Citrate exporter CexA plays a key role in the production of citric acid in fungi; however, its role in intracellular metabolism has remained unclear. In this study, we comparably characterized homologous cexA genes in the white koji fungus Aspergillus luchuensis mut. kawachii and the yellow koji fungus Aspergillus oryzae, which exhibit high and low abilities, respectively, to produce citric acid. Disruption of cexA caused a significant decline of both extracellular and intracellular citric acid accumulation in Aspergillus kawachii, while overexpression of the A. kawachii cexA gene (AkcexA) into A. oryzae significantly enhanced both extracellular and intracellular citric acid accumulation in A. oryzae to a level comparable to that of A. kawachii. In addition, overexpression of two intrinsic cexA homologs (AocexA and AocexB) in A. oryzae also enhanced its extracellular and intracellular citric acid accumulation. Comprehensive analysis of intracellular metabolites from an AkcexA-overexpressing strain of A. oryzae compared with its control strain identified metabolic changes associated with intracellular citric acid accumulation via the glycolytic pathway, pentose phosphate pathway, and tricarboxylic acid cycle. Our results indicate that citric acid export enhances not only extracellular citric acid accumulation but also intracellular metabolic fluxes to generate citric acid.

柠檬酸盐出口商CexA在真菌柠檬酸生产中起着关键作用。然而，其在细胞内代谢中的作用仍不清楚。在这项研究中，我们比较了白曲霉真菌曲霉曲霉的同源cexA基因的特征。kawachii和黄色曲霉米曲霉分别具有高和低的柠檬酸生产能力。cexA的破坏导致kawachii曲霉胞外和细胞内柠檬酸积累显着下降，而kawachii cexA基因（AkcexA）过度表达到米曲霉中。显着增强了米曲霉细胞外和细胞内柠檬酸的积累，达到了与川崎曲霉相当的水平。此外，米曲霉中两个固有的cexA同源物（AocexA和AocexB）的过表达也增强了其胞外和胞内柠檬酸的积累。过量表达米曲霉AkcexA菌株细胞内代谢产物的综合分析与它的对照菌株相比，通过糖酵解途径，磷酸戊糖途径和三羧酸循环确定了与细胞内柠檬酸积累有关的代谢变化。我们的结果表明，柠檬酸的输出不仅增强了细胞外柠檬酸的积累，而且还增强了细胞内代谢通量以产生柠檬酸。