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

2018 ◽  
Author(s):  
Chihiro Kadooka ◽  
Kosuke Izumitsu ◽  
Masahira Onoue ◽  
Kayu Okutsu ◽  
Yumiko Yoshizaki ◽  
...  
Keyword(s):  
Citric Acid ◽  
Acid Production ◽  
Hyphal Growth ◽  
Lysine Biosynthesis ◽  
Citric Acid Production ◽  
Growth Defect ◽  
Citrate Transport ◽  
Severe Growth Defect ◽  
Severe Growth ◽  
Aspergillus Luchuensis

ABSTRACTAspergillus 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.

scholarly journals Mitochondrial Citrate Transporters CtpA and YhmA Are Required for Extracellular Citric Acid Accumulation and Contribute to Cytosolic Acetyl Coenzyme A Generation inAspergillus luchuensismut.kawachii

2019 ◽  
Vol 85 (8) ◽  
Author(s):  
Chihiro Kadooka ◽  
Kosuke Izumitsu ◽  
Masahira Onoue ◽  
Kayu Okutsu ◽  
Yumiko Yoshizaki ◽  
...  
Keyword(s):  
Citric Acid ◽  
Hyphal Growth ◽  
Growth Defect ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Content Type ◽  
Conidium Formation ◽  
Severe Growth Defect ◽  
Severe Growth ◽  
Aspergillus Luchuensis

ABSTRACTAspergillus 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.

scholarly journals LaeA controls citric acid production through regulation of a citrate exporter encoding cexA in Aspergillus luchuensis mut. kawachii

2019 ◽  
Author(s):  
Chihiro Kadooka ◽  
Eri Nakamura ◽  
Kazuki Mori ◽  
Kayu Okutsu ◽  
Yumiko Yoshizaki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Citric Acid ◽  
Epigenetic Regulation ◽  
Chromatin Immunoprecipitation ◽  
Acid Production ◽  
Citric Acid Production ◽  
Complementation Test ◽  
Development Processes ◽  
Cap Analysis ◽  
Aspergillus Luchuensis

ABSTRACTThe putative methyltransferase LaeA is a global regulator of metabolic and development processes in filamentous fungi. We characterized the homologous laeA genes of the white koji fungus Aspergillus luchuensis mut. kawachii (A. kawachii) to determine their role in the hyperproduction of citric acid. The ΔlaeA strain exhibited a significant reduction in citric acid productivity. Cap-analysis gene expression (CAGE) revealed that laeA is required for the gene expression of a putative citrate exporter encoding cexA, which is critical for citric acid production. The deficit in the productivity of citric acid by the ΔlaeA strain was rescued by the overexpression of cexA to a level comparable with that of the cexA-overexpressing ΔcexA strain. In addition, chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) analysis indicated that LaeA regulates the expression of cexA via methylation levels of the histones H3K4 and H3K9. These results indicate that LaeA is involved in citric acid production through epigenetic regulation of cexA in A. kawachii.IMPORTANCEA. kawachii has been traditionally used for production of the distilled spirit shochu in Japan. Citric acid produced by A. kawachii plays an important role in preventing microbial contamination during the shochu fermentation process. This study characterized homologous laeA genes; using CAGE, complementation test, and ChIP-qPCR, it was found that laeA is required for citric acid production though regulation of cexA in A. kawachii. The epigenetic regulation of citrate production elucidated in this study will be useful for controlling the fermentation processes of shochu.

scholarly journals LaeA Controls Citric Acid Production through Regulation of the Citrate Exporter-Encoding cexA Gene in Aspergillus luchuensis mut. kawachii

2019 ◽  
Vol 86 (5) ◽  
Author(s):  
Chihiro Kadooka ◽  
Eri Nakamura ◽  
Kazuki Mori ◽  
Kayu Okutsu ◽  
Yumiko Yoshizaki ◽  
...  
Keyword(s):  
Citric Acid ◽  
Epigenetic Regulation ◽  
Chromatin Immunoprecipitation ◽  
Acid Production ◽  
Citric Acid Production ◽  
Global Regulator ◽  
Qpcr Analysis ◽  
Development Processes ◽  
Cap Analysis ◽  
Aspergillus Luchuensis

ABSTRACT The putative methyltransferase LaeA is a global regulator of metabolic and development processes in filamentous fungi. We characterized the homologous laeA genes of the white koji fungus Aspergillus luchuensis mut. kawachii (A. kawachii) to determine their role in citric acid hyperproduction. The ΔlaeA strain exhibited a significant reduction in citric acid production. Cap analysis gene expression (CAGE) revealed that laeA is required for the expression of a putative citrate exporter-encoding cexA gene, which is critical for citric acid production. Deficient citric acid production by a ΔlaeA strain was rescued by the overexpression of cexA to a level comparable with that of a cexA-overexpressing ΔcexA strain. In addition, chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) analysis indicated that LaeA regulates the expression of cexA via methylation levels of the histones H3K4 and H3K9. These results indicate that LaeA is involved in citric acid production through epigenetic regulation of cexA in A. kawachii. IMPORTANCE A. kawachii has been traditionally used for production of the distilled spirit shochu in Japan. Citric acid produced by A. kawachii plays an important role in preventing microbial contamination during the shochu fermentation process. This study characterized homologous laeA genes; using CAGE, complementation tests, and ChIP-qPCR, it was found that laeA is required for citric acid production through the regulation of cexA in A. kawachii. The epigenetic regulation of citric acid production elucidated in this study will be useful for controlling the fermentation processes of shochu.

scholarly journals The Effect of Lipids on Citric Acid Production by an Aspergillus niger Mutant

1963 ◽  
Vol 30 (3) ◽  
pp. 365-379 ◽  
Author(s):  
N. F. MILLIS ◽  
B. H. TRUMPY ◽  
B. M. PALMER
Keyword(s):  
Citric Acid ◽  
Aspergillus Niger ◽  
Acid Production ◽  
Citric Acid Production
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