Mitochondrial Citrate Transporters CtpA and YhmA Are Required for Extracellular Citric Acid Accumulation and Contribute to Cytosolic Acetyl Coenzyme A Generation in Aspergillus luchuensis mut. kawachii

Abstract

Aspergillus luchuensismut.kawachii(A. kawachii) produces 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 counterexchange transport activity; CtpA transported citrate using countersubstrates, especiallycis-aconitate and malate, whereas YhmA transported citrate using a wider variety of countersubstrates, including citrate, 2-oxoglutarate, malate,cis-aconitate, and succinate. Disruption ofctpAandyhmAcaused deficient hyphal growth and conidium formation with reduced mycelial weight-normalized citrate production. Because we could not obtain a ΔctpAΔyhmAstrain, we constructed an S-taggedctpA(ctpA-S) conditional expression strain in the ΔyhmAbackground using the Tet-On promoter system. Knockdown ofctpA-Sin ΔyhmAresulted in a severe growth defect on minimal medium with significantly reduced acetyl coenzyme A (acetyl-CoA) and lysine levels, indicating that double disruption ofctpAandyhmAleads 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 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, conidium 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.

LaeA Controls Citric Acid Production through Regulation of the Citrate Exporter-EncodingcexAGene in Aspergillus luchuensis mut.kawachii

“…For the evaluation of citric acid production, A. kawachii strains were cultivated in CAP medium after precultivation in M medium. CAP medium was used for enhancing citric acid production concomitant with reduced hyphal growth (24). Fungal pellet morphology is known to affect the production of organic acids, proteins, and secondary metabolites (39), and elevated citric acid production occurs with shorter hyphae or hyperbranched phenotypes in A. niger (40,41).…”

“…Next, we focused on the citric acid export process, as it is considered to play a significant role in the high citric acid production by A. niger (21-23) ( Table 2). Our group previously reported that the two mitochondrial citrate transporters CtpA (ctpA [AKAW_03754]) and YhmA (yhmA [AKAW_06280]) (24) are involved in the transport of citric acid from mitochondria to cytosol: however, their gene expression levels did not significantly change. Conversely, a significant reduction in gene expression (Ϫ6.09 log 2 fold) of the putative citrate exporter-encoding cexA (AKAW_07989).…”

Plant Mitochondrial Carriers: Molecular Gatekeepers That Help to Regulate Plant Central Carbon Metabolism

“…The most widely used system for studying the transport properties of MCFs is through expression-purification-reconstitution assays [18]. In this approach, the candidate gene is cloned and homologously [84] or heterologously expressed in either E. coli [85,86], Lactococcus lactis [87], or yeast [85,87]. The overexpressed protein is isolated and reconstituted into liposomes and the transport activity is measured by direct uptake or export of radioactively labeled substrates.…”

LaeA controls citric acid production through regulation of a citrate exporter encodingcexAinAspergillus luchuensismut.kawachii

“…Next, we focused on the citric acid export process as it is considered to play a significant role in the high citric acid production of A. niger (21–23) (Table 2). Our group previously reported that the two mitochondrial citrate transporters ctpA (AKAW_03754) and yhmA (AKAW_06280) (24) are involved in the transport of citric acid from mitochondria to cytosol: however, their gene expression levels did not significantly change. Conversely, a significant change in gene expression (0.01-fold reduction) of the putative citrate exporter encoding cexA (AKAW_07989) was identified as a citrate exporter from cytosol to extracellular in A. niger (15, 16).…”

“…Levels of extracellular and intracellular organic acids were measured as described previously (24). Briefly, 2 × 10 7 conidia cells of A. kawachii were inoculated into 100 ml M medium, precultured with shaking (180 rpm) at 30°C for 36 h, and then transferred into 50 ml CAP medium and further cultured with shaking (163 rpm) at 30°C for 48 h. The culture supernatant was harvested as the extracellular fraction.…”