Transcriptomics identify the triggering of citrate export as the key event caused by manganese deficiency in Aspergillus niger.

For over a century, the filamentous Ascomycete fungus Aspergillus niger has played a pivotal role in the industrial production of citric acid. A critical fermentation parameter that sustains high-yield citric acid accumulation is the suboptimal concentration of manganese(II) ions in the culture broth at the early stages of the process. However, the requirement for this deficiency has not been investigated on a functional genomics level. In this study, we compared the transcriptome of the citric acid hyper-producer A. niger NRRL2270 strain grown under citric acid-producing conditions in 6-L scale bioreactors at Mn2+ ion-deficient (5 ppb) and Mn2+ ion-sufficient (100 ppb) conditions at three early time points of cultivation. Of the 11,846 genes in the genome, 963 genes (8.1% of the total) were identified as significantly differentially expressed under these conditions. Disproportionately high number of differentially regulated genes encode predicted extracellular and membrane proteins. The most abundant gene group that was upregulated in Mn2+ ion deficiency condition encodes enzymes acting on polysaccharides. In contrast, six clusters of genes encoding secondary metabolites showed downregulation under manganese deficiency. Mn2+ deficiency also triggers upregulation of the cexA gene, which encodes the citrate exporter. We provide functional evidence that the upregulation of cexA is caused by the intracellular accumulation of citrate or acetyl-CoA and is a major factor in triggering citrate overflow. IMPORTANCE: Citric acid is produced on industrial scale by batch fermentation of the filamentous fungus Aspergillus niger. High-yield citric acid production requires a low (<5 ppb) manganese(II) ion concentration in the culture broth. However, the requirement for this deficiency has not been investigated on a functional genomics level. Here, we compared the transcriptome of a citric acid hyper-producer A. niger strain grown under citric acid-producing conditions in 6-L scale bioreactors at Mn2+ ion-deficient (5 ppb) and Mn2+ ion-sufficient (100 ppb) conditions at three early time points of cultivation. We observed that Mn2+ deficiency triggers an upregulation of the citrate exporter gene cexA and provides functional evidence that this event is responsible for citrate overflow. In addition to the industrial relevance, this is the first study that examined the role of Mn2+ ion deficiency in a heterotrophic eukaryotic cell on a genome-wide scale.

Bioreactor as the root cause of the "manganese effect" during Aspergillus niger citric acid fermentations.

High-yield citric acid production by the filamentous Ascomycete fungus Aspergillus niger requires a combination of extreme nutritional conditions, of which maintaining a low manganese (II) ion concentration (<5 µg L-1) is a key feature. Technical-scale production of citric acid predominantly uses stainless-steel tank fermenters, but glass bioreactors used for strain improvement and manufacturing process development also contain stainless steel components, in which manganese is an essential alloying element. We show here that during citric acid fermentations manganese (II) ions were leaching from the bioreactor into the growth media, resulting in altered fungal physiology and morphology, and significant reduction of citric acid yields. The leaching of manganese (II) ions was dependent on the fermentation time, the acidity of the culture broth and the sterilization protocol applied. Manganese (II) ion leaching was partially mitigated by electrochemical polishing of stainless steel components of the bioreactor. High concentrations of manganese (II) ions during early cultivation led to a reduction in citric acid yield. However, the effect of manganese (II) ions on the reduction of citric acid yield diminished towards the second half of the fermentation. Since maintaining low concentrations of manganese (II) ions is costly, the results of this study can potentially be used to modify protocols to reduce the cost of citric acid production.