Inhibition of citric acid accumulation by manganese ions in Aspergillus niger mutants with reduced citrate control of phosphofructokinase

Abstract

Mutant strains of Aspergillus niger with reduced citrate control of carbohydrate catabolism (cic mutants) grow faster than the parent strain on media containing 5% (wt/vol) citrate. The mutants tolerated a higher intracellular citrate concentration than the parent strain. One mutant (cic-713) contained phosphofructokinase activity significantly less sensitive towards citrate than the enzyme from the parent strain. When this mutant was grown under citrate-accumulating conditions, acidogenesis was far less sensitive to inhibition by Mn2+ than in the parent strain. Some of the cic mutants also showed altered citrate inhibition of NADP-specific isocitrate dehydrogenase.The accumulation of large amounts of citrate by the filamentous fungus Aspergillus niger is known to depend on a variety of environmental factors, among which the level of Mn2+ ions in the nutrient medium plays a key role. Only when the Mn2+ concentration is below 0.02 mM (which does not yet affect growth rate or yield) will citrate accumulate in high amounts (17).A biochemical explanation of this manganese effect has been presented recently. Mn2+ deficiency leads to an increase in protein turnover, which ultimately leads to a high intracellular concentration of NH4+ (7, 9); this level of NH4+ is able to counteract the inhibition of phosphofructokinase by citrate (4, 5), thereby guaranteeing a high rate of glucose breakdown by the fructose biphosphate pathway, which is necessary for citric acid accumulation (17).According to this model, Mn2+ deficiency is mainly required for eliminating glycolytic feedback control by citrate. If this model is correct, mutants lacking citrate inhibition of phosphofructokinase should accumulate citrate even in the presence of Mn2+.To test this hypothesis, A. niger B60 (7) was propagated on potato dextrose agar for 7 days, and the conidia were harvested, diluted in sterile water containing 0.1% (wt/vol) Tween 80 to give a final density of 2 x 106 conidia per ml, and shaken vigorously to break clumps. Of this suspension, 10 ml was then placed in an open petri dish 14 cm below a germicidal UV lamp and stirred magnetically for 5 min. Subsequently, the suspension was covered with aluminium foil for 1 h and used for the isolation of potential mutants. Their identification was based on the assumption that mutants lacking citrate control of phosphofructokinase would grow faster on a sucrose medium containing citrate, as discussed below. For this reason, the diluted, mutagenized conidium suspensions were plated on solid medium containing 20% (wt/vol) sucrose and 5% (wt/vol) citrate as carbon sources. Other inorganic nutrients and nitrogen source were essentially as used in the citric acid production medium (8), except that the pH was adjusted to 3.5 with H2SO4. Oxgall (0.5% [wt/vol]; Sigma Chemical Co., St. Louis, Mo.) was added to the medium to restrict growth to small, defined * Corresponding author. colonies (15). The fastest-growing colonies were considered to be less affected in carbohydrate breakdown by citrate and thus we...

Changes in primary metabolism leading to citric acid overflow in Aspergillus niger

“…Although proteases are normally strictly compartmentalised in the cells and are activated from their pre-pro forms only in the vacuoles, some leakage through the tonoplast into the cytosol must occur, since cleavage of the native Pfk1 enzyme takes place. In A. niger cells, increased protein degradation occurs under Mn 2+ -deficient conditions, which was also reflected by increased intracellular proteinase activities (Ma et al 1985;Schreferl et al 1986). For efficient citric acid fermentation, lack of trace metal ions, particularly Mn 2+ from the medium, is of major importance.…”

“…Six organic allosteric ligands either increase or decrease substrate binding affinity and concomitantly determine overall enzyme activity (Poorman et al 1984). Pfk1 from A. niger cells was a subject of various studies (Habison et al 1983;Schreferl et al 1986;Arts et al 1987;Legiša and Benčina 1994;Mesojednik and Legiša 2005;Mlakar and Legiša 2006). The enzyme attracted the interest of investigators, due to its ability to maintain a high glycolytic flux in spite of elevated intracellular concentrations of citrate, a well-known inhibitor of Pfk1, which was reported to reach concentrations between 4 mM (Rö hr and Kubicek 1981) and 10 mM (Legiša and Kidrič 1989).…”

Citrate Inhibition-Resistant Form of 6-Phosphofructo-1-Kinase fromAspergillus niger

“…Much has been reported on PFK1 characteristics from the filamentous fungus A. niger in the past (1,5,6,17,20,27). The enzyme attracted the interest of investigators, due to its ability to maintain a high glycolytic flux in spite of elevated intracellular concentrations of citrate during the process of the secretion of citric acid, which was reported to reach a concentration between 4 mM (23) and 7 mM (13).…”

“…The regulatory properties of PFK1 attracted the interest of investigators in order to explain the phenomenon (1, 5, 6), since this enzyme was shown to be a major control point of A. niger glycolysis (19,27). By studying the kinetics on a partially purified enzyme, it was proposed that the citrate inhibition of PFK1 was counteracted by NH 4 ϩ ions and AMP in A. niger cells (5, 6), while fructose-2,6-bisphosphate significantly stimulated the activity of the enzyme and relieved the inhibition caused by ATP (1).…”

Identification of Genes Associated with Morphology in Aspergillus niger by Using Suppression Subtractive Hybridization

“…3). The effects of Mn 2ϩ concentration on citric acid production have been reported previously (21,44,45), but in the present study, the effects of Mn 2ϩ on A. niger morphology and citric acid production were examined concurrently. Low Mn 2ϩ concentrations (10 to 13 ppb) also significantly suppressed the biomass accumulation (data not shown).…”