Xylitol production from passion fruit peel hydrolysate: Optimization of hydrolysis and fermentation processes.

The passion fruit peel (PFP) has a high cellulose and hemicellulose content, which can be used to produce fermentable sugars. In this context, this study aims to optimize the release of xylose and the production of xylitol from PFP. The optimized conditions were 0.71 M dilute sulfuric acid and a 21.84-minute treatment, yielding 19.03 g/L of xylose (PFP-1). Different PFP hydrolysates were evaluated to improve xylitol production by the yeast Kluyveromyces marxianus ATCC 36907: PFP-2 (PFP1 treated with Ca(OH)2), PFP-3 (PFP-1 treated with Ca(OH)2 and activated carbon), PFP-4 (PFP-3 with biological elimination of glucose with S. cerevisiae, and concentrated at different xylose concentrations). The applied methods resulted in higher xylitol production (14.97 g/L), when PFP hydrolysate was detoxified with Ca(OH)2, treated with activated charcoal for 1 h, biotreated for glucose removal, and concentrated to 40 g/L of xylose.

Metabolic and biotechnological insights on the analysis of the Pdh bypass and acetate production in the yeast Dekkera bruxellensis.

The advancement of knowledge about the physiology of Dekkera bruxellensis has shown its potential for the production of fuel ethanol very close to the conventional fermenting yeast S. cerevisiae. However, some aspects of its metabolism remain uncovered. In the present study, the respiro-fermentative parameters of D. bruxellensis GDB 248 were evaluated under different cultivation conditions. The results showed that sucrose was more efficiently converted to ethanol than glucose, regardless the nitrogen source, which points out for the industrial efficiency of this yeast in sucrose-based substrate. The blockage of the cytosolic acetate production incremented the yeast fermentative efficiency by 27% (in glucose) and 14% (in sucrose). On the other hand, the presence of nitrate as inducer of acetate production reducing the production of ethanol. Altogether, these results settled the hypothesis that acetate metabolism is the main constraint for ethanol production. Besides, this acetate-generating pathway seems to exert some regulatory action on the flux and distribution of the carbon flowing through the central metabolism. These physiological aspects were corroborated by the relative expression analysis of key genes in the crossroad to ethanol, acetate and biomass formation. All the results were discussed in the light of the industrial potential of this yeast.

Alcohol dehydrogenase 1 participates in the Crabtree effect and connects fermentative and oxidative metabolism in the Zygomycete Mucor circinelloides.

Mucor circinelloides is a dimorphic Zygomycete fungus that produces ethanol under aerobic conditions in the presence of glucose, which indicates that it is a Crabtree-positive fungus. To determine the physiological role of the alcohol dehydrogenase (ADH) activity elicited under these conditions, we obtained and characterized an allyl alcohol-resistant mutant that was defective in ADH activity, and examined the effect of adh mutation on physiological parameters related to carbon and energy metabolism. Compared to the Adh+ strain R7B, the ADH-defective (Adh-) strain M5 was unable to grow under anaerobic conditions, exhibited a considerable reduction in ethanol production in aerobic cultures when incubated with glucose, had markedly reduced growth capacity in the presence of oxygen when ethanol was the sole carbon source, and exhibited very low levels of NAD+-dependent alcohol de-hydrogenase activity in the cytosolic fraction. Further characterization of the M5 strain showed that it contains a 10-bp deletion that interrupts the coding region of the adhl gene. Complementation with the wild-type allele adh1+ by transformation of M5 remedied all the defects caused by the adh1 mutation. These findings indicate that in M. circinelloides, the product of the adh1 gene mediates the Crabtree effect, and can act as either a fermentative or an oxidative enzyme, depending on the nutritional conditions, thereby participating in the association between fermentative and oxidative metabolism. It was found that the spores of M. circinelloides possess low mRNA levels of the ethanol assimilation genes (adl2 and acs2), which could explain their inability to grow in the alcohol.

SNF1 controls the glycolytic flux and mitochondrial respiration.

The switch between mitochondrial respiration and fermentation as the main ATP production pathway through an increase glycolytic flux is known as the Crabtree effect. The elucidation of the molecular mechanism of the Crabtree effect may have important applications in ethanol production and lay the groundwork for the Warburg effect, which is essential in the molecular etiology of cancer. A key piece in this mechanism could be Snf1p, which is a protein that participates in the nutritional response including glucose metabolism. Thus, this work aimed to recognize the role of the SNF1 gene on the glycolytic flux and mitochondrial respiration through the glucose concentration variation to gain insights about its relationship with the Crabtree effect. Herein, we found that SNF1 deletion in Saccharomyces cerevisiae cells grown at 1% glucose, decreased glycolytic flux, increased NAD(P)H concentration, enhanced HXK2 gene transcription, and decreased mitochondrial respiration. Meanwhile, the same deletion increased the mitochondrial respiration of cells grown at 10% glucose. Altogether, these findings indicate that SNF1 is important to respond to glucose concentration variation and is involved in the switch between mitochondrial respiration and fermentation.

Biological diversity of carbon assimilation among isolates of the yeast Dekkera bruxellensis from wine and fuel-ethanol industrial processes.

Dekkera bruxellensis is considered a spoilage yeast in winemaking, brewing and fuel-ethanol production. However, there is growing evidence in the literature of its biotechnological potential. In this work, we surveyed 29 D. bruxellensis isolates from three countries and two different industrial origins (winemaking and fuel-ethanol production) for the metabolization of industrially relevant sugars. The isolates were characterized by the determination of their maximum specific growth rates, and by testing their ability to grow in the presence of 2-deoxy-d-glucose and antimycin A. Great diversity was observed among the isolates, with fuel-ethanol isolates showing overall higher specific growth rates than wine isolates. Preferences for galactose (three wine isolates) and for cellobiose or lactose (some fuel-ethanol isolates) were observed. Fuel-ethanol isolates were less sensitive than wine isolates to glucose catabolite repression (GCR) induction by 2-deoxy-d-glucose. In strictly anaerobic conditions, isolates selected for having high aerobic growth rates were able to ferment glucose, sucrose and cellobiose at fairly high rates without supplementation of casamino acids or yeast extract in the culture medium. The phenotypic diversity found among wine and fuel-ethanol isolates suggests adaptation to these environments. A possible application of some of the GCR-insensitive, fast-growing isolates in industrial processes requiring co-assimilation of different sugars is considered.

A common mechanism explains the induction of aerobic fermentation and adaptive antioxidant response in Phaffia rhodozyma.

BACKGROUND: Growth conditions that bring about stress on Phaffia rhodozyma cells encourage the synthesis of astaxanthin, an antioxidant carotenoid, which protects cells against oxidative damage. Using P. rhodozyma cultures performed with and without copper limitation, we examined the kinetics of astaxanthin synthesis along with the expression of asy, the key astaxanthin synthesis gene, as well as aox, which encodes an alternative oxidase protein. RESULTS: Copper deficiency had a detrimental effect on the rates of oxygen consumption and ethanol reassimilation at the diauxic shift. In contrast, copper deficiency prompted alcoholic fermentation under aerobic conditions and had a favorable effect on the astaxanthin content of cells, as well as on aox expression. Both cultures exhibited strong aox expression while consuming ethanol, but particularly when copper was absent. CONCLUSION: We show that the induction of either astaxanthin production, aox expression, or aerobic fermentation exemplifies the crucial role that redox imbalance plays in triggering any of these phenomena. Based on our own results and data from others, we propose a mechanism that rationalizes the central role played by changes of respiratory activity, which lead to redox imbalances, in triggering both the short-term antioxidant response as well as fermentation in yeasts and other cell types.

Anaplerotic Role of Glucose in the Oxidation of Endogenous Fatty Acids during Dengue Virus Infection.

Dengue virus (DENV) is among the most important human arboviruses and is clinically and experimentally associated with lipid metabolism disorders. Using high-resolution respirometry, we analyzed the metabolic switches induced by DENV in a human hepatic cell line. This experimental approach allowed us to determine the contribution of fatty acids, glutamine, glucose, and pyruvate to mitochondrial bioenergetics, shedding light on the mechanisms involved in DENV-induced metabolic alterations. We found that while infection strongly inhibits glutamine oxidation, it increases the cellular capacity of metabolizing glucose; remarkably, though, this substrate, instead being used as an energy source, performs an anaplerotic role in the oxidation of endogenous lipids. Fatty acids become the main energetic substrate in infected cell, and through the pharmacological modulation of ß-oxidation we demonstrated that this pathway is essential for virus replication. Interestingly, infected cells were much less susceptible to the Crabtree effect, i.e., the glucose-mediated inhibition of mitochondrial oxygen consumption, suggesting that infection favors cellular respiration by increasing ADP availability. IMPORTANCE Dengue virus infection is a major cause of human arbovirosis, for which clinical and experimental evidence supports the idea that liver dysfunction and lipid metabolism disorders are characteristics of severe disease. Analyzing mitochondrial bioenergetics, here we show that infection of hepatic cells with dengue virus favors the cellular capacity of metabolizing glucose, impairing the normal metabolic flexibility that allows the oxidative machinery to switch among the main energetic substrates. However, instead of being used as an energy source, glucose performs an anaplerotic role in the oxidation of endogenous fatty acids, which become the main energetic substrate during infection. Taken together, the results shed light on metabolic mechanisms that may explain the profound alterations in lipid metabolism for severe dengue patients, contributing to the understanding of dengue physiopathology.

In Saccharomyces cerevisiae fructose-1,6-bisphosphate contributes to the Crabtree effect through closure of the mitochondrial unspecific channel.

In Saccharomyces cerevisiae addition of glucose inhibits oxygen consumption, i.e. S. cerevisiae is Crabtree-positive. During active glycolysis hexoses-phosphate accumulate, and probably interact with mitochondria. In an effort to understand the mechanism underlying the Crabtree effect, the effect of two glycolysis-derived hexoses-phosphate was tested on the S. cerevisiae mitochondrial unspecific channel (ScMUC). Glucose-6-phosphate (G6P) promoted partial opening of ScMUC, which led to proton leakage and uncoupling which in turn resulted in, accelerated oxygen consumption. In contrast, fructose-1,6-bisphosphate (F1,6BP) closed ScMUC and thus inhibited the rate of oxygen consumption. When added together, F1,6BP reverted the mild G6P-induced effects. F1,6BP is proposed to be an important modulator of ScMUC, whose closure contributes to the "Crabtree effect".

Production of functional killer protein in batch cultures upon a shift from aerobic to anaerobic conditions

The aim of this work was to study the production of functional protein in yeast culture. The cells of Saccharomyces cerevisiae Embrapa 1B (K+R+) killed a strain of Saccharomyces cerevisiae Embrapa 26B (K-R-)in grape must and YEPD media. The lethal effect of toxin-containing supernatant and the effect of aeration upon functional killer production and the correlation between the products of anaerobic metabolism and the functional toxin formation were evaluated. The results showed that at low sugar concentration, the toxin of the killer strain of Sacch. cerevisiae was only produced under anaerobic conditions . The system of killer protein production showed to be regulated by Pasteur and Crabtree effects. As soon as the ethanol was formed, the functional killer toxin was produced. The synthesis of the active killer toxin seemed to be somewhat associated with the switch to fermentation process and with concomitant alcohol dehydrogenase (ADH) activity.