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 Mn 2+ 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 Mn 2+ 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 Mn 2+ at low (5 µg L −1) external Mn 2+ concentration, and reduced the intake of Mn 2+ at high (> 100 µg L −1) external Mn 2+ concentration. Compared to the parent strain, overexpression of dmtA increased Mn 2+ intake at both low and high external Mn 2+ concentrations. Cultivation of the parent strain under Mn 2+ 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 Mn 2+ , while overexpression of dmtA led to enhanced germination and normal hyphal morphology at limiting Mn 2+ 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 Mn 2+ , 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 Mn 2+. Conclusions: Our results demonstrate that dmtA is physiologically important in the transport of Mn 2+ ions in A. niger, and manipulation of its expression modulates citric acid overflow.

Identification and characterization of Nramp transporter AoNramp1 in Aspergillus oryzae

“…In A. oryzae, AoNramp1 deletion and AoNramp1 overexpressing strains had a similar response to metal-deplete and -replete conditions (Fig. 5), whereas dmtA-disrupted mutant had different phenotypes with dmtA overexpression strain under Mn excess and deficient conditions (Fejes et al 2020). Notably, the similar response of AoNramp1 overexpression and deletion strains to Zn and Mn treatments in growth only includes mycelium growth and conidiation formation (Fig.…”

“…A possible explanation for this is whether overexpression AoNramp1 or knockout of AoNramp1 both disrupt metal ion homeostasis and A. oryzae is sensitive to the variety of metal ions. In line, overexpression and disruption of Nramp gene bring about changes in metal ion concentration of cells and metal ions have a great influence on the growth of A. oryzae (Fejes et al 2020;Zhang et al 2019;Ishimaru et al 2012;Mori et al 2018;Cohen et al 2000).…”

“…For example, our previous study has found that the zinc/iron-regulated transporters are involved in the production of the secondary metabolite kojic acid in A. oryzae (Zhang et al 2019). In A. niger, manganese deficiency contributes to the production of citric acid, which is closely related to the Nramp-family transporter DmtA (Fejes et al 2020). However, the information on the Nramp protein as a metal transporter is unavailable in A. oryzae.…”

“…In Candida albicans, SMF12 is proposed to be an orthologue of Smf1p and may have functions in the assimilation of Mn under alkaline conditions (Wehmeier et al 2020;Bensen et al 2004). In Aspergillus niger, the NRAMP-family transporter DmtA has transport activity for Mn, and deletion of dmtA caused defects in germination and hyphal morphology (Fejes et al 2020). These findings enrich our knowledge that Nramp transporters influence not only metal transport but also many other biological processes.…”

The Role of Metal Ions in Fungal Organic Acid Accumulation

“…In contrast to these standard chemical engineering methods that have been wellpractised in the industry for several decades now, the identification of the major manganese transporter DmtA of A. niger, whose knock-out results in Mn 2+ -insensitive production of citric acid [49], unfolded a novel strategy to address the problem. Furthermore, manipulation of dmtA challenged the notion that the effect of Mn 2+ deficiency on citric acid accumulation and hyphal morphology is a consequence of insufficient availability of this metal ion.…”