Publications

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

  Odoni D.I., Vazquez-Vilar M., van Gaal M.P., Schonewille T., Martins Dos Santos V.A.P., Tamayo-Ramos J.A., Suarez-Diez M., Schaap P.J.

  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 ... >> More

  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. << Less

  FEMS Microbiol. Lett. 366:0-0(2019) [PubMed] [EuropePMC]

• Identification of the citrate exporter Cex1 of Yarrowia lipolytica.

  Erian A.M., Egermeier M., Rassinger A., Marx H., Sauer M.

  Yarrowia lipolytica is a yeast with many talents, one of them being the production of citric acid. Although the citrate biosynthesis is well studied, little is known about the transport mechanism by which citrate is exported. To gain better insight into this mechanism, we set out to identify a tra ... >> More

  Yarrowia lipolytica is a yeast with many talents, one of them being the production of citric acid. Although the citrate biosynthesis is well studied, little is known about the transport mechanism by which citrate is exported. To gain better insight into this mechanism, we set out to identify a transporter involved in citrate export of Y. lipolytica. A total of five proteins were selected for analysis based on their similarity to a known citrate exporter, but neither a citrate transport activity nor any other phenotypic function could be attributed to them. Differential gene expression analysis of two strains with a distinct citrate productivity revealed another three putative transporters, one of which is YALI0D20196p. Disrupting YALI0D20196g in Y. lipolytica abolished citrate production, while extrachromosomal expression enhanced citrate production 5.2-fold in a low producing wildtype. Furthermore, heterologous expression of YALI0D20196p in the non-citrate secreting yeast Saccharomyces cerevisiae facilitated citrate export. Likewise, expression of YALI0D20196p complemented the ability to secrete citrate in an export-deficient strain of Aspergillus niger, confirming a citrate export function of YALI0D20196p. This report on the identification of the first citrate exporter in Y. lipolytica, termed Cex1, represents a valuable starting point for further investigations of the complex transport processes in yeasts. << Less

  FEMS Yeast Res. 0:0-0(2020) [PubMed] [EuropePMC]

• Engineering of the citrate exporter protein enables high citric acid production in Aspergillus niger.

  Steiger M.G., Rassinger A., Mattanovich D., Sauer M.

  Aspergillus niger was engineered using a gene responsible for citric acid transport, which has a significant impact on citric acid secretion when overexpressed. The transport gene was identified by a homology search using an itaconic acid transporter from Ustilago maydis as template. The encoding ... >> More

  Aspergillus niger was engineered using a gene responsible for citric acid transport, which has a significant impact on citric acid secretion when overexpressed. The transport gene was identified by a homology search using an itaconic acid transporter from Ustilago maydis as template. The encoding homologous protein CexA belongs to the major facilitator superfamily subclass DHA1 and members of this family work as drug-H⁺ antiporter. The disruption of this gene completely abolishes citric acid secretion, which indicates that this protein is the main citric acid transporter in A. niger. In the disruption strain, the metabolism is re-routed mainly to oxalic acid, which is a known by-product during citric acid production. The gene can be heterologously expressed in Saccharomyces cerevisiae, which leads to the secretion of citric acid during the growth on glucose. These results confirm the functionality of CexA as the main transporter for citric acid of A. niger. Overexpression of cexA leads to a significant increase in secreted citric acid. Thereby, striking differences between a strong constitutive expression system using pmbfA as a promoter and an inducible expression system using ptet-on can be observed. The inducible system significantly outcompetes the constitutive expression system yielding up to 109 g/L citric acid, which is 5 times higher compared to the parental wild-type strain and 3 times higher compared to the constitutive expression system. These results demonstrate the importance of the cellular transport system for an efficient production of metabolites. By overexpressing a single gene, it is possible to significantly improve the citric acid secretion capability of a moderately producing parental strain. << Less

  Metab. Eng. 52:224-231(2019) [PubMed] [EuropePMC]

• Uptake and export of citric acid by Aspergillus niger is reciprocally regulated by manganese ions.

  Netik A., Torres N.V., Riol J.M., Kubicek C.P.

  The uptake as well as the export of citric acid by Aspergillus niger occur by active, deltapH-driven, H(+)-symport dependent systems. They are inhibited by nonmetabolizable tricarboxylic acid analogues and phthalic acid, and by several other mono-, di- and tribasic organic acids. However, citrate ... >> More

  The uptake as well as the export of citric acid by Aspergillus niger occur by active, deltapH-driven, H(+)-symport dependent systems. They are inhibited by nonmetabolizable tricarboxylic acid analogues and phthalic acid, and by several other mono-, di- and tribasic organic acids. However, citrate export could only be demonstrated in a mycelium cultivated under manganese-deficient growth conditions, whereas the uptake of citrate from the medium was only detectable upon precultivation of A. niger in a medium supplemented with Mn2+ ions. In addition, the uptake of citrate was dependent on the presence of Mn2+ ions in the assay, and inhibited by EDTA. This requirement for Mn2+ could also be partially fulfilled by Mg2+, Fe2+ or Zn2+, whereas Cu2+ ions inhibited citrate transport. The observed divergent effects of manganese ions on citrate uptake and citrate export may be a major reason for the well documented requirement for manganese deficiency of citric acid accumulation. << Less

  Biochim Biophys Acta 1326:287-294(1997) [PubMed] [EuropePMC]