Mitochondrial citrate transporters CtpA and YhmA are required for extracellular citric acid accumulation and contribute to cytosolic acetyl-CoA generation in Aspergillus luchuensis mut. kawachii .

Aspergillus luchuensis mut . kawachii produces a large amount of citric acid during the process of fermenting shochu, a traditional Japanese distilled spirit. In this study, we characterized A. kawachii CtpA and YhmA, which are homologous to the yeast Saccharomyces cerevisiae mitochondrial citrate transporters Ctp1 and Yhm2, respectively. CtpA and YhmA were purified from A. kawachii and reconstituted into liposomes. The proteoliposomes exhibited only counter-exchange transport activity; CtpA transported citrate using counter substrates especially for cis -aconitate and malate, whereas YhmA transported citrate using a wider variety of counter substrates, including citrate, 2-oxoglutarate, malate, cis -aconitate, and succinate. Disruption of ctpA and yhmA caused deficient hyphal growth and conidia formation with reduced mycelial weight-normalized citrate production. Because we could not obtain a Δ ctpA Δ yhmA strain, we constructed a ctpA-S conditional expression strain in the Δ yhmA background using the Tet-On promoter system. Knockdown of ctpA - S in Δ yhmA resulted in a severe growth defect on minimal medium with a significantly reduced acetyl-CoA and lysine levels, indicating that double disruption of ctpA and yhmA leads to synthetic lethality; however, we subsequently found that the severe growth defect was relieved by addition of acetate or lysine, which could remedy the acetyl-CoA level. 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 acetyl-CoA biogenesis in A. kawachii IMPORTANCE Citrate 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 from Aspergillus luchuensis mut . kawachii Biochemical and gene disruption analyses showed that CtpA and YhmA are mitochondrial citrate transporters required for normal hyphal growth, conidia formation, cytosolic acetyl-CoA synthesis, and citric acid production. 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.