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The development of clean, sustainable, and effective extraction techniques has received much attention in recent years due to the growing interest in green extraction. For downstream processing (extraction) of biomolecules, natural deep eutectic solvents (NADES) are considered as efficient, adaptable, and significant new green solvents. Their advantages include low-cost constitutive elements such as choline chloride, which serves as a representative hydrogen bond acceptor (HBA), and naturally occurring hydrogen bond donors (HBDs), including different sugars, alcohols, and amides. Because of their stability, biodegradability, non-toxicity, and low volatility, NADES are of particular interest and are considered as "green solvents" within the context of green chemistry. The aqueous two-phase system (ATPS) based on NADES was investigated as a possible extraction medium for the purification of Aspergillus oryzae lipase from raw liquid enzyme formulations. Choline-chloride was selected as the hydrogen bond acceptor while fructose, glucose, glycerol, malonic acid, urea, thiourea and ethylene glycole were considered as hydrogen bond donors. After the NADESs were prepared and characterized (pH, density, and viscosity) the σ- profile for each NADES was determined. The extraction process was carried out in a batch extractor and the extraction efficiency and enzyme activity in each NADES based aqueous two phase system (ATPS) were determined. Based on the experimental results and σ-profiles, a model for the prediction of enzyme activity and extraction efficiency in different ATPSs was developed using the COSMO-RS program. The obtained model was validated on a set of independent experiments performed with ATPS based on different NADES.

The nuclear RNA exosome is a conserved complex involved in 3'-5' RNA degradation and processing in eukaryotic cells. In yeast, its 9-subunit structural core and catalytic subunit Dis3 are essential for viability, while the absence of the second catalytic subunit Rrp6 is inviable only at high temperature. In this work, we show that RNA exosome mutants undergo osmoremedial cell lysis at high temperature or upon treatment with cell wall stressors (published in Novačić A et al. (2021) Mol. Biol. Cell 32(5), 363-375). The catalytic subunit Dis3 provides exoribonuclease catalytic activity needed for maintenance of cell wall stability in S. cerevisiae and is supported by a non-catalytic function of the Rrp6 subunit and the RNA exosome cofactors Rrp47 and the TRAMP complex. Moreover, we identify the mechanism leading to cell wall destabilization, by showing that genes encoding proteins involved in the early steps of protein glycosylation pathway are downregulated in RNA exosome mutants at high temperature, through mechanisms involving increased accumulation of non-coding RNAs transcribed at their gene loci. Consistent with this, overexpression of the essential enzyme Psa1, which catalyzes the synthesis of the mannosylation precursor, suppresses temperature sensitivity and aberrant morphology of these cells. Additionally, we show that the reason why the cell wall instability phenotype of RNA exosome mutants is most pronounced for the commonly used W303 laboratory strain is a mutation that inactivates the SSD1 gene, which encodes an RNA-binding protein involved in regulating the translation of cell wall-related transcripts.

RNA-vezujući protein Ssd1 regulira ekspresiju gena koji kodiraju za proteine uključene u stabilizaciju stanične stijenke kvasca. U očuvanju integriteta stanične stijenke bitnu ulogu ima i RNA egzosom, kompleks koji procesira i degradira RNA supstrate egzoribonukleaznom aktivnošću katalitičkih podjedinica Dis3 i Rrp6. Stanice mutanata rrp6 pokazuju temperaturnu osjetljivost koja je posljedica kompromitirane stabilnosti stanične stijenke, a ovaj fenotip je najizraženiji kod linije sojeva kvasca W303. Budući da sojevi ove linije nose nefunkcionalnu inačicu alela koji kodira za Ssd1 (ssd1-d), ispitan je utjecaj ekspresije funkcionalnog alela SSD1-V s odgovarajućeg genomskog lokusa i centromernog plazmida u stanicama mutanata W303 rrp6 i dis3 exo-. Ekspresija SSD1-V suprimirala je defekte u rastu i morfologiji stanica ovih mutanata u odnosu na odgovarajuće ssd1-d stanice pri djelovanju povišene temperature i agenasa koji oštećuju staničnu stijenku. Ovi rezultati upućuju da RNA egzosom i Ssd1 djeluju sinergistički pri održavanju integriteta stanične stijenke kvasca. The RNA-binding protein Ssd1 regulates the expression of genes encoding yeast cell wall morphogenesis proteins. Maintenance of yeast cell wall integrity is also mediated by the RNA exosome, a complex which processes and degrades RNA substrates through exoribonuclease activities of its catalytic subunits, Dis3 and Rrp6. Yeast rrp6 mutant cells display a temperature-sensitive phenotype as a result of impaired cell wall stability, which is most pronounced in the W303 genetic background. Since strains of this background harbour a non-functional form of the allele encoding Ssd1 (ssd1-d), in this work we tested the effect of the expression of the active SSD1-V allele from its corresponding genomic locus and from a centromeric plasmid in W303 rrp6 and dis3 exo- mutant cells. Expression of SSD1-V suppressed growth and morphology defects upon exposure to high temperature and cell wall stressors compared with the corresponding ssd1- d cells. These results suggest that RNA exosome and Ssd1 act synergistically during the maintenance of cell wall integrity in yeast.