31500317

RESUMO

d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin C with a two enzymes system. DAAO from the yeast Trigonopsis variabilis (TvDAAO) shows the best catalytic parameters with cephalosporin C among all known DAAOs. We prepared and characterized multipoint TvDAAO mutants to improve their activity towards cephalosporin C and increase stability. All TvDAAO mutants showed better properties in comparison with the wild-type enzyme. The best mutant was TvDAAO with amino acid changes E32R/F33D/F54S/C108F/M156L/C298N. Compared to wild-type TvDAAO, the mutant enzyme exhibits a 4 times higher catalytic constant for cephalosporin C oxidation and 8- and 20-fold better stability against hydrogen peroxide inactivation and thermal denaturation, respectively. This makes this mutant promising for use in biotechnology. The paper also presents the comparison of TvDAAO catalytic properties with cephalosporin C reported by others.


Assuntos
Substituição de Aminoácidos , Cefalosporinas/metabolismo , D-Aminoácido Oxidase/genética , Saccharomycetales/enzimologia , Biocatálise , D-Aminoácido Oxidase/química , D-Aminoácido Oxidase/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Peróxido de Hidrogênio/farmacologia , Mutação Puntual , Saccharomycetales/genética , Termodinâmica
13.
J Biotechnol ; 304: 10-15, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31400343

RESUMO

Huimcola insolens cutinase (HiC) was heterologously expressed in Pichia pastoris. To avoid a carbon starvation step, fermentation was conducted using combinations of sorbitol with glycerol and methanol in the cell growth and induction phases, respectively. The cutinase productivity (27.71 U mL-1 h-1) was 9.93 U mL-1 h-1 greater than that achieved using traditional two-phase methods, and a cutinase activity of 2660 U mL-1, using p-nitrophenyl butyrate as substrate, was achieved after only 96 h in a 3-L bioreactor. Subsequently, the combination of HiC with Thermobifida fusca cutinase (TfC) in cotton fabric bioscouring was evaluated by monitoring the wettability and dyeability of the fabric. Treatment with 20 U mL-1 of HiC at 80 °C for 5 min followed by 30 U mL-1 of TfC at 50 °C for 1 h gave the best results. The total treatment time was shorter and performance was better than those seen with the alkali method.


Assuntos
Hidrolases de Éster Carboxílico/genética , Pichia/crescimento & desenvolvimento , Saccharomycetales/enzimologia , Biodegradação Ambiental , Reatores Biológicos/microbiologia , Carbono/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Fibra de Algodão , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Engenharia Genética , Pichia/genética , Pichia/metabolismo , Saccharomycetales/genética , Têxteis
14.
J Biotechnol ; 304: 28-30, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31415788

RESUMO

The possibility of using active dry microbial preparations in biotechnological processes is essential for the development of new modern industrial technologies. In this study, we show the possibility of obtaining such preparations of the genetically engineered yeast strain Ogataea (Hansenula) polymorpha with glutathione overproduction. Special pre-treatment involving the gradual rehydration of dry cells in water vapour led to the restoration/reactivation of almost 100% of dehydrated cells. Furthermore, dry cells do not lose their viability during storage at room temperatures. Application of dry cells as the inoculum provides the same levels of glutathione synthesis as that of a native yeast culture.


Assuntos
Glutationa Sintase/genética , Glutationa/biossíntese , Saccharomycetales/crescimento & desenvolvimento , Fatores de Transcrição de Zíper de Leucina Básica/genética , Dessecação , Hidratação , Engenharia Genética , Glutationa Sintase/metabolismo , Viabilidade Microbiana , Saccharomycetales/genética , Saccharomycetales/metabolismo
15.
J Biotechnol ; 304: 44-51, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31419454

RESUMO

A novel type III fungal CAR was identified from the organism Thermothelomyces thermophila. High expression levels were observed in E. coli using the pETDuet-1 plasmid system in combination with an autoinduction protocol. A broad substrate scope ranging from aromatic to aliphatic carboxylic acids was tested and TtCAR showed activity for all substrates. High specific activities for aromatic substrates and short chain aliphatic substrates were observed, comparable to those of NcCAR, the first type III fungal CAR. TtCAR's pH and temperature optima were at 6.5 and 30 °C, respectively. Up to 20% (v/v) cosolvents did not show a decrease in specific activity of TtCAR using (E)-cinnamic acid as a substrate. Its half-life at 40 °C was determined to be 8.25 h and its melting temperature (Tm) was 56 °C. In vitro reactions with TtCAR reduced 95.2% of 10 mM vanillic acid, which correlated to a titer of 1.4 g L-1 of vanillin. The space time yield of 0.029 g L-1 h-1 indicates that further improvements would be necessary for an industrially relevant application. This would be especially important when competing against de novo synthesis of bio vanillin by microbial strains producing >30 g L-1. In de novo and in vivo biosynthesis systems, by-products are fairly common. By contrast, we were pleased to observe less than 0.7% of vanillyl alcohol formed, making the cell-free acid reduction in the envisaged sequential two-step bioconversion from eugenol to vanillin very attractive.


Assuntos
Benzaldeídos/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Saccharomycetales/enzimologia , Cinamatos/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Oxirredutases/química , Conformação Proteica , Engenharia de Proteínas , Saccharomycetales/genética , Especificidade por Substrato , Termodinâmica , Ácido Vanílico/metabolismo
16.
G3 (Bethesda) ; 9(10): 3345-3358, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31427453

RESUMO

The enzyme telomerase ensures the integrity of linear chromosomes by maintaining telomere length. As a hallmark of cancer, cell immortalization and unlimited proliferation is gained by reactivation of telomerase. However, a significant fraction of cancer cells instead uses alternative telomere lengthening mechanisms to ensure telomere function, collectively known as Alternative Lengthening of Telomeres (ALT). Although the budding yeast Naumovozyma castellii (Saccharomyces castellii) has a proficient telomerase activity, we demonstrate here that telomeres in N. castellii are efficiently maintained by a novel ALT mechanism after telomerase knockout. Remarkably, telomerase-negative cells proliferate indefinitely without any major growth crisis and display wild-type colony morphology. Moreover, ALT cells maintain linear chromosomes and preserve a wild-type DNA organization at the chromosome termini, including a short stretch of terminal telomeric sequence. Notably, ALT telomeres are elongated by the addition of ∼275 bp repeats containing a short telomeric sequence and the subtelomeric DNA located just internally (TelKO element). Although telomeres may be elongated by several TelKO repeats, no dramatic genome-wide amplification occurs, thus indicating that the repeat addition may be regulated. Intriguingly, a short interstitial telomeric sequence (ITS) functions as the initiation point for the addition of the TelKO element. This implies that N. castellii telomeres are structurally predisposed to efficiently switch to the ALT mechanism as a response to telomerase dysfunction.


Assuntos
Saccharomycetales/genética , Homeostase do Telômero , Telômero/genética , Cromossomos Fúngicos , Genoma Fúngico , Genômica/métodos , Humanos , Telomerase/metabolismo
17.
J Basic Microbiol ; 59(8): 792-806, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31368594

RESUMO

The aim of this study was to examine four strains of two yeast species in relation to their capability for assimilating alkanes in the presence of heavy metals (HMs). The four strains tested were Candida pseudoglaebosa ENCB-7 and Kodamaea ohmeri ENCB-8R, ENCB-23, and ENCB-VIK. Determination was made of the expression of CYP52 genes involved in alkane hydroxylation. When exposed to Cu2+ , Zn2+ , Pb2+ , Cd2+ , and As3+ at pH 3 and 5, all four strains could assimilate several n-alkanes having at least six carbon atoms. The three K. ohmeri strains could also utilize branched alkanes, cycloalkanes, and n-octanol as sole carbon sources. Kinetic assays demonstrated greater biomass production and specific growth of the yeasts exposed to long-chain n-alkanes. Fragments of paralogous CYP52 genes of C. pseudoglaebosa ENCB-7 and K. ohmeri ENCB-23 were amplified, sequenced, and phylogenetically evaluated. Reverse-transcription polymerase chain reaction revealed that n-nonane and n-decane induced to CpCYP52-G3, CpCYP52-G9, and CpCYP52-G10. KoCYP52-G3 was induced with n-decane and n-octanol. Also, CpCYP52-G3 and CpCYP52-G9 were induced by glucose. In conclusion, C. pseudoglaebosa and K. ohmeri were able to degrade several alkanes in the presence of HMs and under acidic conditions. These yeasts harbor paralogous alkane-induced CYP52 genes, which display different profiles of transcriptional expression.


Assuntos
Alcanos/metabolismo , Metais Pesados/metabolismo , Saccharomycetales/metabolismo , Alcanos/química , Biodegradação Ambiental , Biomassa , Candida/classificação , Candida/genética , Candida/crescimento & desenvolvimento , Candida/metabolismo , Sistema Enzimático do Citocromo P-450/genética , DNA Ribossômico/genética , Proteínas Fúngicas/genética , Concentração de Íons de Hidrogênio , Cinética , Filogenia , Saccharomycetales/classificação , Saccharomycetales/genética , Saccharomycetales/crescimento & desenvolvimento
18.
PLoS Genet ; 15(7): e1008304, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31365533

RESUMO

Variation in synonymous codon usage is abundant across multiple levels of organization: between codons of an amino acid, between genes in a genome, and between genomes of different species. It is now well understood that variation in synonymous codon usage is influenced by mutational bias coupled with both natural selection for translational efficiency and genetic drift, but how these processes shape patterns of codon usage bias across entire lineages remains unexplored. To address this question, we used a rich genomic data set of 327 species that covers nearly one third of the known biodiversity of the budding yeast subphylum Saccharomycotina. We found that, while genome-wide relative synonymous codon usage (RSCU) for all codons was highly correlated with the GC content of the third codon position (GC3), the usage of codons for the amino acids proline, arginine, and glycine was inconsistent with the neutral expectation where mutational bias coupled with genetic drift drive codon usage. Examination between genes' effective numbers of codons and their GC3 contents in individual genomes revealed that nearly a quarter of genes (381,174/1,683,203; 23%), as well as most genomes (308/327; 94%), significantly deviate from the neutral expectation. Finally, by evaluating the imprint of translational selection on codon usage, measured as the degree to which genes' adaptiveness to the tRNA pool were correlated with selective pressure, we show that translational selection is widespread in budding yeast genomes (264/327; 81%). These results suggest that the contribution of translational selection and drift to patterns of synonymous codon usage across budding yeasts varies across codons, genes, and genomes; whereas drift is the primary driver of global codon usage across the subphylum, the codon bias of large numbers of genes in the majority of genomes is influenced by translational selection.


Assuntos
Uso do Códon , Saccharomycetales/genética , Viés , Variação Genética , Genoma Fúngico , Seleção Genética
19.
PLoS One ; 14(8): e0221228, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31412087

RESUMO

Early and accurate diagnosis of coccidioidomycosis, also known as Valley fever, is critical for appropriate disease treatment and management. Current serodiagnosis is based on the detection of patient serum antibodies that react with tube precipitin (TP) and complement fixation (CF) antigens of Coccidioides. IgM is the first class of antibodies produced by hosts in response to coccidioidal insults. The highly glycosylated ß-glucosidase 2 (BGL2) is a major active component of the TP antigen that stimulates IgM antibody responses during early Coccidioides infection. The predominant IgM epitope on BGL2 is a unique 3-O-methyl-mannose moiety that is not produced by commonly used protein expression systems. We genetically engineered and expressed a recombinant BGL2 (rBGL2ur), derived from Coccidioides, in non-pathogenic Uncinocarpus reesii, a fungus phylogenetically related to the Coccidioides pathogen. The rBGL2ur protein was purified from the culture medium of transformed U. reesii by nickel affinity chromatography, and the presence of 3-O-methyl mannose was demonstrated by gas chromatography. Seroreactivity of the purified rBGL2ur protein was tested by enzyme-linked immunosorbent assays using sera from 90 patients with coccidioidomycosis and 134 control individuals. The sensitivity and specificity of the assay with rBGL2ur were 78.8% and 87.3%, respectively. These results were comparable to those obtained using a proprietary MiraVista Diagnostic (MVD) IgM (63.3% sensitivity; 96.3% specificity), but significantly better than the ID-TP assay using non-concentrated patient sera (33.3% sensitivity; 100% specificity). Expression of rBGL2ur in U. reesii retains its antigenicity for coccidioidomycosis serodiagnosis and greatly reduces biosafety concerns for antigen production, as Coccidioides spp. are biological safety level 3 agents.


Assuntos
Anticorpos Antifúngicos , Antígenos de Fungos/imunologia , Coccidioides , Coccidioidomicose , Precipitinas , Saccharomycetales , Testes Sorológicos , Anticorpos Antifúngicos/química , Anticorpos Antifúngicos/imunologia , Coccidioides/química , Coccidioides/genética , Coccidioides/imunologia , Coccidioidomicose/diagnóstico , Coccidioidomicose/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Precipitinas/química , Precipitinas/imunologia , Saccharomycetales/química , Saccharomycetales/genética
20.
J Biol Chem ; 294(37): 13629-13637, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31320474

RESUMO

The Mag1 and Tpa1 proteins from budding yeast (Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA. Mag1 initiates the base excision repair pathway by removing alkylated bases from DNA, and Tpa1 has been proposed to directly repair alkylated bases as does the prototypical oxidative dealkylase AlkB from Escherichia coli However, we found that in vivo repair of methyl methanesulfonate (MMS)-induced alkylation damage in DNA involves Mag1 but not Tpa1. We observed that yeast strains without tpa1 are no more sensitive to MMS than WT yeast, whereas mag1-deficient yeast are ∼500-fold more sensitive to MMS. We therefore investigated the substrate specificity of Mag1 and found that it excises alkylated bases that are known AlkB substrates. In contrast, purified recombinant Tpa1 did not repair these alkylated DNA substrates, but it did exhibit the prolyl hydroxylase activity that has also been ascribed to it. A comparison of several of the kinetic parameters of Mag1 and its E. coli homolog AlkA revealed that Mag1 catalyzes base excision from known AlkB substrates with greater efficiency than does AlkA, consistent with an expanded role of yeast Mag1 in repair of alkylation damage. Our results challenge the proposal that Tpa1 directly functions in DNA repair and suggest that Mag1-initiated base excision repair compensates for the absence of oxidative dealkylation of alkylated nucleobases in budding yeast. This expanded role of Mag1, as compared with alkylation repair glycosylases in other organisms, could explain the extreme sensitivity of Mag1-deficient S. cerevisiae toward alkylation damage.


Assuntos
Proteínas de Transporte/metabolismo , DNA Glicosilases/metabolismo , Reparo do DNA/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alquilantes , Alquilação/genética , Proteínas de Transporte/genética , Dano ao DNA/efeitos dos fármacos , DNA Glicosilases/genética , DNA Fúngico/metabolismo , Remoção de Radical Alquila/genética , Endodesoxirribonucleases/genética , Escherichia coli/metabolismo , Metanossulfonato de Metila/farmacologia , Mutagênicos/farmacologia , Mutação , Estresse Oxidativo/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Especificidade por Substrato
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