RESUMO
Yeast-based bioethanol production from lignocellulosic hydrolysates (LH) is an attractive and sustainable alternative for biofuel production. However, the presence of acetic acid (AA) in LH is still a major problem. Indeed, above certain concentrations, AA inhibits yeast fermentation and triggers a regulated cell death (RCD) process mediated by the mitochondria and vacuole. Understanding the mechanisms involved in AA-induced RCD (AA-RCD) may thus help select robust fermentative yeast strains, providing novel insights to improve lignocellulosic ethanol (LE) production. Herein, we hypothesized that zinc vacuolar transporters are involved in vacuole-mediated AA-RCD, since zinc enhances ethanol production and zinc-dependent catalase and superoxide dismutase protect from AA-RCD. In this work, zinc limitation sensitized wild-type cells to AA-RCD, while zinc supplementation resulted in a small protective effect. Cells lacking the vacuolar zinc transporter Zrt3 were highly resistant to AA-RCD, exhibiting reduced vacuolar dysfunction. Moreover, zrt3Δ cells displayed higher ethanol productivity than their wild-type counterparts, both when cultivated in rich medium with AA (0.29 g L-1 h-1 versus 0.11 g L-1 h-1) and in an LH (0.73 g L-1 h-1 versus 0.55 g L-1 h-1). Overall, the deletion of ZRT3 emerges as a promising strategy to increase strain robustness in LE industrial production.
RESUMO
Different positive pharmacological effects have been attributed to the natural product resveratrol (RSV), including antioxidant, antiaging, and cancer chemopreventive properties. However, its low bioavailability and rapid metabolic degradation has led to the suspicion that many of the biological activities of this compound observed in vitro may not be attainable in humans. To improve its bioavailability and pharmacokinetic profile, attempts have been made to encapsulate RSV into lipid-based nanocarrier systems. Here, the dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) liposomal system (1:2) loaded with RSV revealed appropriate characteristics for drug release purposes: reduced size for cellular uptake (157 ± 23 nm), stability up to 80 days, positive surface charge (ζ ≈ +40 mV), and a controlled biphasic release of RSV from the lipid nanocarriers over a period of almost 50 h at pH 5.0 and 7.4. Moreover, the encapsulation efficiency of the nanocarrier ranged from 70% to 92% and its RSV loading capacity from 9% to 14%, when [RSV] was between 100 and 200 µM. The partition coefficient ( Kp) of RSV between lipid and aqueous phase was log Kp = 3.37 ± 0.10, suggesting moderate to high lipophilicity of this natural compound and reinforcing the lipid nanocarriers' suitability for RSV incorporation. The thermodynamic parameters of RSV partitioning in the lipid nanocarriers at 37 °C (Δ H = 43.76 ± 5.68 kJ mol-1; Δ S = 0.20 ± 0.005 kJ mol-1; and Δ G = -18.46 ± 3.48 kJ mol-1) reflected the spontaneity of the process and the establishment of hydrophobic interactions. The cellular uptake mechanism of the RSV-loaded nanocarriers labeled with the lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was studied in the eukaryotic model system Saccharomyces cerevisiae. Thirty minutes after incubation, yeast cells readily internalized nanocarriers and the spots of blue fluorescence of DPH clustered around the central vacuole in lipid droplets colocalized with the green fluorescence of the lipophilic endocytosis probe FM1-43. Subsequent studies with the endocytosis defective yeast deletion mutant ( end3Δ) and with the endocytosis inhibitor methyl-ß-cyclodextrin supported the involvement of an endocytic pathway. This novel nanotechnology approach opens good perspectives for medical applications.