Meyerozyma caribbica Isolated from Vinasse-Irrigated Sugarcane Plantation Soil: A Promising Yeast for Ethanol and Xylitol Production in Biorefineries.

The production of fuels and other industrial products from renewable sources has intensified the search for new substrates or for the expansion of the use of substrates already in use, as well as the search for microorganisms with different metabolic capacities. In the present work, we isolated and tested a yeast from the soil of sugarcane irrigated with vinasse, that is, with high mineral content and acidic pH. The strain of Meyerozyma caribbica URM 8365 was able to ferment glucose, but the use of xylose occurred when some oxygenation was provided. However, some fermentation of xylose to ethanol in oxygen limitation also occurs if glucose was present. This strain was able to produce ethanol from molasses substrate with 76% efficiency, showing its tolerance to possible inhibitors. High ethanol production efficiencies were also observed in acidic hydrolysates of each bagasse, sorghum, and cactus pear biomass. Mixtures of these substrates were tested and the best composition was found for the use of excess plant biomass in supplementation of primary substrates. It was also possible to verify the production of xylitol from xylose when the acetic acid concentration is reduced. Finally, the proposed metabolic model allowed calculating how much of the xylose carbon can be directed to the production of ethanol and/or xylitol in the presence of glucose. With this, it is possible to design an industrial plant that combines the production of ethanol and/or xylitol using combinations of primary substrates with hydrolysates of their biomass.

A comparative study between Fusarium solani and Neocosmospora vasinfecta revealed differential profile of fructooligosaccharide production.

Fructooligosaccharides (FOS) are fructose-based oligosaccharides employed as additives to improve the food's nutritional and technological properties. The rhizosphere of plants that accumulate fructopolysaccharides as inulin has been revealed as a source of filamentous fungi. These fungi can produce FOS either by inulin hydrolysis or by biosynthesis from sucrose, including unusual FOS with enhanced prebiotic properties. Here, we investigated the ability of Fusarium solani and Neocosmospora vasinfecta to produce FOS from different carbon sources. Fusarium solani and N. vasinfecta grew preferentially in inulin instead of sucrose, resulting in the FOS production as the result of endo-inulinase activities. N. vasinfecta was also able to produce the FOS 1-kestose and 6-kestose from sucrose, indicating transfructosylating activity, absent in F. solani. Moreover, the results showed how these carbon sources affected fungal cell wall composition and the expression of genes encoding for ß-1,3-glucan synthase and chitin synthase. Inulin and fructose promoted changes in fungal macroscopic characteristics partially explained by alterations in cell wall composition. However, these alterations were not directly correlated with the expression of genes related to cell wall synthesis. Altogether, the results pointed to the potential of both F. solani and N. vasinfecta to produce FOS at specific profiles.

The Saccharomyces cerevisiae Ncw2 protein works on the chitin/ß-glucan organisation of the cell wall.

The NCW2 gene was recently described as encoding a GPI-bounded protein that assists in the re-modelling of the Saccharomyces cerevisiae cell wall (CW) and in the repair of damage caused by the polyhexamethylene biguanide (PHMB) polymer to the cell wall. Its absence produces a re-organization of the CW structure that result in resistance to lysis by glucanase. Hence, the present study aimed to extend the analysis of the Ncw2 protein (Ncw2p) to determine its physiological role in the yeast cell surface. The results showed that Ncw2p is transported to the cell surface upon O-mannosylation mediated by the Pmt1p-Pmt2p enzyme complex. It co-localises with the yeast bud scars, a region in cell surface formed by chitin deposition. Once there, Ncw2p enables correct chitin/ß-glucan structuring during the exponential growth. The increase in molecular mass by hyper-mannosylation coincides with the increasing in chitin deposition, and leads to glucanase resistance. Treatment of the yeast cells with PHMB produced the same biological effects observed for the passage from exponential to stationary growth phase. This might be a possible mechanism of yeast protection against cationic biocides. In conclusion, we propose that Ncw2p takes part in the mechanism involved in the control of cell surface rigidity by aiding on the linkage between chitin and glucan layers in the modelling of the cell wall during cell growth.

Gene regulation of the Lactobacillus vini in response to industrial stress in the fuel ethanol production.

The industrial ethanol fermentation imposes several stresses to microorganisms. However, some bacterial species are well adapted and manage to endure these harmful conditions. Lactobacillus vini is one of the most found bacteria in these environments, indicating the existence of efficient tolerance mechanisms. In view of this premise, the present study aimed to describe the tolerance of L. vini to several stressing agents encounter in industrial environments and the genetic components of the stress response. In general, L. vini showed significant tolerance to stressors commonly found in fuel-ethanol fermentations, and only doses higher than normally reached in processes restrained its growth. The lag phase and the growth rate were the most responsive kinetic parameter affected. Gene expression analysis revealed that uspII gene positively responded to all conditions tested, a typical profile of a general stress response gene. In addition, the results also revealed aspects of regulatory modules of co-expressed genes responding to different stresses, and also the similarities of response mechanism with basis in common cellular damages. Altogether, these data contribute to uncover the factors that could favour L. vini in the industrial fermentation which could be shared with other well adapted species and reports the first stress response genes in this bacterium.

The effects of the Ncw2 protein of Saccharomyces cerevisiae on the positioning of chitin in response to cell wall damage.

The recently described NCW2 gene encodes a protein that is assumed to be located in the cell wall (CW). This protein was proposed to participate in the repair of CW damages induced by polyhexamethylene biguanide (PHMB). However, much of the information on the biological function(s) of Ncw2p still remains unclear. In view of this, this study seeks to extend the analysis of this gene in light of the way its protein functions in the Cell Wall Integrity (CWI) mechanism. Deletion of the NCW2 gene led to constitutive overexpression of some key CWI genes and increased chitin deposition in the walls of cells exposed to PHMB. This means the lack of Ncw2p might activate a compensatory mechanism that upregulates glucan CWI genes for cell protection by stiffening the CW. This condition seems to alleviate the response through the HOG pathway and makes cells sensitive to osmotic stress. However, Ncw2p may not have been directly involved in tolerance to osmotic stress itself. The results obtained definitely place the NCW2 gene in the list of CWI genes of S. cerevisiae and indicate that its protein has an auxiliary function in the maintenance of the glucan/chitin balance and ensuring the correct structure of the yeast cell wall.

Lactobacillus vini: mechanistic response to stress by medium acidification.

This work describes the response of Lactobacillusvini, a bacterium found as a contaminant in winemaking and fuel ethanol fermentation processes, to acid stress caused by inorganic or weak organic acids. First, we observed for the first time that bacterial cells become resistant to lysis by lysozyme when submitted to acidic stress. Then, the predicted intracellular acidification can be reversed by the presence of arginine, histidine and glutamine. However, these molecules were not able to reverse the effect of resistance to lysis, indicating the independence of these mechanisms. In general, a reduction in the expression of the main genes involved in the synthesis and deposition of material in the cell wall was observed, whereas the genes involved in the reabsorption of this structure showed increased expression. These data suggested that L. vini responds to the acidification of the medium through early entry into the stationary phase, firing two signals for cell wall remodelling and maintenance of intracellular pHin a coordinated way, most probably by alkalization and the proton extrusion process. If this picture is conserved among lactobacilli, it may not only have an impact on research associated with fermentation processes, but also on that associated with probiotic improvement.

Respiratory deficiency in yeast mevalonate kinase deficient may explain MKD-associate metabolic disorder in humans.

Mevalonate kinase deficiency (MKD) an orphan drug rare disease affecting humans with different clinical presentations, is still lacking information about its pathogenesis; no animal or cell model mimicking the genetic defect, mutations at MVK gene, and its consequences on the mevalonate pathway is available. Trying to clarify the effects of MVK gene impairment on the mevalonate pathway we used a yeast model, the erg12-d mutant strain Saccharomyces cerevisiae (orthologous of MKV) retaining only 10% of mevalonate kinase (MK) activity, to describe the effects of reduced MK activity on the mevalonate pathway. Since shortage of isoprenoids has been described in MKD, we checked this observation using a physiologic approach: while normally growing on glucose, erg12-d showed growth deficiency in glycerol, a respirable carbon source, that was not rescued by supplementation with non-sterol isoprenoids, such as farnesol, geraniol nor geranylgeraniol, produced by the mevalonate pathway. Erg12-d whole genome expression analysis revealed specific downregulation of RSF2 gene encoding general transcription factor for respiratory genes, explaining the absence of growth on glycerol. Moreover, we observed the upregulation of genes involved in sulphur amino acids biosynthesis that coincided with the increasing in the amount of proteins containing sulfhydryl groups; upregulation of ubiquinone biosynthesis genes was also detected. Our findings demonstrated that the shortage of isoprenoids is not the main mechanism involved in the respiratory deficit and mitochondrial malfunctioning of MK-defective cells, while the scarcity of ubiquinone plays an important role, as already observed in MKD patients.

NCW2, a Gene Involved in the Tolerance to Polyhexamethylene Biguanide (PHMB), May Help in the Organisation of ß-1,3-Glucan Structure of Saccharomyces cerevisiae Cell Wall.

In the present work, we provide biological evidences supporting the participation of NCW2 gene in the mechanism responsible for cell tolerance to polyhexamethylene biguanide (PHMB), an antifungal agent. The growth rate of yeast cells exposed to this agent was significantly reduced in ∆ncw2 strain and the mRNA levels of NCW2 gene in the presence of PHMB showed a 7-fold up-regulation. Moreover, lack of NCW2 gene turns yeast cell more resistant to zymolyase treatment, indicating that alterations in the ß-glucan network do occur when Ncw2p is absent. Computational analysis of the translated protein indicated neither catalytic nor transmembrane sites and reinforced the hypothesis of secretion and anchoring to cell surface. Altogether, these results indicated that NCW2 gene codes for a protein which participates in the cell wall biogenesis in yeasts and that Ncw2p might play a role in the organisation of the ß-glucan assembly.

Evidence for a Role for the Plasma Membrane in the Nanomechanical Properties of the Cell Wall as Revealed by an Atomic Force Microscopy Study of the Response of Saccharomyces cerevisiae to Ethanol Stress.

UNLABELLED: A wealth of biochemical and molecular data have been reported regarding ethanol toxicity in the yeast Saccharomyces cerevisiae However, direct physical data on the effects of ethanol stress on yeast cells are almost nonexistent. This lack of information can now be addressed by using atomic force microscopy (AFM) technology. In this report, we show that the stiffness of glucose-grown yeast cells challenged with 9% (vol/vol) ethanol for 5 h was dramatically reduced, as shown by a 5-fold drop of Young's modulus. Quite unexpectedly, a mutant deficient in the Msn2/Msn4 transcription factor, which is known to mediate the ethanol stress response, exhibited a low level of stiffness similar to that of ethanol-treated wild-type cells. Reciprocally, the stiffness of yeast cells overexpressing MSN2 was about 35% higher than that of the wild type but was nevertheless reduced 3- to 4-fold upon exposure to ethanol. Based on these and other data presented herein, we postulated that the effect of ethanol on cell stiffness may not be mediated through Msn2/Msn4, even though this transcription factor appears to be a determinant in the nanomechanical properties of the cell wall. On the other hand, we found that as with ethanol, the treatment of yeast with the antifungal amphotericin B caused a significant reduction of cell wall stiffness. Since both this drug and ethanol are known to alter, albeit by different means, the fluidity and structure of the plasma membrane, these data led to the proposition that the cell membrane contributes to the biophysical properties of yeast cells. IMPORTANCE: Ethanol is the main product of yeast fermentation but is also a toxic compound for this process. Understanding the mechanism of this toxicity is of great importance for industrial applications. While most research has focused on genomic studies of ethanol tolerance, we investigated the effects of ethanol at the biophysical level and found that ethanol causes a strong reduction of the cell wall rigidity (or stiffness). We ascribed this effect to the action of ethanol perturbing the cell membrane integrity and hence proposed that the cell membrane contributes to the cell wall nanomechanical properties.

Transcriptomic response of Saccharomyces cerevisiae for its adaptation to sulphuric acid-induced stress.

In bioethanol production plants, yeast cells are generally recycled between fermentation batches by using a treatment with sulphuric acid at a pH ranging from 2.0 to 2.5. We have previously shown that Saccharomyces cerevisiae cells exposed to sulphuric acid treatment induce the general stress response pathway, fail to activate the protein kinase A signalling cascade and requires the mechanisms of cell wall integrity and high osmolarity glycerol pathways in order to survive in this stressful condition. In the present work, we used transcriptome-wide analysis as well as physiological assays to identify the transient metabolic responses of S. cerevisiae under sulphuric acid treatment. The results presented herein indicate that survival depends on a metabolic reprogramming of the yeast cells in order to assure the yeast cell viability by preventing cell growth under this harmful condition. It involves the differential expression of a subset of genes related to cell wall composition and integrity, oxidation-reduction processes, carbohydrate metabolism, ATP synthesis and iron uptake. These results open prospects for application of this knowledge in the improvement of industrial processes based on metabolic engineering to select yeasts resistant to acid treatment.

Genetic Interaction between HOG1 and SLT2 Genes in Signalling the Cellular Stress Caused by Sulphuric Acid in Saccharomyces cerevisiae.

In fuel ethanol production, recycling of yeast biomass includes treatment of cells with diluted sulphuric acid in order to control bacterial population. However, this strategy might lead to a loss of cell viability, with potential negative consequences to the fermentation yield. In a recent paper we showed that the proteins Slt2 and Hog1 are essential for yeast tolerance to sulphuric acid. As a complement of the aforementioned work, we used DNA microarray technology to search for differentially expressed genes in hog1Δ and slt2Δ deletion mutants after treatment with sulphuric acid. Our results show how Slt2p and Hog1p could coordinate the interplay among protein kinase A (PKA), protein kinase C and high-osmolarity glycerol pathways. Moreover, the SSK22 and KDX1 genes may be part of this network, although their proteins were shown to be non-essential for cell growth/survival at low pH. These proteins might work by enhancing the signal which downregulates the PKA pathway leading to cell cycle arrest, in order to regenerate the integrity of yeast cell wall and cell homeostasis under acid shock.

The resistance of the yeast Saccharomyces cerevisiae to the biocide polyhexamethylene biguanide: involvement of cell wall integrity pathway and emerging role for YAP1.

BACKGROUND: Polyhexamethylene biguanide (PHMB) is an antiseptic polymer that is mainly used for cleaning hospitals and pools and combating Acantamoeba infection. Its fungicide activity was recently shown by its lethal effect on yeasts that contaminate the industrial ethanol process, and on the PE-2 strain of Saccharomyces cerevisiae, one of the main fermenting yeasts in Brazil. This pointed to the need to know the molecular mechanism that lay behind the cell resistance to this compound. In this study, we examined the factors involved in PHMB-cell interaction and the mechanisms that respond to the damage caused by this interaction. To achieve this, two research strategies were employed: the expression of some genes by RT-qPCR and the analysis of mutant strains. RESULTS: Cell Wall integrity (CWI) genes were induced in the PHMB-resistant Saccharomyces cerevisiae strain JP-1, although they are poorly expressed in the PHMB-sensitive Saccharomyces cerevisiae PE2 strain. This suggested that PHMB damages the glucan structure on the yeast cell wall. It was also confirmed by the observed sensitivity of the yeast deletion strains, Δslg1, Δrom2, Δmkk2, Δslt2, Δknr4, Δswi4 and Δswi4, which showed that the protein kinase C (PKC) regulatory mechanism is involved in the response and resistance to PHMB. The sensitivity of the Δhog1 mutant was also observed. Furthermore, the cytotoxicity assay and gene expression analysis showed that the part played by YAP1 and CTT1 genes in cell resistance to PHMB is unrelated to oxidative stress response. Thus, we suggested that Yap1p can play a role in cell wall maintenance by controlling the expression of the CWI genes. CONCLUSION: The PHMB treatment of the yeast cells activates the PKC1/Slt2 (CWI) pathway. In addition, it is suggested that HOG1 and YAP1 can play a role in the regulation of CWI genes.

Polyhexamethyl biguanide can eliminate contaminant yeasts from fuel-ethanol fermentation process.

Industrial ethanol fermentation is a non-sterile process and contaminant microorganisms can lead to a decrease in industrial productivity and significant economic loss. Nowadays, some distilleries in Northeastern Brazil deal with bacterial contamination by decreasing must pH and adding bactericides. Alternatively, contamination can be challenged by adding a pure batch of Saccharomyces cerevisiae-a time-consuming and costly process. A better strategy might involve the development of a fungicide that kills contaminant yeasts while preserving S. cerevisiae cells. Here, we show that polyhexamethyl biguanide (PHMB) inhibits and kills the most important contaminant yeasts detected in the distilleries of Northeastern Brazil without affecting the cell viability and fermentation capacity of S. cerevisiae. Moreover, some physiological data suggest that PHMB acts through interaction with the yeast membrane. These results support the development of a new strategy for controlling contaminant yeast population whilst keeping industrial yields high.