Isolation of xylose-assimilating yeasts and optimization of xylitol production by a new Meyerozyma guilliermondii strain.

Production of xylitol from lignocellulosic biomass is of interest to modern biorefineries, because this biomass should be processed into a spectrum of chemicals (bio-based products) and not only energy. The isolation of new yeast strains capable of efficiently converting xylose into xylitol and withstanding inhibitors released from biomass hydrolysis can contribute to making its production feasible in biorefineries. Forty-three out of 128 yeast strains isolated from the gut of Passalidae beetles were capable of assimilating xylose as the sole carbon source. Meyerozyma guilliermondii UFV-1 was selected due to its ability to grow and ferment D-xylose in a synthetic medium. This yeast assimilated the broad range of sugars present in lignocellulosic biomass hydrolysates, such as xylose, raffinose, cellobiose, rhamnose, arabinose, and glucose. Its optimum growth conditions were pH 8.0 and a temperature of 30 °C. In concentrations of 0.07 mol/L acetic acid, 0.05 mol/L 5-hydroximethylfurfural, and 0.04 mol/L furfural, M. guilliermondii UFV-1 did not grow. Maximum xylitol production in aerobiosis and hypoxia were 51.88 and 27.73 g/L, respectively. Under aerobic condition, xylose concentration and agitation rate were the factors which were statistically significant, while only the agitation rate was significant in hypoxia. We fitted a response surface (RS) that estimated the best agitation rate (113.33 rpm) and xylose concentration (90 g/L) for maximum xylitol production in aerobiosis. Therefore, M. guilliermondii UFV-1 displays potential for being used for xylitol production in biorefineries.

Assessment of ethanol tolerance of Kluyveromyces marxianus CCT 7735 selected by adaptive laboratory evolution.

Kluyveromyces marxianus CCT 7735 shows potential for producing ethanol from lactose; however, its low ethanol tolerance is a drawback for its industrial application. The first aim of this study was to obtain four ethanol-tolerant K. marxianus CCT 7735 strains (ETS1, ETS2, ETS3, and ETS4) by adaptive laboratory evolution. The second aim was to select among them the strain that stood out and to evaluate metabolic changes associated with the improved ethanol tolerance in this strain. The ETS4 was selected for displaying a specific growth rate higher than the parental strain under ethanol stress (122%) and specific ethanol production rate (0.26 g/g/h) higher than those presented by the ETS1 (0.22 g/g/h), ETS2 (0.17 g/g/h), and ETS3 (0.17 g/g/h) under non-stress condition. Further analyses were performed with the ETS4 in comparison with its parental strain in order to characterize metabolic changes. Accumulation of valine and metabolites of the citric acid cycle (isocitric acid, citric acid, and cis-aconitic acid) was observed only in the ETS4 subjected to ethanol stress. Their accumulation in this strain may have been important to increase ethanol tolerance. Furthermore, the contents of fatty acid methyl esters and ergosterol were higher in the ETS4 than in the parental strain. These differences likely contributed to enhance ethanol tolerance in the ETS4. KEY POINTS: • K. marxianus ethanol-tolerant strains were selected by adaptive laboratory evolution. • Valine and metabolites of the TCA cycle were accumulated in the ETS4. • High contents of fatty acids and ergosterol contributed to enhance ethanol tolerance.

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.

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.

Cachaça yeast strains: alternative starters to produce beer and bioethanol.

This 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.

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.

Biotechnological potential of yeast isolates from cachaça: the Brazilian spirit.

This study identified phenotypic traits appropriate for biotechnological applications of 118 yeasts isolated from cachaça distilleries. Different properties were verified: capacity to use alternative carbon sources; ability to tolerate high concentrations of sucrose, ethanol, methanol, aluminum and zinc as well as different pH values and foam production. Pichia guilliermondii and Pichia anomala strains were identified as the most promising ones for application in the second-generation biofuel industry, showing ability to grow on high glycerol concentrations. Other isolates, identified as Saccharomyces cerevisiae, produced bioethanol comparable to the industrial strains, and were therefore ideal for use in the first-generation ethanol industry. Some of these strains also showed high resistance to aluminum, as observed in sugarcane juice, and to inter-cycle washings with diluted sulphuric acid, as performed in the industrial bioethanol production process. In summary, yeast isolates from cachaça distilleries displayed robustness and phenotypic plasticity, which makes them interesting for biotechnological applications.

Genomic analysis and lactose transporter expression in Kluyveromyces marxianus CCT 7735.

Kluyveromyces marxianus CCT 7735 has been used to produce ethanol, aromatic compounds, enzymes and heterologous proteins besides assimilates lactose as carbon source. Its genome has 10.7 Mb and encodes 4787 genes distributed in 8 nuclear chromosomes and one mitochondrial. Contrary to Kluyveromyces lactis, which has a unique LAC12 gene (encodes lactose permease), K. marxianus possesses four. The presence of degenerated copies and Solo-LTRs related to retrotransposon TKM close to the LAC12 genes in K. marxianus indicates ectopic recombinations. The Lac12 permeases of K. marxianus and K. lactis are conserved, however the conservation is higher between the copy of the left side of the chromosome three and the unique copy of K. lactis, indicating that this copy is the ancestor. The expression of the four LAC12 genes occurred in aerobiosis and hypoxia. Notably, the high lactose consumption in hypoxia seems to be related to the high expression of the LAC12 genes.