Metabolism of lactose by Kluyveromyces marxianus UFV-3 and Kluyveromyces lactis JA6

O metabolismo fermentativo de lactose em Kluyveromyces marxianus UFV-3 foi comparado ao de Kluyveromyces lactis JA6 em aerobiose e hipoxia por meio da análise de cinéticas de crescimento, consumo de substrato, produção de etanol e formação de biomassa, em função da concentração de lactose. Determinou-se ainda a expressão gênica e atividade de enzimas-chave do metabolismo de lactose em aerobiose e hipoxia. Nas fermentações conduzidas em aerobiose e hipoxia em concentrações de lactose de 0,25 a 64 mM, K. marxianus UFV-3 apresentou maior velocidade específica de crescimento e maior concentração de biomassa após 24 horas de cultivo, em relação a K. lactis JA6. Nas fermentações com 64 mM de lactose em aerobiose e hipoxia K. marxianus UFV-3 mostrou maior rendimento de etanol por lactose que K. lactis JA6, demonstrando que alta concentração de substrato favorece metabolismo fermentativo em K. marxianus UFV-3. Sob aerobiose K. marxianus UFV-3 possui maior atividade de piruvato desidrogenase que K. lactis JA6, justificando a maior formação de biomassa e maior velocidade de crescimento verificada em K. marxianus UFV-3 em aerobiose. Sob hipoxia as maiores atividades de β-galactosidase e de piruvato descarboxilase de K. marxianus UFV-3 em relação à K. lactis JA6 parecem ser os fatores que contribuem para a maior formação de etanol de K. marxianus UFV-3 quando comparado a K. lactis JA6. Em hipoxia K. marxianus apresentou maior expressão de genes que codificam enzimas para a via de Leilor, para a enzima β-gal e piruvato descarboxilase quando comparado à aerobiose. Indicando que estes genes são importantes para o metabolismo de lactose em hipoxia. Já em K. lactis JA6 nenhum destes genes apresentou expressão aumentada em hipoxia. Tanto a maior expressão de genes que codificam enzimas chaves do metabolismo de lactose quanto à maior atividade enzimática parecem ser os responsáveis pela capacidade fermentativa de K. marxianus UFV-3.The fermentative metabolism of lactose in Kluyveromyces marxianus UFV-3 was compared to the Kluyveromyces lactis JA6 under aerobic and hypoxia through the analysis of kinetics of growth, consumption of substrate, production of ethanol and formation of biomass, depending on the concentration of lactose. It was determinated the gene expression and activity of key enzymes of metabolism of lactose under aerobic and hypoxia. In fermentations carried out under aerobic and hypoxia in lactose concentrations of 0.25 to 64 mM, K. marxianus UFV-3 showed higher specific growth rate and higher concentration of biomass after 24 hours of cultivation, for K. lactis JA6. In fermentations with 64 mM lactose under aerobic and hypoxia K. marxianus UFV-3 showed higher yield to ethanol by K. lactis JA6, demonstrating that high concentrations of substrate metabolism favors fermentation in K. marxianus UFV-3. Under aerobic K. marxianus UFV-3 has more activity of pyruvate dehydrogenase that K. lactis JA6, justifying the formation of higher biomass and higher growth rate observed in K. marxianus UFV-3. Under hypoxia the highest activities of pyruvate decarboxylase and β- galactosidase of K. marxianus UFV-3 in relation to K. lactis JA6 seem to be the factors that contribute to the increased formation of ethanol in K. marxianus UFV-3 when compared to K. lactis JA6. In hypoxia K. marxianus showed higher expression of genes that encode enzymes β-galactosidase, pyruvate decarboxylase and the Leilor pathway for when compared to aerobic. Indicating that these genes are important for the metabolism of lactose in hypoxia. Already in K. lactis JA6 none of these genes showed increased expression in hypoxia. It appears the higher expression of genes encoding key enzymes of metabolism of lactose as the major enzymatic activity is responsible for fermentation potential of K. marxianus UFV-3.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Optimization of the fermentation and secretome analysis of Kluyveromyces marxianus UFV-3 in medium containing lactose in different forms of cultivation

Kluyveromyces marxianus UFV-3 é uma levedura que possui metabolismo respiro-fermentativo, ou seja, a fermentação e respiração coexistem, contudo a via fermentativa ou respiratória pode ser favorecida dependendo da concentração de oxigênio e carboidrato no meio de cultivo. Considerando esta característica das leveduras do gênero Kluyveromyces o objetivo deste trabalho foi otimizar a produção de etanol a partir de permeado de soro de queijo (PSQ) e caracterizar o secretoma de Kluyveromyces marxianus UFV-3, tendo em vista o uso desta levedura como uma hospedeira para produção de proteínas heterólogas. Para otimizar a produção de etanol em PSQ foi utilizada a metodologia de superfície resposta (MSR) com delineamento composto central rotacionado (DCCR) avaliando os efeitos de pH (4,5-6,5), temperatura (30-45 ºC), concentração de lactose (50-250 g L-1) e biomassa celular seca (1-2 g L-1). Foram realizadas 29 fermentações em hipoxia além de 7 fermentações para validar a equação obtida na MSR. Temperatura foi o fator mais significante na produção de etanol, seguido de pH, biomassa celular e concentração de lactose. As condições para produção de etanol com rendimentos superiores a 90% foram: temperatura entre 33,3-38,3 ºC, pH entre 4,7-5,7, biomassa celular entre 1,26-1,68 g L-1 e concentração de lactose entre 50-108 g L-1. A equação gerada no processo de otimização foi validada podendo, deste modo, ser utilizada para futuros processos de escalonamento da produção de etanol utilizando K. marxianus UFV-3. Para analisar como as condições de cultivo influenciam a secreção de proteínas de K. marxianus UFV-3, esta levedura foi cultivada em batelada, batelada alimentada (taxa de alimentação inicial Do - 0.05 e 0.1 h-1) e cultura contínua (diluição - D - 0.1 e 0.3 h-1). Os sobrenadantes dos cultivos foram analisados em SDS-PAGE e em todos os cultivos observou-se baixa diversidade de proteínas secretadas. Entretanto nos cultivos em batelada alimentada (Do=0.05 h-1) e cultivo contínuo (D=0.1 h-1) detectou-se uma maior concentração e diversidade de proteínas que nos demais cultivos, além de, em ambos os cultivos, obtivemos uma proteína secretada em maior proporção que as demais. Para identificar as proteínas do secretoma K. marxianus UFV-3 foi cultivada em cultura contínua (D=0.1 h-1) devido à reprodutibilidade deste método, além de, maior controle e homogeneidade dos cultivos. As amostras dos cultivos contínuos foram fracionadas por tamanho em um gel de digestão seguido de separação reversa dos peptídeos em um sistema de nano-LC e posterior identificação dos peptídeos em MALDI-TOF/TOF. De um total de nove proteínas, oito proteínas foram identificadas como sendo estruturais ou de membrana e a proteína que foi diferencialmente secretada no meio foi identificada como endopoligalacturonase endoPG (EC: 3.2.1.15). EndoPG é uma proteína de interesse biotecnológico e foi caracterizada com atividade ótima na temperatura de 59,5 ºC e no pH 5,1. Além disso, verificou-se que esta enzima não é secretada em meio contendo glicose, por outro lado endoPG é secretada em meio contendo lactose, galactose e principalmente em meio contendo glicerol.Kluyveromyces marxianus UFV-3 is yeast that has respirofermentative metabolism, i.e., respiration and fermentation coexists, however fermentative or respiratory pathway can be favored depending on the oxygen and carbohydrate concentration in the culture medium. Considering this characteristic of yeasts of the genus Kluyveromyces the objective of this study was to optimize the production of ethanol from cheese whey permeate (CWP) and characterize the secretome of K. marxianus UFV-3 in view of the use of this yeast as a host for producing heterologous proteins. To optimize the production of ethanol we used the response surface method (RSM) with a central composite rotational design (CCRD) to evaluate the effects of pH (4.5-6.5), temperature (30-45 ºC), lactose concentration (50-250 g L-1), and dry cell mass concentration (1-2 g L-1). We performed 29 fermentations under hypoxia in CWP and 7 fermentations for the validation of the equation obtained via RSM. Temperature was the most significant factor in optimizing ethanol production, followed by pH, cell biomass concentration, and lactose concentration. The conditions for producing ethanol at yields above 90% were as follows: temperature between 33.3-38.5 ºC, pH between 4.7-5.7, dry cell mass concentration between 1.26-1.68 g L-1, and lactose concentration between 50-108 g L-1. The equation generated from the optimization process was validated and exhibited excellent bias and accuracy values for the future use of this model in scaling up the fermentation process. To analyze how culture conditions influencing protein secretion in K. marxianus UFV-3, this yeast was cultivated in five different conditions: batch, fed-batch (initial feed rate of 0.05 and 0.1 h-1) and continuous culture (0.3 and 0.1 h-1) in medium containing lactose. The supernatant of the cultures were analyzed on SDSPAGE and all the samples had low diversity of proteins. However, in continuous cultures with dilution of 0.1 h-1 and in fed batch 0.05 h-1 detected a higher concentration and diversity of proteins in other cultures, and in both cultures, we obtained a protein secreted in greater proportion than the others. To identify proteins secretome of K. marxianus was cultivated in continuous culture because it has more reproducible results and control of fermentation parameters when compared with fed batch. Samples of the continuous culture supernatant were subjected to protein size fractioning and in-gel digestion, followed by reversed-phase peptide separation in the nano-LC system and subsequent peptide identification by MALDI-TOF/TOF. Eight proteins were identified as structural proteins or membrane proteins, and the protein was secreted differentially was identified as an endopolygalacturonase - endoPG - (EC 3.2.1.15). EndoPG was characterized with optimum activity enzymatic at a temperature of 59.5 ºC and pH 5.1. Furthermore, it was found that apparently is repressed by glucose and shows intense secretion in batch cultures in medium containing glycerol as the carbon source, and cultures on lactose and galactose.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Screening of yeasts isolated from brazilian environments for the 2-Phenylethanol (2-PE) production

Phenylethanol 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

Screening of yeasts isolated from brazilian environments for the 2-Phenylethanol (2-PE) production

Phenylethanol 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

Sugar transport systems in Kluyveromyces marxianus CCT 7735.

The 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

Lpx1p links glucose-induced calcium signaling and plasma membrane H+-ATPase activation in Saccharomyces cerevisiae cells.

In 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

Optimizing ethanol production by thermotolerant Kluyveromyces marxianus CCT 7735 in a mixture of sugarcane bagasse and ricotta whey

The 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

Lpx1p links glucose-induced calcium signaling and plasma membrane H+-ATPase activation in Saccharomyces cerevisiae cells

In 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, no protein that could be regulated by calcium in this context has been identified. Lpx1p, a serine-protease that is also involved in the glucose-induced activation of the plasma membrane H+-ATPase, could be a candidate to respond to intracellular calcium signaling involved in this process. In this work, by using different approaches, we obtained 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 maintains 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 accessible for phosphorylation by at least one protein kinase

Transcriptome analysis of the thermotolerant yeast Kluyveromyces marxianus CCT 7735 under ethanol stress

The 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

Ethanol stress responses of Kluyveromyces marxianus CCT 7735 revealed by proteomic and metabolomic analyses

Kluyveromyces 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