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.

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.

23Currently, there is no consensus regarding the mechanism underlying Aspergillus niger citrate 24 biosynthesis and secretion, although it is amongst the most studied biotechnological production 25 processes. Carbon excess relative to various other medium constituents is key, but the complex 26 interplay between the limiting factors required for extracellular citrate accumulation remains 27 elusive. It is thought that one of the industrial bottlenecks for citrate production is citrate export, 28 however, no A. niger citrate exporter has yet been identified. Here, we show that the phenotype 29 of increased extracellular citrate accumulation can have fundamentally different underlying 30 mechanisms, depending on how this response is triggered, and that combining gene expression 31 analyses of the different conditions can lead to the compilation of a shortlist of the most 32 promising citrate exporter candidates. Specifically, we found that varying the amount and type 33 of supplement of an arginine auxotrophic A. niger strain shows down-regulation of citrate 34 metabolising enzymes in the condition in which more citrate is accumulated extracellularly. 35This contrasts with the transcriptional adaptations triggered by iron limitation, which also 36 induces increased A. niger citrate production. By combining data obtained from these two 37 manners of inducing comparatively high extracellular citrate accumulation, we were able to 38 compile a shortlist of the most likely citrate transporter candidates. Two of the most promising 39 candidates were tested in the yeast Saccharomyces cerevisiae, one of which showed the ability 40 to secrete citrate. Deletion of the endogenous A. niger gene encoding the corresponding 41 transporter abolished the ability of this fungus to secrete citrate. Instead, under conditions that 42 usually favour A. niger citrate production, we found increased accumulation of extracellular 43 oxalate. Our findings provide steps in untangling the complex interplay of different 44 mechanisms underlying A. niger citrate accumulation, and we identify, for the first time, a 45 3 fungal citrate exporter, offering a valuable tool for improvement of A. niger as biotechnological 46 cell-factory for organic acid production. 47 Author Summary 48Citrate is widely applied as acidifier, flavouring and chelating agent. Industrial citrate 49 production currently relies on the filamentous fungus Aspergillus niger. Although the industrial 50 production process using A. niger has vastly improved since initiated almost 100 years ago, 51 citrate export remains a bottleneck. Here, we studied the gene expression pattern of A. niger 52 under various citrate producing conditions. Using these expression patterns and different 53 computational approaches, we compiled a shortlist of putative citrate exporter candidates. In 54 this way, we were able to identify a gene encoding a transporter protein capable of citrate 55 export. We show that the yeast Saccharomyces cerevisiae, normally a citrate non-producer, 56 secretes detectab...