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

Abstract

Background: 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 Mn²⁺ ions in the medium at or below 5 µg L^-1. Understanding manganese metabolism in A. niger is therefore of critical importance to citric acid production. To this end, we investigated transport of Mn²⁺ ions in A. niger NRRL2270.

Results: 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 Mn²⁺ at low (5 µg L^-1) external Mn²⁺ concentration, and reduced the intake of Mn²⁺ at high (> 100 µg L^-1) external Mn²⁺ concentration. Compared to the parent strain, overexpression of dmtA increased Mn²⁺ intake at both low and high external Mn²⁺ concentrations. Cultivation of the parent strain under Mn²⁺ ions limitation conditions (5 µg L^-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 µg L^-1 Mn²⁺, while overexpression of dmtA led to enhanced germination and normal hyphal morphology at limiting Mn²⁺ concentration. Growth of both the parent and the deletion strains under citric acid producing conditions resulted in molar yields (Y_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 µg L^-1 Mn²⁺, whereas production by the parent strain was reduced by 60%. The Y_p/s of the overexpressing strain was 17% of that of the parent strain, irrespective of the concentrations of external Mn²⁺.

Conclusions: Our results demonstrate that dmtA is physiologically important in the transport of Mn²⁺ ions in A. niger, and manipulation of its expression modulates citric acid overflow.

Keywords: Aspergillus niger; Citric acid; Manganese ions; Manganese transporter.