Mitochondrial citrate transporters CtpA and YhmA are involved in lysine biosynthesis in the white koji fungus,Aspergillus luchuensismut. kawachii

Aspergillus luchuensismut. kawachiiproduces a large amount of citric acid during the process of fermenting shochu, a traditional Japanese distilled spirit. In this study, we characterizedA. kawachiiCtpA and YhmA, which are homologous to the yeastSaccharomyces cerevisiaemitochondrial citrate transporters Ctp1 and Yhm2, respectively. CtpA and YhmA were purified fromA. kawachiiand reconstituted into liposomes. The proteoliposomes exhibited only counter-exchange transport activity; CtpA transported citrate using counter substrates especially forcis-aconitate and malate, whereas YhmA transported citrate using a wider variety of counter substrates, including citrate, 2-oxoglutarate, malate,cis-aconitate, and succinate. Disruption ofctpAandyhmAcaused deficient hyphal growth and conidia formation with reduced mycelial weight–normalized citrate production. Because we could not obtain a ΔctpAΔyhmAstrain, we constructed actpA-Sconditional expression strain in the ΔyhmAbackground using the Tet-On promoter system. Knockdown ofctpA-Sin ΔyhmAresulted in a severe growth defect on minimal medium, indicating that double disruption ofctpAandyhmAleads to synthetic lethality; however, we subsequently found that the severe growth defect was relieved by addition of lysine. Our results indicate that CtpA and YhmA are mitochondrial citrate transporters involved in citric acid production and that transport of citrate from mitochondria to the cytosol plays an important role in lysine biogenesis inA. kawachii.IMPORTANCECitrate transport is believed to play a significant role in citrate production by filamentous fungi; however, details of the process remain unclear. This study characterized two citrate transporters fromAspergillus luchuensismut. kawachii. Biochemical and gene disruption analyses showed that CtpA and YhmA are mitochondrial citrate transporters required for normal hyphal growth, conidia formation, and citric acid production. In addition, this study provided insights into the links between citrate transport and lysine biosynthesis. The characteristics of fungal citrate transporters elucidated in this study will help expand our understanding of the citrate production mechanism and facilitate the development and optimization of industrial organic acid fermentation processes.