The effects of external Mn2+ concentration on hyphal morphology and citric acid production are mediated primarily by the NRAMP-family transporter DmtA in Aspergillus niger

Citric acid, a commodity product of industrial biotechnology, is produced by fermentation of the filamentous fungus Aspergillus niger. A requirement for high-yield citric acid production is keeping the concentration of Mn.sup.2+ ions in the medium at or below 5 [micro]g L.sup.-1. Understanding manganese metabolism in A. niger is therefore of critical importance to citric acid production. To this end, we investigated transport of Mn.sup.2+ ions in A. niger NRRL2270. we identified an A. niger gene (dmtA; NRRL3_07789), predicted to encode a transmembrane protein, with high sequence identity to the yeast manganese transporters Smf1p and Smf2p. Deletion of dmtA in A. niger eliminated the intake of Mn.sup.2+ at low (5 [micro]g L.sup.-1) external Mn.sup.2+ concentration, and reduced the intake of Mn.sup.2+ at high (> 100 [micro]g L.sup.-1) external Mn.sup.2+ concentration. Compared to the parent strain, overexpression of dmtA increased Mn.sup.2+ intake at both low and high external Mn.sup.2+ concentrations. Cultivation of the parent strain under Mn.sup.2+ ions limitation conditions (5 [micro]g L.sup.-1) reduced germination and led to the formation of stubby, swollen hyphae that formed compact pellets. Deletion of dmtA caused defects in germination and hyphal morphology even in the presence of 100 [micro]g L.sup.-1 Mn.sup.2+, while overexpression of dmtA led to enhanced germination and normal hyphal morphology at limiting Mn.sup.2+ concentration. Growth of both the parent and the deletion strains under citric acid producing conditions resulted in molar yields (Y.sub.p/s) of citric acid of > 0.8, although the deletion strain produced ~ 30% less biomass. This yield was reduced only by 20% in the presence of 100 [micro]g L.sup.-1 Mn.sup.2+, whereas production by the parent strain was reduced by 60%. The Y.sub.p/s of the overexpressing strain was 17% of that of the parent strain, irrespective of the concentrations of external Mn.sup.2+. Our results demonstrate that dmtA is physiologically important in the transport of Mn.sup.2+ ions in A. niger, and manipulation of its expression modulates citric acid overflow.