DEFAR - Artigos publicados em periódicos
URI permanente para esta coleçãohttp://www.hml.repositorio.ufop.br/handle/123456789/531
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Item Moniliophthora perniciosa, the causal agent of cacao witches’ broom disease is killed in vitro by Saccharomyces cerevisiae and Wickerhamomyces anomalus yeasts.(2021) Ferraz, Pedro; Brandão, Rogélio Lopes; Cássio, Fernanda; Lucas, CândidaCacao plantations from South America have been afflicted with the severe fungal disease known as Witches’ Broom Disease (WBD), caused by the basidiomycete Moniliophthora perniciosa. Yeasts are increasingly recognized as good fungal biocides, although their application is still mostly restricted to the postharvest control of plant and fruit decay. Their possible utilization in the field, in a preharvest phase, is nevertheless promising, particularly if the strains are locally adapted and evolved and if they belong to species considered safe for man and the environment. In this work, a group of yeast strains originating from sugarcane-based fermentative processes in Brazil, the cacao-producing country where the disease is most severe, were tested for their ability to antagonize M. perniciosa in vitro. Wickerhamomyces anomalus LBCM1105 and Saccharomyces cerevisiae strains LBCM1112 from spontaneous fermentations used to produce cachaça, and PE2 widely used in Brazil in the industrial production of bioethanol, efficiently antagonized six strains of M. perniciosa, originating from several South American countries. The two fastest growing fungal strains, both originating from Brazil, were further used to assess the mechanisms underlying the yeasts’ antagonism. Yeasts were able to inhibit fungal growth and kill the fungus at three different temperatures, under starvation, at different culture stages, or using an inoculum from old yeast cultures. Moreover, SEM analysis revealed that W. anomalus and S. cerevisiae PE2 cluster and adhere to the hyphae, push their surface, and fuse to them, ultimately draining the cells. This behavior concurs with that classified as necrotrophic parasitism/mycoparasitism. In particular, W. anomalus within the adhered clusters appear to be ligated to each other through roundish groups of fimbriae-like structures filled with bundles of microtubule-sized formations, which appear to close after cells detach, leaving a scar. SEM also revealed the formation of tube-like structures apparently connecting yeast to hypha. This evidence suggests W. anomalus cells form a network of yeast cells connecting with each other and with hyphae, supporting a possible cooperative collective killing and feeding strategy. The present results provide an initial step toward the formulation of a new eco-friendly and effective alternative for controlling cacao WBD using live yeast biocides.Item On‐site produced enzyme cocktails for saccharifcation and ethanol production from sugarcane bagasse fractionated by hydrothermal and alkaline pretreatments.(2022) Rodrigues, Patrísia de Oliveira; Barreto, Elisa da Silva; Brandão, Rogélio Lopes; Gurgel, Leandro Vinícius Alves; Pasquini, Daniel; Baffi, Milla AlvesEnzymatic blends produced by fungal monocultures and consortia cultured in solid-state fermentation (SSF), using sugarcane bagasse (SB) and wheat bran as substrates (1:1, w/w), were evaluated for saccharifcation of sugarcane bagasse pretreated by autohydrolysis (hydrothermal pretreatment—HP) and alkaline delignifcation (HP-Soda). The highest glucose releases were obtained after saccharifcations of SB pretreated by HP using enzyme cocktails produced by Aspergillus niger and by the consortium among A. fumigatus, Ganoderma lucidum and Trametes versicolor, with 10.8 and 9.8 g L−1, respectively. For SB pretreated by HP-Soda, the hydrolysate 10 (extract from A. niger, G. lucidum and Pleurotus ostreatus consortium) achieved maximal glucose concentration (11.92 g L−1). After alcoholic fermentation of the hydrolysates, the greatest ethanol yield in relation to the maximum theoretical yield (60.8%) was obtained in the fermentation of hydrolysate 1 (A. niger) obtained from SB pretreated by HP-Soda. These results demonstrated that on-site produced enzyme cocktails can be applied for sac- charifcation of pretreated sugarcane bagasse and also contribute to cost reduction of bioconversion processes.Item Small scale screening of yeast strains enables high-throughput evaluation of performance in lignocellulose hydrolysates.(2020) Dijk, Marlous van; Trollmann, Ignis; Saraiva, Margarete Alice Fontes; Brandão, Rogélio Lopes; Olsson, Lisbeth; Nygård, YvonneSecond generation biorefineries demand efficient lignocellulosic hydrolysate fermenting strains and recent advances in strain isolation and engineering have progressed the bottleneck in developing production hosts from generation of strains into testing these under relevant conditions. In this paper, we introduce a methodology for high-throughput analysis of yeast strains directly in lignocellulosic hydrolysates. The Biolector platform was used to assess aerobic and anaerobic growth of 12 Saccharomyces cerevisiae strains and their ΔPdr12 mutants in wheat straw hydrolysate. The strains evaluated included lab, industrial and wild type strains and the screening could capture significant differences in growth and ethanol production among the strains. The methodology was also demonstrated with corn stover hydrolysate and the results were in line with shake flask cultures. Our study demonstrates that growth in lignocellulosic hydrolysates could be rapidly monitored using 1 ml cultures and that measuring growth and product formation under relevant conditions are crucial for evaluating strain performance.Item Fractionation of sugarcane bagasse using hydrothermal and advanced oxidative pretreatments for bioethanol and biogas production in lignocellulose biorefineries.(2019) Bittencourt, Gustavo Amaro; Barreto, Elisa da Silva; Brandão, Rogélio Lopes; Baeta, Bruno Eduardo Lobo; Gurgel, Leandro Vinícius AlvesThe fractionation of sugarcane bagasse (SB) by hydrothermal pretreatment (HP, autohydrolysis) followed by alkaline extraction (AE) and advanced oxidative pretreatment (AOP) for production of second-generation ethanol and biogas was investigated. The AOP of SB was optimized using a Doehlert design, varying the applied H2O2 load, liquid-to-solid ratio (LSR), and time. The responses evaluated were yield (Y), residual cellulose (RC), delignification (DE), and enzymatic conversion (EC). The AE of SB pretreated by HP led to 61.8% DE (using 0.2 mol L−1 NaOH). This high lignin removal enabled substantial savings of H2O2 in the AOP. The optimized AOP conditions led to 78% Y, 82.2% RC, 42.7% DE, and 88.9% EC (overall glucose yield of 60.9%). Fermentation of the enzymatic hydrolysate with Saccharomyces cerevisiae yielded 190.8 Lethanol tonSB−1. Biogas production by anaerobic digestion of residual liquid streams of the pretreatment steps yielded 27.46 NLCH4 kgSB−1. An energy balance was estimated for the SB fractionation.Item High‑affinity transport, cyanide‑resistant respiration, and ethanol production under aerobiosis underlying efficient high glycerol consumption by Wickerhamomyces anomalus.(2019) Cunha, Aureliano Claret da; Gomes, Lorena Soares; Santos, Fernanda Godoy; Oliveira, Fábio Faria; Teixeira, Janaina Aparecida; Sampaio, Geraldo Magela Santos; Trópia, Maria José Magalhães; Castro, Ieso de Miranda; Lucas, Cândida Manuel Ribeiro Simões; Brandão, Rogélio LopesWickerhamomyces anomalus strain LBCM1105 was originally isolated from the wort of cachaça (the Brazilian fermented sugarcane juice-derived Brazilian spirit) and has been shown to grow exceptionally well at high amounts of glycerol. This paramount residue from the biodiesel industry is a promising cheap carbon source for yeast biotechnology. The assessment of the physiological traits underlying the W. anomalus glycerol consumption ability in opposition to Saccharomyces cerevisiae is presented. A new WaStl1 concentrative glycerol-H+ symporter with twice the affinity of S. cerevisiae was identified. As in this yeast, WaSTL1 is repressed by glucose and derepressed/induced by glycerol but much more highly expressed. Moreover, LBCM1105 aerobically growing on glycerol was found to produce ethanol, providing a redox escape to compensate the redox imbalance at the level of cyanide-resistant respiration (CRR) and glycerol 3P shuttle. This work is critical for understanding the utilization of glycerol by non-Saccharomyces yeasts being indispensable to consider their industrial application feeding on biodiesel residue.Item Sugar transport systems in Kluyveromyces marxianus CCT 7735.(2019) Silveira, Fernando Augusto da; Diniz, Raphael Hermano Santos; Sampaio, Geraldo Magela Santos; Brandão, Rogélio Lopes; Silveira, Wendel Batista da; Castro, Ieso de MirandaThe pattern of glucose repression in most Kluyveromyces marxianus strains does not correlate with fermentative behaviour; however, glucose repression and fermentative metabolism appear to be linked to the kinetics of sugar uptake. In this work, we show that lactose transport in K. marxianus CCT 7735 by lactose-grown cells is mediated by a low-affinity H+-sugar symporter. This system is glucose repressed and able to transport galactose with low affinity. We also observed the activity of a distinct lactose transporter in response to raffinose. Regarding glucose uptake, specificities of at least three low-affinity systems rely on the carbon source available in a given growth medium. Interestingly, it was observed only one high-affinity system is able to transport both glucose and galactose. We also showed that K. marxianus CCT 7735 regulates the expression of sugar transport systems in response to glucose availability.Item Cachaça yeast strains : alternative starters to produce beer and bioethanol.(2018) Araújo, Thalita Macedo; Souza, Magalhaes Teixeira de; Diniz, Raphael Hermano Santos; Yamakawa, Celina Kiyomi; Soares, Lauren Bergmann; Lenczak, Jaciane Lutz; Oliveira, Juliana Velasco de Castro; Goldman, Gustavo Henrique; Barbosa, Edilene Alves; Campos, Anna Clara Silva; Castro, Ieso de Miranda; Brandão, Rogélio LopesThis work was performed to verify the potential of yeast strains isolated from cachaça distilleries for two specific biotechnological applications: beer and bioethanol production. In the beer production, the strains were tested for characteristics required in brewery practices, such as: capacity to ferment maltose and maltotriose, ability to grow at lowest temperatures, low H2S production, and flocculation profile. Among the strains tested, two of them showed appropriate characteristics to produce two different beer styles: lager and ale. Moreover, both strains were tested for cachaça production and the results confirmed the capacity of these strains to improve the quality of cachaça. In the bioethanol production, the fermentation process was performed similarly to that used by bioethanol industries: recycling of yeast biomass in the fermentative process with sulfuric acid washings (pH 2.0). The production of ethanol, glycerol, organic acids, dry cell weight, carbohydrate consumption, and cellular viability were analyzed. One strain presented fermentative parameters similar to PE2, industrial/commercial strain, with equivalent ethanol yields and cellular viability during all fermentative cycles. This work demonstrates that cachaça distilleries seem to be an interesting environment to select new yeast strains to be used in biotechnology applications as beer and bioethanol production.Item Identification of novel alleles conferring superior production of rose flavor phenylethyl acetate using polygenic analysis in yeast.(2017) Carvalho, Bruna Trindade de; Holt, Sylvester; Souffriau, Ben; Brandão, Rogélio Lopes; Moreno, Maria Remédios Foulquié; Theveleina, Johan M.Flavor compound metabolism is one of the last areas in metabolism where multiple genes encoding biosynthetic enzymes are still unknown. A major challenge is the involvement of side activities of enzymes having their main function in other areas of metabolism. We have applied pooled-segregant whole-genome sequence analysis to identify novel Saccharomyces cerevisiae genes affecting production of phenylethyl acetate (2-PEAc). This is a desirable flavor compound of major importance in alcoholic beverages imparting rose- and honey-like aromas, with production of high 2-PEAc levels considered a superior trait. Four quantitative trait loci (QTLs) responsible for high 2-PEAc production were identified, with two loci each showing linkage to the genomes of the BTC.1D and ER18 parents. The first two loci were investigated further. The causative genes were identified by reciprocal allele swapping into both parents using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. The superior allele of the first major causative gene, FAS2, was dominant and contained two unique single nucleotide polymorphisms (SNPs) responsible for high 2-PEAc production that were not present in other sequenced yeast strains. FAS2 encodes the alpha subunit of the fatty acid synthetase complex. Surprisingly, the second causative gene was a mutant allele of TOR1, a gene involved in nitrogen regulation. Exchange of both superior alleles in the ER18 parent strain increased 2-PEAc production 70%, nearly to the same level as in the best superior segregant. Our results show that polygenic analysis combined with CRISPR/ Cas9-mediated allele exchange is a powerful tool for identification of genes encoding missing metabolic enzymes and for development of industrial yeast strains generating novel flavor profiles in alcoholic beverages.Item Lpx1p links glucose-induced calcium signaling and plasma membrane H+-ATPase activation in Saccharomyces cerevisiae cells.(2017) Castanheira, Diogo Dias; Santana, Eduardo Perovano; Santos, Fernanda Godoy; Diniz, Raphael Hermano Santos; Oliveira, Fábio Faria; Pereira, Renata Rebeca; Trópia, Maria José Magalhães; Castro, Ieso de Miranda; Brandão, Rogélio LopesIn yeast, as in other eukaryotes, calcium plays an essential role in signaling transduction to regulate different processes. Many pieces of evidence suggest that glucose-induced activation of plasma membrane H+-ATPase, essential for yeast physiology, is related to calcium signaling. Until now, it was not identified any protein that could be regulated by calcium in this context. Lpx1p, a serine-protease that is also involved in the glucose-induced activation of the plasma membrane H+-ATPase activation, could be a candidate to respond to intracellular calcium signaling involved in this process. In this work, and by using different approaches, we showed many pieces of evidence suggesting that the requirement of calcium signaling for activation of the plasma membrane H+-ATPase is due to its requirement for activation of Lpx1p. According to the current model, activation of Lpx1p would cause hydrolysis of an acetylated tubulin that keeps the plasma membrane H+-ATPase in an inactive state. Therefore, after its activation, Lpx1p would hydrolyze the acetylated tubulin making the plasma membrane H+-ATPase phosphorylation accessible for at least one protein kinase.Item New lager brewery strains obtained by crossing techniques using cachaça (Brazilian Spirit) yeasts.(2017) Figueiredo, Bruna Inez Carvalho de; Saraiva, Margarete Alice Fontes; Pimenta, Paloma Patrick de Souza; Testasicca, Miriam Conceição de Souza; Sampaio, Geraldo Magela Santos; Cunha, Aureliano Claret da; Afonso, Luís Carlos Crocco; Queiroz, Marisa Vieira de; Castro, Ieso de Miranda; Brandão, Rogélio LopesThe development of hybrids has been an effective approach to generate novel yeast strains with optimal technological profile for use in beer production. This study describes the generation of a new yeast strain for lager beer production by direct mating between two Saccharomyces cerevisiae strains isolated from cachaça distilleries: one that was strongly flocculent, and the other with higher production of acetate esters. The first step in this procedure was to analyze the sporulation ability and reproductive cycle of strains belonging to a specific collection of yeasts isolated from cachaça fermentation vats. Most strains showed high rates of sporulation, spore viability, and homothallic behavior. In order to obtain new yeast strains with desirable properties useful for lager beer production, we compare haploid-to-haploid and diploid-to-diploid mating procedures. Moreover, an assessment of parental phenotype traits showed that the segregant diploid C2-1d generated from a diploid-to-diploid mating experiment showed good fermentation performance at low temperature, high flocculation capacity, and desirable production of acetate esters that was significantly better than that of one type lager strain. Therefore, strain C2-1d might be an important candidate for the production of lager beer, with distinct fruit traces and originating using a non-genetically modified organism (GMO) approach.