Aspergillus nigercitrate exporter revealed by comparison of two alternative citrate producing conditions
Authors: Odoni Dorett I , Laothanachareon Thanaporn , Vazquez-Vilar Marta , van Gaal Merlijn P , Schonewille Tom , Bruinsma Lyon , Martins dos Santos Vitor AP , Tamayo-Ramos Juan Antonio , Suarez-Diez Maria , Schaap Peter J 
Published: April 2018

Currently, there is no consensus regarding the mechanism underlyingAspergillus nigercitrate 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, noA. nigercitrate 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 auxotrophicA. nigerstrain 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 increasedA. nigercitrate 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 yeastSaccharomyces cerevisiae, one of which showed the ability to secrete citrate. Deletion of the endogenousA. nigergene encoding the corresponding transporter abolished the ability of this fungus to secrete citrate. Instead, under conditions that usually favourA. nigercitrate production, we found increased accumulation of extracellular oxalate. Our findings provide steps in untangling the complex interplay of different mechanisms underlyingA. nigercitrate accumulation, and we identify, for the first time, a fungal citrate exporter, offering a valuable tool for improvement ofA. nigeras biotechnological cell-factory for organic acid production.

Author Summary

Citrate is widely applied as acidifier, flavouring and chelating agent. Industrial citrate production currently relies on the filamentous fungusAspergillus niger. Although the industrial production process usingA. niger has vastly improved since initiated almost 100 years ago, citrate export remains a bottleneck. Here, we studied the gene expression pattern ofA. nigerunder 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 yeastSaccharomyces 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 inA. nigeritself, 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 ofA. nigeras biotechnological cell-factory for the production of other organic acids.