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

Abstract

Currently, there is no consensus regarding the mechanism underlying Aspergillus niger citrate biosynthesis and secretion, although it is amongst the most studied biotechnological production processes. Carbon excess relative to various other medium constituents is key, but the complex interplay between the limiting factors required for extracellular citrate accumulation remains elusive. It is thought that one of the industrial bottlenecks for citrate production is citrate export, however, no A. niger citrate exporter has yet been identified. Here, we show that the phenotype of increased extracellular citrate accumulation can have fundamentally different underlying mechanisms, depending on how this response is triggered, and that combining gene expression analyses of the different conditions can lead to the compilation of a shortlist of the most promising citrate exporter candidates. Specifically, we found that varying the amount and type of supplement of an arginine auxotrophic A. niger strain shows down-regulation of citrate metabolising enzymes in the condition in which more citrate is accumulated extracellularly. This contrasts with the transcriptional adaptations triggered by iron limitation, which also induces increased A. niger citrate production. By combining data obtained from these two manners of inducing comparatively high extracellular citrate accumulation, we were able to compile a shortlist of the most likely citrate transporter candidates. Two of the most promising candidates were tested in the yeast Saccharomyces cerevisiae, one of which showed the ability to secrete citrate. Deletion of the endogenous A. niger gene encoding the corresponding transporter abolished the ability of this fungus to secrete citrate. Instead, under conditions that usually favour A. niger citrate production, we found increased accumulation of extracellular oxalate. Our findings provide steps in untangling the complex interplay of different mechanisms underlying A. niger citrate accumulation, and we identify, for the first time, a fungal citrate exporter, offering a valuable tool for improvement of A. niger as biotechnological cell-factory for organic acid production.\n\nAuthor SummaryCitrate is widely applied as acidifier, flavouring and chelating agent. Industrial citrate production currently relies on the filamentous fungus Aspergillus niger. Although the industrial production process using A. niger has vastly improved since initiated almost 100 years ago, citrate export remains a bottleneck. Here, we studied the gene expression pattern of A. niger under various citrate producing conditions. Using these expression patterns and different computational approaches, we compiled a shortlist of putative citrate exporter candidates. In this way, we were able to identify a gene encoding a transporter protein capable of citrate export. We show that the yeast Saccharomyces cerevisiae, normally a citrate non-producer, secretes detectable amounts of citrate when harbouring this gene. In addition, we verify the biological function of this gene in A. niger itself, as removing this gene resulted in a citrate non-producing phenotype, which is atypical for this fungus. This finding is particularly exciting, as it is the first identification of a eukaryotic citrate exporter. With this, we not only provide a tool for improvement of industrial citrate production, but knowledge of this gene should help develop new methods for improvement of A. niger as biotechnological cell-factory for the production of other organic acids.