Microbiologia
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11840
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Resultados da Pesquisa
Item Ethanol stress responses of Kluyveromyces marxianus CCT 7735 revealed by proteomic and metabolomic analyses(Antonie van Leeuwenhoek, 2019) Alvim, Mariana Caroline Tocantins; Vital, Camilo Elber; Vieira, Nívea Moreira; Silveira, Fernando Augusto da; Balbino, Thércia Rocha; Diniz, Raphael Hermano Santos; Brito, Amanda Fernandes; Bazzolli, Denise Mara Soares; Silveira, Wendel Batista da; Barros, Edvaldo; Ramos, Humberto Josué de OliveiraKluyveromyces marxianus CCT 7735 offers advantages to ethanol production over Saccharomyces cerevisiae, including thermotolerance and the ability to convert lactose to ethanol. However, its growth is impaired at high ethanol concentrations. Herein we report on the protein and intracellular metabolite profiles of K. marxianus at 1 and 4 h under ethanol exposure. The concentration of some amino acids, trehalose and ergosterol were also measured. We observed that proteins and metabolites from carbon pathways and translation were less abundant, mainly at 4 h of ethanol stress. Nevertheless, the concentration of some amino acids and trehalose increased at 8 and 12 h under ethanol stress, indicating an adaptive response. Moreover, our results show that the abundance of proteins and metabolites related to the oxidative stresses responses increased. The results obtained in this study provide insights into understanding the physiological changes in K. marxianus under ethanol stress, indicating possible targets for ethanol tolerant strains construction.Item Screening of yeasts isolated from brazilian environments for the 2-Phenylethanol (2-PE) production(Biotechnology and Bioprocess Engineering, 2018-06) Lima, Lorena Azevedo de; Diniz, Raphael Hermano Santos; Queiroz, Marisa Vieira de; Fietto, Luciano Gomes; Silveira, Wendel Batista daPhenylethanol alcohol, or 2-phenylethanol (2-PE) production by yeasts has been considered a promising alternative to its chemical synthesis. In order to evaluate the potential of yeast strains isolated from different Brazilian environments, we evaluated the 2-PE production of 267 strains. Among them, the Kluyveromyces marxianus CCT 7735 yeast stood out as being the best 2-PE producer. The K. marxianus CCT 7735 growth was impaired by 2-PE; nevertheless, this effect is less pronounced than the inhibition reported for certain Saccharomyces cerevisiae strains. The maximum 2-PE titer obtained under optimized conditions was 3.44 g/L, 28% higher than the titer achieved under unoptimized conditions. The optimized conditions were: 30ºC, and glucose and L-phe concentrations of 3.0 and 4.0 g/L, respectively. Moreover, the specific production rate of 2-PE increased twofold compared to the unoptimized conditions.Item Characterization of a thermotolerant laccase produced by Streptomyces sp. SB086(Annals of Microbiology, 2013-12-28) Fernandes, Tatiana Alves Rigamonte; Silveira, Wendel Batista da; Passos, Flávia Maria Lopes; Zucchi, Tiago DominguesLaccases have become desirable enzymes for application in many industrial processes. Nowadays, most of these enzymes are obtained from fungi. Among prospective studies for bacterial laccase genes, some have included actinomycetes, but only a few studies have characterized the enzyme produced. Thus, we have isolated a laccase-producing actinomycete from forest soil under restoration process and further aimed to characterize its produced enzyme. The isolate SB086 was assigned to the Streptomyces genus by a combination of phenotypical, chemical and phylogenetic properties. Our data indicate that the bacterium produces a thermotolerant laccase. The maximum activity was obtained in the pH range 4.0–5.0 and at 50 °C in reaction mixture containing 5 mM CuSO4; thermal stability was noted at 60 °C and 70 °C—a well-desired characteristic for industry. The active enzyme presented a high molecular mass (over 100 kDa) and was less sensitive to inhibition by metal ions than generally described for bacterial laccases. Our findings support in silico data of bacterial laccase secretion, and reinforce the view that actinomycetes may be a rich source of laccase for industrial application.Item Optimizing ethanol production by thermotolerant Kluyveromyces marxianus CCT 7735 in a mixture of sugarcane bagasse and ricotta wh(Food Science and Biotechnology, 2015-05-01) Ferreira, Priscila Gonçalves; Silveira, Fernando Augusto da; Santos, Raquel Cristina Vieira dos; Genier, Hugo Leonardo André; Diniz, Raphael Hermano Santos; Ribeiro Jr., José Ivo; Fietto, Luciano Gomes; Passos, Flávia Maria Lopes; Silveira, Wendel Batista daThe simultaneous saccharification and fermentation (SSF) process is a promising strategy to obtain ethanol from cellulosic biomass. In this study, sugarcane bagasse was supplemented with ricotta whey to increase the sugar, vitamin, and trace metal concentrations in the fermentation medium. The optimum conditions for SSF ethanol production from a mixture of sugarcane bagasse and ricotta whey produced by Kluyveromyces marxianus CCT 7735 were evaluated considering five factors: cellulase concentration, cellulosic biomass concentration, pH, temperature, and agitation. The highest ethanol yield was 49.65 g/L with a cellulosic biomass of 80 g/L, pH value of 5.05, agitation at 65 rpm and temperature of 39.2°C. The results demonstrated that a mixture of the cellulosic residue of sugarcane bagasse and ricotta whey is promising for ethanol production because the ethanol yield in the mixture was higher than that in single substrate of sugarcane bagasse.Item Applying functional metagenomics to search for novel lignocellulosic enzymes in a microbial consortium derived from a thermophilic composting phase of sugarcane bagasse and cow manure(Antonie van Leeuwenhoek, 2016-06-27) Colombo, Lívia Tavares; Oliveira, Marcelo Nagem Valério de; Carneiro, Deisy Guimarães; Souza, Robson Assis de; Alvim, Mariana Caroline Tocantins; Santos, Josenilda Carlos dos; Silva, Cynthia Canêdo da; Vidigal, Pedro Marcus Pereira; Silveira, Wendel Batista da; Passos, Flávia Maria LopesEnvironments where lignocellulosic biomass is naturally decomposed are sources for discovery of new hydrolytic enzymes that can reduce the high cost of enzymatic cocktails for second-generation ethanol production. Metagenomic analysis was applied to discover genes coding carbohydrate-depleting enzymes from a microbial laboratory subculture using a mix of sugarcane bagasse and cow manure in the thermophilic composting phase. From a fosmid library, 182 clones had the ability to hydrolyse carbohydrate. Sequencing of 30 fosmids resulted in 12 contigs encoding 34 putative carbohydrate-active enzymes belonging to 17 glycosyl hydrolase (GH) families. One third of the putative proteins belong to the GH3 family, which includes β-glucosidase enzymes known to be important in the cellulose-deconstruction process but present with low activity in commercial enzyme preparations. Phylogenetic analysis of the amino acid sequences of seven selected proteins, including three β-glucosidases, showed low relatedness with protein sequences deposited in databases. These findings highlight microbial consortia obtained from a mixture of decomposing biomass residues, such as sugar cane bagasse and cow manure, as a rich resource of novel enzymes potentially useful in biotechnology for saccharification of lignocellulosic substrate.Item Transcriptome analysis of the thermotolerant yeast Kluyveromyces marxianus CCT 7735 under ethanol stress(Applied Microbiology and Biotechnology, 2017-08-03) Diniz, Raphael Hermano Santos; Villada, Juan C.; Alvim, Mariana Caroline Tocantins; Vidigal, Pedro Marcus Pereira; Vieira, Nívea Moreira; Lamas-Maceiras, Mónica; Cerdán, María Esperanza; González-Siso, María-Isabel; Lahtvee, Petri-Jaan; Silveira, Wendel Batista daThe thermotolerant yeast Kluyveromyces marxianus displays a potential to be used for ethanol production from both whey and lignocellulosic biomass at elevated temperatures, which is highly alluring to reduce the cost of the bioprocess. Nevertheless, contrary to Saccharomyces cerevisiae, K. marxianus cannot tolerate high ethanol concentrations. We report the transcriptional profile alterations in K. marxianus under ethanol stress in order to gain insights about mechanisms involved with ethanol response. Time-dependent changes have been characterized under the exposure of 6% ethanol and compared with the unstressed cells prior to the ethanol addition. Our results reveal that the metabolic flow through the central metabolic pathways is impaired under the applied ethanol stress. Consistent with these results, we also observe that genes involved with ribosome biogenesis are downregulated and gene-encoding heat shock proteins are upregulated. Remarkably, the expression of some gene-encoding enzymes related to unsaturated fatty acid and ergosterol biosynthesis decreases upon ethanol exposure, and free fatty acid and ergosterol measurements demonstrate that their content in K. marxianus does not change under this stress. These results are in contrast to the increase previously reported with S. cerevisiae subjected to ethanol stress and suggest that the restructuration of K. marxianus membrane composition differs in the two yeasts which gives important clues to understand the low ethanol tolerance of K. marxianus compared to S. cerevisiae.Item Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1)(Applied Microbiology and Biotechnology, 2010-04-02) Bragança, Caio Roberto Soares; Colombo, Lívia Tavares; Roberti, Alvaro Soares; Alvim, Mariana Caroline Tocantins; Cardoso, Silvia Almeida; Reis, Kledna Constancio Portes; Paula, Sérgio Oliveira de; Silveira, Wendel Batista da; Passos, Flavia Maria LopesThe yeast Kluyveromyces marxianus is a convenient host for industrial synthesis of biomolecules. However, despite its potential, there are few studies reporting the expression of heterologous proteins using this yeast. Here, we report expression of a dengue virus protein in K. marxianus for the first time. The dengue virus type 1 nonstructural protein 1 (NS1) was integrated into the K. marxianus UFV-3 genome at the LAC4 locus using an adapted integrative vector designed for high-level expression of recombinant protein in Kluyveromyces lactis. The NS1 gene sequence was codon-optimized to increase the level of protein expression in yeast. The synthetic gene was cloned in frame with K. lactis α-mating factor signal peptide, and the recombinant plasmid obtained was used to transform K. marxianus UFV-3 by electroporation. The transformed cells, selected in yeast extract peptone dextrose containing 200 μg mL−1 Geneticin, were mitotically stable. Analysis of recombinant strains by RT-PCR and protein detection using blot analysis confirmed both transcription and expression of extracellular NS1 polypeptide. After induction with galactose, the NS1 protein was analyzed by sodium dodecyl sulfate-PAGE and immunogenic detection. Protein production was investigated under two conditions: with galactose and biotin pulses at 24-h intervals during 96 h of induction and without galactose and biotin supplementation. Protease activity was not detected in post-growth medium. Our results indicate that recombinant K. marxianus is a good host for the production of dengue virus NS1 protein, which has potential for diagnostic applications.Item Integrated analysis of individual codon contribution to protein biosynthesis reveals a new approach to improving the basis of rational gene design(DNA Research, 2017-04-24) Villada, Juan C.; Brustolini, Otávio José Bernardes; Silveira, Wendel Batista daGene codon optimization may be impaired by the misinterpretation of frequency and optimality of codons. Although recent studies have revealed the effects of codon usage bias (CUB) on protein biosynthesis, an integrated perspective of the biological role of individual codons remains unknown. Unlike other previous studies, we show, through an integrated framework that attributes of codons such as frequency, optimality and positional dependency should be combined to unveil individual codon contribution for protein biosynthesis. We designed a codon quantification method for assessing CUB as a function of position within genes with a novel constraint: the relativity of position-dependent codon usage shaped by coding sequence length. Thus, we propose a new way of identifying the enrichment, depletion and non-uniform positional distribution of codons in different regions of yeast genes. We clustered codons that shared attributes of frequency and optimality. The cluster of non-optimal codons with rare occurrence displayed two remarkable characteristics: higher codon decoding time than frequent–non-optimal cluster and enrichment at the 5′-end region, where optimal codons with the highest frequency are depleted. Interestingly, frequent codons with non-optimal adaptation to tRNAs are uniformly distributed in the Saccharomyces cerevisiae genes, suggesting their determinant role as a speed regulator in protein elongation.