Inhibition of alternative respiration system of Scheffersomyces stipitis and effect on glucose or xylose fermentation.

Scheffersomyces stipitis is a Crabtree-negative pentose fermenting yeast, which shows a complex respiratory system involving a cytochrome and an alternative salicylhydroxamic acid (SHAM)-sensitive respiration mechanism that is poorly understood. This work aimed to investigate the role of the antimycin A (AA) sensitive respiration and SHAM-sensitive respiration in the metabolism of xylose and glucose by S. stipitis, upon different agitation conditions. Inhibition of the SHAM-sensitive respiration caused a significant (P < 0.05) decrease in glycolytic flux and oxygen consumption when using glucose and xylose under agitation conditions, but without agitation, only a mild reduction was observed. The combination of SHAM and AA abolished respiration, depleting the glycolytic flux using both carbon sources tested, leading to increased ethanol production of 21.05 g/L at 250 rpm for 0.5 M glucose, and 8.3 g/L ethanol using xylose. In contrast, inhibition of only the AA-sensitive respiration, caused increased ethanol production to 30 g/L using 0.5 M glucose at 250 rpm, and 11.3 g/L from 0.5 M xylose without agitation. Results showed that ethanol production can be induced by respiration inhibition, but the active role of SHAM-sensitive respiration should be considered to investigate better conditions to increase and optimize yields.

Effect of cytochrome bc1 complex inhibition during fermentation and growth of Scheffersomyces stipitis using glucose, xylose or arabinose as carbon sources.

Scheffersomyces stipitis shows a high capacity to ferment xylose, with a strong oxygen dependence to allow NAD+ regeneration. However, without oxygen regeneration of NADH occurs by other metabolic pathways like alcoholic fermentation. There are few reports about inhibitors of mitochondrial respiration and their effects on growth and fermentation. This work aimed to explore the effect of cytochrome bc1 complex inhibition by antimycin A (AA), on growth and fermentation of S. stipitis using glucose, xylose and arabinose as carbon sources, at three agitation levels (0, 125 and 250 rpm). It was possible to discriminate between respiratory and fermentative metabolism in these different conditions using xylose or arabinose. Despite the inhibition of mitochondrial respiration, the glycolytic flux was active because S. stipitis metabolized glucose or xylose to produce ATP; on 0.5 M glucose the cells yielded 17-33 g L-1 ethanol. However, more complex results were obtained on xylose, which depended upon agitation conditions where ethanol production without agitation increased up to 11 g L-1. Inhibition of respiratory chain in S. stipitis could therefore be a good strategy to improve ethanol yields.

Glycolytic sequence and respiration of Debaryomyces hansenii as compared to Saccharomyces cerevisiae.

The fermentation and respiration activities of Debaryomyces hansenii were compared with those of Saccharomyces cerevisiae grown to stationary phase with high respiratory activity. It was found that: (a) glucose consumption, fermentation and respiration were lower than for S. cerevisiae; (b) fasting produced a much smaller decrease of respiration; (c) glucose consumed and not transformed to ethanol was higher; (d) in S. cerevisiae, full oxygenation prevented ethanol production but this effect was reversed by CCCP, whereas D. hansenii still showed some ethanol production under aerobiosis, which was moderately increased by CCCP. ATP levels were similar in the two yeasts. Levels of glycolytic intermediaries after glucose addition, and enzyme activities, indicated that the main difference and limiting step to explain the lower fermentation of D. hansenii is phosphofructokinase activity. Respiration and fermentation, which are lower in D. hansenii, compete for the re-oxidation of reduced nicotinamide adenine nucleotides; this competition, in turn, seems to play a role in defining the fermentation rates of the two yeasts. The effect of CCCP on glucose consumption and ethanol production also indicates a role of ADP in both the Pasteur and Crabtree effects in S. cerevisiae but not in D. hansenii. D. hansenii shows an alternative oxidase, which in our experiments did not appear to be coupled to the production of ATP.

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress.

The comparative analysis of growth, intracellular content of Na+ and K+, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na+ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.

Sodium and potassium transport in the halophilic yeast Debaryomyces hansenii.

Debaryomyces hansenii, a halophile yeast found in shallow sea waters and salty food products grows optimally in 0.6 M of either NaCl or KCl, accumulating high concentrations of Na(+) or K(+). After growth in NaCl or KCl, a rapid efflux of either accumulated cation was observed if the cells were incubated in the presence of KCl or NaCl, respectively, accompanied by a slower accumulation of the cation present in the incubation medium. However, a similar, rapid efflux was observed if cells were incubated in buffer, in the absence of external cations. This yeast shows a cation uptake activity of both (86)Rb(+) and (22)Na(+) with saturation kinetics, and much higher affinity for (86)Rb(+) than for (22)Na(+). The pH dependence of the kinetics constants was similar for both cations, and although K(m) values were higher at pH 8.0, there was also an increase in the V(max) values. The accumulation of (22)Na(+) was found to be increased in cells grown in the presence of 0.6 M NaCl. (86)Rb(+) was also accumulated more in these cells, but to a slightly greater extent. The inhibition kinetics of the uptake of (22)Na(+) by K(+), and that of (86)Rb(+) by Na(+) was found to be non-competitive. It can be concluded that Na(+) in D. hansenii is not excluded but instead, its metabolic systems must be resistant to high salt concentrations.

Mitochondrial nitric oxide inhibits ATP synthesis. Effect of free calcium in rat heart.

Nitric oxide is a small potentially toxic molecule and a diatomic free radical. We report the interaction of L-arginine, oxygen and calcium with the synthesis of nitric oxide in heart mitochondria. Nitric oxide synthesis is increased in broken rat heart mitochondria compared with intact and permeabilized mitochondria. Intact mitochondria subjected to hypoxia-reoxygenation conditions accumulated nitric oxide that inhibits oxygen consumption and ATP synthesis. ATPase activity is not affected during this augment of nitric oxide. Physiological free calcium concentrations protected mitochondria from the damage caused by the accumulation of nitric oxide. Higher concentrations of the divalent cation increase the damage exerted by nitric oxide.

Effect of D-amino acids on some mitochondrial functions in rat liver.

We studied the role of the D-amino acids (D-aa) D-serine, D-alanine, D-methionine, D-aspartate, D-tyrosine and D-arginine on rat liver mitochondria. The stability of D-amino acids, mitochondrial swelling, transmembrane potential and oxygen consumption were studied under oxidative stress conditions in rat liver mitochondria. In the presence of glutamate-malate all D-aas salts increased mitochondrial swelling, while in the presence of succinate plus rotenone only D-ala, D-arg and D-ser, induced mitochondrial swelling. The transmembrane potential (deltapsi) was decreased in the presence of 1 microM Ca(2+). The D-aas inhibited oxygen consumption in state 3. The D-aa studied exerted effects on mitochondria via an increase of free radicals production.

Regulation of the rate of synthesis of nitric oxide by Mg(2+) and hypoxia. Studies in rat heart mitochondria.

In isolated rat heart mitochondria, L-arginine is oxidized by a nitric oxide synthase (mtNOS) achieving maximal rates at 1 mM L-arginine. The NOS inhibitor N(G)-nitro-L-arginine methyl ester (NAME) inhibits the increase in NO production. Extramitochondrial free magnesium inhibited NOS production by 59% at 3.2 mM. The mitochondrial free Mg(2+) concentration increased to different extents in the presence of L-arginine (29%), the NO donor (S-nitroso-N-acetylpenicillamine) (105%) or the NOS inhibitors L-NAME (48%) or N(G)-nitro-L-arginine methyl ester, N(G)-monomethyl-L-arginine (L-NMMA) (53%). Under hypoxic conditions, mtNOS activity was inhibited by Mg(2+) by up to 50% after 30 min of incubation. Reoxygenation restored the activity of the mtNOS to pre-hypoxia levels. The results suggest that in heart mitochondria there is an interaction between Mg(2+) levels and mtNOS activity which in turn is modified by hypoxia and reoxygenation.

Effect of Ca(2+) and Mg(2+) on the Mn-superoxide dismutase from rat liver and heart mitochondria.

The manganese superoxide dismutase (Mn-SOD) converts superoxide anions to hydrogen peroxide plus oxygen, providing the first line of defense against oxidative stress in mitochondria. Heart mitochondria exhibited higher Mn-SOD activity than liver mitochondria. In mitochondria from both tissues Mn-SOD activity decreased after incubation at low oxygen concentration (hypoxic mitochondria). The effects of free Ca(2+) ([Ca(2+)](f)) and free Mg(2+) ([Mg(2+)](f)) on normoxic and hypoxic mitochondria from either organ were tested. In normoxic mitochondria from either tissue, both [Ca(2+)](f) and [Mg(2+)](f) activated the enzyme, although [Mg(2+)](f) was less efficient as an activator and the effect was lower in heart than in liver mitochondria. When added simultaneously, high [Ca(2+)](f) and [Mg(2+)](f) exhibited additive effects which were more pronounced in heart mitochondria and were observed regardless of whether mitochondria had been incubated under normal or low oxygen. The data suggest that [Ca(2+)](f) plays a role in regulating Mn-SOD in concert with the activation of aerobic metabolism.