College Papers

Debaryomyces the mitochondrial cytochrome pathway establishing a Radical

Debaryomyces hansenii, a marine, halotolerant and oleaginous yeast, has a branched mitochondrial respiratory chain (Adler L et al., 1985). Besides the canonical pathway composes by complexes I-IV, the electron flux can be driven through alternative routes that include a cyanide-resistant alternative oxidase (AOX), an external-NADH dehydrogenase (NDH2e) and a glycerol phosphate dehydrogenase (MitGPDH) (Cabrera-Orefice A. et al., 2014), Fig. 1.In yeasts, the physiological purpose of alternative oxidoreductases is not entirely known, but probably avoid electron flux congestion on the mitochondrial cytochrome pathway establishing a Radical Oxygen Species-narrowing method (El-Khoury R. et al., 2013). Protection from ROS could be explained due to this redox enzymes maintain a high oxygen consumption without a proton pumping activity, uncoupling the oxidative phosphorylation despite a raised transmembrane potential (Guerrero-Castillo et al., 2011) (Joseph-Horne et al., 2001).In contrast to NDH2e and MitGPDH constitutive expression (Cabrera-Orefice et al., 2014), AOX appears at the stationary growth phase (Veiga et al., 2003). Nevertheless, data from our group suggest that the expression of AOX may also depend on the carbon source added to the culture medium. Therefore, we analysed cyanide-resistant respiration (CRR) in whole yeast cells cultivated on different carbon sources.5. Materials and methods5.1 ChemicalsD-glucose, D-galactose, D-sorbitol, glycerol, malic acid, NaCl, Trizma® base (Tris), ADP, propyl-gallate, and antifoam A were from Sigma Chem Co. Bovine serum albumin was from Millipore. Yeast extract and peptone were from BD Bioxon. DL-lactic acid, H3PO4, NaCN, KCl, and MgCl2 were from J.T. Baker. 5.2 BiologicalsD. hansenii Y7426 strain was stored in YP Galactose 1 M NaCl plate cultures (1% yeast extract, 2% peptone, 2% D-galactose, 1 M NaCl and 2% agar).  5.3 Culture conditionsCells were grown in 150 mL pre-cultures of five different culture mediums (YP Dextrose 0.6 M NaCl, YP Galactose 0.6 M NaCl, YP Succinate 0.6 M NaCl (pH 5.5), YP Lactate 0.6 M NaCl (pH 5.5), and YP Glycerol 0.6 M NaCl) for 24 h under orbital agitation in a shaker at 250 rpm at 30 °C. Later, 750 mL of the same medium was inoculated with each pre-culture. Incubation continued following the same conditions until the cells reached the sought growth stage. 5.4 Growth curvesWe determined the optical density of the cultures during 96 h with a  .Exponential phase develops from 15 until 24 h, and the stationary phase from 48 until 96 h in all the carbon sources. Accordingly, we performed the oxygen consumption measurements with the cells in logarithmic mean (18h) and at 72h of the stationary phase. 5.5 Biomass quantificationYeast cells were harvested from 150 mL of each liquid media (n=3) and washed once with sterile bidistilled water. The samples were dried at 90°C for 24h to report grammes of dry weight/mL. 5.6 Protein quantification Total protein concentration was measured by the Biuret method (Gornall A et al., 1949) in a Beckman DU-50 spectrophotometer at A540 nm to determine one mg/ mL of cells per assay. Bovine serum albumin was used to develop the calibration curve. 5.7 OximetryThe oxygen consumption rate of complete cells was determined in a water chamber at 30 °C with an SI-5300 Oxygraph equipped with a Clark electrode. Yeasts were added to a reaction mixture composed by 1 M sorbitol, ten mM maleate (pH adjusted to 6.8 with Tris), ten mM Tris-phosphate, one mM MgCl2 and 75 mM KCl, to get a final volume of 1.5 mL per assay. Respiratory control rates were obtained dividing the phosphorylating state (III), induced with 0.5 mM ADP, between state IV, produced without adding ADP.To specifically inhibit the components of the respiratory chain, various inhibitors, whose concentrations are specified in the footnote of each figure, were used. 5.9 Statistical analysisThe statistical analysis was made with Prism 6 (GraphPad Software, Inc.) Dry biomass was determined in triplicates. Oxygen consumption data came from five independent experiments. The results are presented as mean ± standard deviation.  Mean values are reported and compared by one-way ANOVA. The difference among the observed averages was tested using the Tukey test. Those results with p <0.05 were considered statistically significant.  6. Results 6.1 Effect of various carbon sources on growth of Debaryomyces hansenii.Dry weights of yeast cells cultures were quantified. Maximum peak in the biomass content was observed in cultures supplied with fermentative carbon sources and during the stationary growth phase, Fig. 2. Remarkable was the biomass production in YP Glycerol (0.401 ± 0.02 g/mL), a non-fermentative carbon source, which was similar to the yield obtained in cultures with glucose (0.462 ± 0.01 g/mL) or galactose (0.354 ± 0.02 g/mL). Not a high biomass production was measured in cultures provided with lactate or succinate, other non-fermentative carbon sources.6.2 AOX activity of D. hansenii cultures supplied with different carbon sources.To characterise the AOX activity of D. hansenii, yeasts were cultured in either fermentable (YP D-Glucose and YP Galactose) or non-fermentable (YP Lactose, YP Succinate and YP Glycerol) media and subsequently harvested at mid-logarithmic phase or after 72h of growth. Then, the oxygen consumption rate of the endogenous respiration (red column) was determined and also after the inhibition of the cytochrome pathway with 500 microM NaCN (blue column), Fig. 3. The remaining activity associated with the AOX, the Cyanide Resistant Respiration (CRR), was inhibited adding one mM propyl-gallate (PG) (green column). AOX activity showed to be dependent on the carbon source. In cells grown in glucose, CRR seemed to be higher in the exponential phase, Fig. 3B. In contrast, cells aged in lactate or succinate presented an active CRR since the exponential phase and comprised between 40-50% of the basal respiration in both cut-points, Fig. 3.Noticeably, AOX activity in glycerol media cultures did not change throughout growth even in the stationary phase, when it is expected to increase in the needing of physiological uncoupling. In fact, after 72h cells showed a low CRR, a distinctive response compared with other non-fermentative sources, Fig. 3. 7. Discussion The oxygen consumption rate of the branched mitochondrial respiratory chain from D. hansenii was analysed in whole cells cultured in different carbon sources.  In agreement with the results obtained by Veiga A. et al. in 2002, we found CRR induced during the stationary growth phase. Ubiquinone feeds cyanide-resistant-AOX directly with electrons skipping proton-pumping activities of canonical complexes (Guerrero-Castillo et al., 2011) that had been suggested as an adaptation mechanism to the variable energy demands. Alternative-oxidoreductases expression in the stationary phase, a nondividing resting state, reduces ATP production independently of high oxygen consumption (low ATP/O ratio) (Veiga A., et al., 2003).   In addition to the growth phase, changes in carbon source supplies modify AOX activity. Non-fermentative substrates like lactate or succinate may saturate cytochrome route, and an active CRR would provide an alternative route to electron flux. However, in glycerol-containing media, also a non-fermentative carbon source, AOX activity was poorly detected during the whole growth curve. Besides, biomass yield in glycerol-grown cells was higher than in other three carbon sources, except glucose, and could be related to biosynthetic pathways, and lipid storage carried out by oleaginous yeast species (Adler L. et al., 1985). Probably D. hansenii did not express AOX to keep the Oxidative Phosphorylation highly coupled to support ATP needs for glycerol-promoted lipid biosynthesis (Luévano-Martínez LA. et al., 2010) an idea supported by the observation of lipid supernatant in cytosolic extracts from yeast cells grown in YP Glycerol 0.6M NaCl, Fig 4.   This result is of interest since crude glycerol, a byproduct of biodiesel production, could be used and reincorporated to the energy production or for generation of value-added chemicals (Sestric R. et al., 2014).   8. Conclusion In D. hansenii, AOX has different activity pattern related to the carbon source in the media and the growth phase. Besides, lipid metabolism is promoted in this species with glycerol-grown cells, a relevant finding for biotechnological purposes.