Aspergillus niger citrate exporter revealed by comparison of two alternative citrate producing conditions

Currently, there is no consensus regarding the mechanism underlying Aspergillus niger citrate biosynthesis and secretion. We hypothesise that depending on the experimental setup, extracellular citrate accumulation can have fundamentally different underlying transcriptomic landscapes. We show that varying the amount and type of supplement of an arginine auxotrophic A. niger strain results in transcriptional down-regulation of citrate metabolising enzymes in the condition in which more citrate is accumulated extracellularly. This contrasts with the transcriptional adaptations when increased citrate production is triggered by iron limitation. By combining gene expression data obtained from these two very distinct experimental setups with hidden Markov models and transporter homology approaches, we were able to compile a shortlist of the most likely citrate transporter candidates. Two candidates (An17g01710 and An09g06720m.01) were heterologously expressed in the yeast Saccharomyces cerevisiae, and one of the resultant mutants showed the ability to secrete citrate. Our findings provide steps in untangling the complex interplay of different mechanisms underlying A. niger citrate accumulation, and we demonstrate how a comparative transcriptomics approach complemented with further bioinformatics analyses can be used to pinpoint a fungal citrate exporter.grant in the framework of the BE-BASIC program F01.011 Transport processes in the production of organic acids by Aspergillus niger, and the WUR IPOP Systems Biology program KB-17-003.02.026 Genome-wide metabolic modelling and data integration of organic acid production in filamentous fungi

Aspergillus niger secretes citrate to increase iron bioavailability

Aspergillus niger has an innate ability to secrete various organic acids, including citrate. The conditions required for A. niger citrate overproduction are well described, but the physiological reasons underlying extracellular citrate accumulation are not yet fully understood. One of the less understood culture conditions is the requirement of growth-limiting iron concentrations. While this has been attributed to iron-dependent citrate metabolizing enzymes, this straightforward relationship does not always hold true. Here, we show that an increase in citrate secretion under iron limited conditions is a physiological response consistent with a role of citrate as A. niger iron siderophore. We found that A. niger citrate secretion increases with decreasing amounts of iron added to the culture medium and, in contrast to previous findings, this response is independent of the nitrogen source. Differential transcriptomics analyses of the two A. niger mutants NW305 (gluconate non-producer) and NW186 (gluconate and oxalate non-producer) revealed up-regulation of the citrate biosynthesis gene citA under iron limited conditions compared to iron replete conditions. In addition, we show that A. niger can utilize Fe(III) citrate as iron source. Finally, we discuss our findings in the general context of the pH-dependency of A. niger organic acid production, offering an explanation, besides competition, for why A. niger organic acid production is a sequential process influenced by the external pH of the culture medium

Aspergillus niger N402 derivatives grown with different amounts of iron in the medium

Aspergillus niger N402 derivatives were grown with either no iron added to the medium, or 10g/L Fe(II)SO4 added to the medium

Aspergillus niger N402 derivatives grown with different amounts of iron in the medium

Aspergillus niger N402 derivatives were grown with either no iron added to the medium, or 10g/L Fe(II)SO4 added to the medium

Aspergillus niger N402 derivatives grown with different amounts of iron in the medium

Aspergillus niger N402 derivatives were grown with either no iron added to the medium, or 10g/L Fe(II)SO4 added to the medium

Aspergillus niger N402 derivatives grown with different supplements

Aspergillus niger N402 derivatives, harbouring an argB deletion and thus making them arginine auxothrophs, were grown with either 1.1 mM arginine or 5 mM citrulline added to the medium

Aspergillus niger N402 derivatives grown with different supplements

Aspergillus niger N402 derivatives, harbouring an argB deletion and thus making them arginine auxothrophs, were grown with either 1.1 mM arginine or 5 mM citrulline added to the medium