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

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.