Changes in Energy Status of Saccharomyces cerevisiae Cells during Dehydration and Rehydration.

Anhydrobiosis is the state of life when cells are exposed to waterless conditions and gradually cease their metabolism. In this study, we determined the sequence of events in Saccharomyces cerevisiae energy metabolism during processes of dehydration and rehydration. The intensities of respiration and acidification of the medium, the amounts of phenyldicarbaundecaborane (PCB-) bound to yeast membranes, and the capabilities of cells to accumulate K+ were assayed using an electrochemical monitoring system, and the intracellular content of ATP was measured using a bioluminescence assay. Mesophilic, semi-resistant to desiccation S. cerevisiae strain 14 and thermotolerant, very resistant to desiccation S. cerevisiae strain 77 cells were compared. After 22 h of drying, it was possible to restore the respiration activity of very resistant to desiccation strain 77 cells, especially when glucose was available. PCB- binding also indicated considerably higher metabolic activity of dehydrated S. cerevisiae strain 77 cells. Electrochemical K+ content and medium acidification assays indicated that permeabilization of the plasma membrane in cells of both strains started almost simultaneously, after 8-10 h of desiccation, but semi-resistant strain 14 cells maintained the K+ gradient for longer and more strongly acidified the medium. For both cells, the fast rehydration in water was less efficient compared to reactivation in the growth medium, indicating the need for nutrients for the recovery. Higher viability of strain 77 cells after rehydration could be due to the higher stability of their mitochondria.

Sustainable green strategy for recovery of glucose from end-of-life euro banknotes.

Usually, Euro banknotes are made from cotton substrates and their waste is disposed of in landfill or is incinerated. In order to valorize the end-of-life euro banknotes (ELEBs), the substrates were used in this research for cellulase production via submerged fungal fermentation (SFF), and the resultant fungal cellulase w s used in ELEBs hydrolysis process for extraction of glucose. The experiments were started by exposing the ELEBs to different types of pretreatments, including milling process, alkali (NaOH/urea solution), and acid leaching to remove any contamination (e.g. dyes) and to decrease the crystallinity of cellulose (the main element in cotton substrate) thus increasing the degradation rate during the fermentation process. The effect of pretreatments on the morphology and chemical composition of ELEBs was observed using Scanning Electron Microscope and Energy Dispersive Spectrometry. Afterwards, Trichoderma reesei-DSM76 was used for cellulase production from the treated ELEBs with high cellulase activity (12.97 FPU/g). The resultant cellulase was upscaled in a bioreactor and used in ELEBs hydrolysis. Finally, the results showed that the optimized pretreatment methods (milling followed by leaching process) significantly improved the cellulase activity and glucose recovery, which was estimated by 96%. According to the obtained results, the developed strategy has a great potential for conversion of ELEBs into a glucose product that could be used in biofuels and bioplastics applications.

Antifungal activity of styrylpyridinium compounds against Candida albicans.

We synthesized a set of 13 new and earlier described styrylpyridinium compounds (N-alkyl styrylpyridinium salts with bromide or tosylate anions) in order to evaluate antifungal activity against C. albicans cells, to assay the possible synergism with fluconazole, and to estimate cytotoxicity to mammalian cells. All compounds were synthesized according to a well-known two-step procedure involving alkylation of γ-picoline with appropriate alkyl bromide and further condensation with substituted benzaldehyde. Compounds with long N-alkyl chains (C18 H37 -C20 H41 ) had no antifungal activity against the cells of all tested C. albicans strains. Other styrylpyridinium compounds were able to inhibit yeast growth at the concentrations of 0.06-16 µg/ml. At fungicidal concentrations, the compound with the CN- group was least toxic to mammalian cells, showed the most effective synergism with fluconazole, and only slightly inhibited the respiration of C. albicans. The compound with the 4'-diethylamino group exhibited the strongest fungicidal properties and effectively blocked the respiration of C. albicans cells. However, toxicity to mammalian cells was also high. Summarizing, the results of our study indicate that styrylpyridinium compounds are promising candidates in the development of new antifungal drugs.

Engineering of sugar transporters for improvement of xylose utilization during high-temperature alcoholic fermentation in Ogataea polymorpha yeast.

BACKGROUND: Xylose transport is one of the bottlenecks in the conversion of lignocellulosic biomass to ethanol. Xylose consumption by the wild-type strains of xylose-utilizing yeasts occurs once glucose is depleted resulting in a long fermentation process and overall slow and incomplete conversion of sugars liberated from lignocellulosic hydrolysates. Therefore, the engineering of endogenous transporters for the facilitation of glucose-xylose co-consumption is an important prerequisite for efficient ethanol production from lignocellulosic hydrolysates. RESULTS: In this study, several engineering approaches formerly used for the low-affinity glucose transporters in Saccharomyces cerevisiae, were successfully applied for earlier identified transporter Hxt1 in Ogataea polymorpha to improve xylose consumption (engineering involved asparagine substitution to alanine at position 358 and replacement of N-terminal lysine residues predicted to be the target of ubiquitination for arginine residues). Moreover, the modified versions of S. cerevisiae Hxt7 and Gal2 transporters also led to improved xylose fermentation when expressed in O. polymorpha. CONCLUSIONS: The O. polymorpha strains with modified Hxt1 were characterized by simultaneous utilization of both glucose and xylose, in contrast to the wild-type and parental strain with elevated ethanol production from xylose. When the engineered Hxt1 transporter was introduced into constructed earlier advanced ethanol producer form xylose, the resulting strain showed further increase in ethanol accumulation during xylose fermentation. The overexpression of heterologous S. cerevisiae Gal2 had a less profound positive effects on sugars uptake rate, while overexpression of Hxt7 revealed the least impact on sugars consumption.