Vacuoles were isolated from Yarrowia lipolytica yeast cells taken at various growth phases under carbon or nitrogen limitation. Vacuoles from the cells at the logarithmic growth phase showed a high activity of vacuolar H(+)-ATPase (0.9-1.1 U/mg protein) and efficiently generated chemical proton gradient and membrane potential across the tonoplast. Ca(2+)- and citrate transport were found to be maximal at this growth phase. At growth retardation and then in the stationary phase all the parameters studied decreased irrespective of the method of growth limitation. The citrate-transporting activity of vacuoles completely disappeared at growth retardation, also irrespective of the limitation method and irrespective of whether yeast cells overproduced citrate in the culture medium. The citrate-transporting system of Y. lipolytica vacuolar membrane is concluded not to be involved in citrate efflux and this efflux is probably performed by the plasmalemma transport system.
[Show abstract][Hide abstract] ABSTRACT: Glycerol, a major by-product of ethanol fermentation by Saccharomyces cerevisiae, is of significant importance to the wine, beer, and ethanol production industries. To gain a clearer understanding of and
to quantify the extent to which parameters of the pathway affect glycerol flux in S. cerevisiae, a kinetic model of the glycerol synthesis pathway has been constructed. Kinetic parameters were collected from published
values. Maximal enzyme activities and intracellular effector concentrations were determined experimentally. The model was
validated by comparing experimental results on the rate of glycerol production to the rate calculated by the model. Values
calculated by the model agreed well with those measured in independent experiments. The model also mimics the changes in the
rate of glycerol synthesis at different phases of growth. Metabolic control analysis values calculated by the model indicate
that the NAD+-dependent glycerol 3-phosphate dehydrogenase-catalyzed reaction has a flux control coefficient (Cv1J) of approximately 0.85
and exercises the majority of the control of flux through the pathway. Response coefficients of parameter metabolites indicate
that flux through the pathway is most responsive to dihydroxyacetone phosphate concentration (RDHAPJ = 0.48 to 0.69), followed by ATP concentration (RATPJ = −0.21 to −0.50). Interestingly, the pathway responds weakly to NADH concentration (RNADHJ = 0.03 to 0.08). The model indicates that the best strategy to increase flux through the pathway is not to increase enzyme
activity, substrate concentration, or coenzyme concentration alone but to increase all of these parameters in conjunction
with each other.
[Show abstract][Hide abstract] ABSTRACT: The effect of aluminum on dimorphic fungi Yarrowia lipolytica was investigated. High aluminum (0.5-1.0 mM AlK(SO(4))(2)) inhibits yeast-hypha transition. Both vanadate-sensitive H(+) transport and ATPase activities were increased in total membranes isolated from aluminum-treated cells, indicating that a plasma membrane H(+) pump was stimulated by aluminum. Furthermore, Al-treated cells showed a stronger H(+) efflux in solid medium. The present results suggest that alterations in the plasma membrane H(+) transport might underline a pH signaling required for yeast/hyphal development. The data point to the cell surface pH as a determinant of morphogenesis of Y. lipolytica and the plasma membrane H(+)-ATPase as a key factor of this process.
[Show abstract][Hide abstract] ABSTRACT: The optimal cultivation conditions ensuring the maximal rate of citric acid (CA) biosynthesis by glycerol-grown mutant Yarrowia lipolytica NG40/UV7 were found to be as follows: growth limitation by inorganic nutrients (nitrogen, phosphorus, or sulfur), 28 °C, pH 5.0, dissolved oxygen concentration (pO2) of 50 % (of air saturation), and pulsed addition of glycerol from 20 to 80 g L(-1) depending on the rate of medium titration. Under optimal conditions of fed-batch cultivation, in the medium with pure glycerol, strain Y. lipolytica NG40/UV7 produced 115 g L(-1) of CA with the mass yield coefficient of 0.64 g g(-1) and isocitric acid (ICA) amounted to 4.6 g L(-1); in the medium with raw glycerol, CA production was 112 g L(-1) with the mass yield coefficient of 0.90 g g(-1) and ICA amounted to 5.3 g L(-1). Based on the activities of enzymes involved in the initial stages of raw glycerol assimilation, the tricarboxylic acid cycle and the glyoxylate cycle, the mechanism of increased CA yield from glycerol-containing substrates in Y. lipolytica yeast was explained.
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