Functional analysis of the putative citrate transporter CitT
responsible for the ability to produce citric acid in koji fungi
Taiki FUTAGAMI
Education and Research Center for Fermentation Studies,
Faculty of Agriculture, Kagoshima University
Research aims
The white koji fungus, Aspergillus luchuensis mut. kawachii, has been used for theproduction of shochu, a Japanese traditional distilled spirit. Koji fungi play an important rolein producing amylolytic enzymes for the production of fermented foods. In addition, thewhite koji fungus produces a large amount of citric acid, which decreases the pH of“moromi” (mash) and prevents microbial contamination during the fermentation process.
This ability is a characteristic feature of the white and black koji fungus (Aspergillusluchuensis) among the koji fungi because the yellow koji fungus, Aspergillus oryzae, and theshoyu koji fungus, Aspergillus sojae, do not produce such large amounts of citric acid. In
this study, we characterized the citrate exporter CexA (referred as CitT in the ResearchSubject) in A. kawachii and A. oryzae to elucidate the differences in the citric acidproductivity of koji fungi. We also investigated the epigenetic regulation of cexA in A.
kawachii.
Methods
The cexA homolog of white koji fungus was designated as AkcexA (AKAW_07989). Inaddition, cexA homologs of yellow koji fungus were designated as AocexA(AO090009000645) and AocexB (AO090023000271).
We constructed an AkcexA disruptant strain and an AkcexA overexpression strain (usingthe tefA promoter) in A. kawachii. We also constructed A. oryzae strains overexpressing
AkcexA, AocexA, and AocexB using the amyB promoter. We then made rice koji using thesestrains, and measured the resulting citric acid concentrations. Citric acid productivity wasevaluated per estimated mycelial weight in rice koji based on the nucleic acid content. Inaddition, the intracellular metabolites of the A. oryzae control and AkcexA overexpression
strains were comprehensively analyzed using CE-MS.
We found that the transcriptional level of AkcexA was significantly reduced by thedisruption of a putative methyltransferase encoding laeA in A. kawachii. Thus, weconstructed an AkcexA overexpression strain in the A. kawachii laeA disruptant strain using
the gpdA promoter, which is not controlled by LaeA. We also performed chromatinimmunoprecipitation coupled with quantitative PCR (ChIP-qPCR) to analyze theeuchromatin/heterochromatin ratios of the AkcexA promoter region in A. kawachii.
Results
Recently the citrate exporter CexA was identified in Aspergillus niger and excretion of
intracellular citric acid was demonstrated as a significantly important step for the high citricacid productivity1,2). Thus, we analyzed the relationship between the citric acid productivityof koji fungi and the expression level of cexA. The A. kawachii AkcexA disruptant strain
showed significantly lower (2.3%) citric acid productivity compared to the control strain3). Incontrast, the A. kawachii AkcexA overexpression strain showed significantly higher(2.6-fold) citric acid productivity compared to the control strain. These results suggest thatAkCexA is a major citrate transporter and that the citrate export process is a rate-limiting
step in A. kawachii. Next, we examined whether cexA overexpression could enhance thecitric acid production in A. oryzae. Interestingly, the A. oryzae AkcexA overexpression strain
showed citric acid productivity similar to that of A. kawachii. In addition, A. oryzae AocexAand AocexB overexpression strains also showed significantly higher citric acid production
compared to the control strain, indicating that AoCexA and AoCexB are citrate transporters
in A. oryzae. Moreover, the intracellular citrate concentration was decreased in the A.
kawachii AkcexA disruptant strain, whereas it was increased in the A. oryzae AkcexA, AocexA,and AocexB overexpression strains. This result indicates that CexA enhances both
extracellular and intracellular citric acid accumulation in A. kawachii and A. oryzae. Taken
together, these results suggest that CexA is a significantly important factor in determining
the citrate production ability of koji fungi.
Next, we analyzed the regulatory mechanism of AkcexA gene expression in A. kawachii.
Disruption of a putative methyltransferase encoding laeA gene wqas reported to cause adefect in the citric acid production by A. niger4). Disruption of laeA also markedly reduced
citrate production in A. kawachii5). Because LaeA is known as an epigenetic regulator, weanalyzed the gene expression changes caused by laeA disruption using the Cap Analysis
Gene Expression (CAGE)-sequence. We found that AkcexA expression was significantly
reduced in the laeA disruptant strain and that the forced expression of AkcexA in the laeAdisruptant strain remedied the citric acid productivity. This suggests that LaeA is involved incitric acid production through gene regulation of AkcexA. LaeA is believed to regulate geneexpression by controlling the methylation level of histones. To determine the mechanism
underlying LaeA-dependent AkcexA expression, we performed ChIP-qPCR analysis ofhistone H3, histone H3K4me3, and histone H3K9me3 in A. kawachii control and laeAdisruptant strains. H3K4me3 and H3K9me3 are known as euchromatin and heterochromatinmarkers, respectively. The histone H3 occupancy at the AkcexA promoter did not changebetween the control and AklaeA disruptant strains. However, the euchromatin marker
H3K4me3 at the AkcexA promoter was decreased to a level similar to that of the negative
control in the laeA disruptant strain. Further, the heterochromatin marker H3K9me3 wassignificantly enriched in the laeA disruptant strain compared with that in the control strain.
These results indicate that LaeA controls AkcexA expression by modulating
euchromatin/heterochromatin ratios at the AkcexA promoter region in A. kawachii.
Conclusion
Overexpression of the A. kawachii AkcexA in A. oryzae significantly enhanced citricacid accumulation to a level comparable with that in A. kawachii3). This suggests that CexA
is a significantly important factor determining the citric acid productivity of koji fungi. Inaddition, we revealed that AkcexA expression was regulated by LaeA in A. kawachii5). The
epigenetic regulation of citric acid production elucidated in this study will be useful forcontrolling the citric acid production of koji fungi.
References
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citric acid accumulation in the koji fungi Aspergillus luchuensis mut. kawachii andAspergillus oryzae. J. Biosci. Bioeng. doi: 10.1016/j.jbiosc.2020.09.002 (publishedonline)
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niger by systems genetics: LaeA is required for citric acid production and regulates theformation of some secondary metabolites. G3 (Bethesda) 6: 193-204.
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Environ. Microbiol. 86: e01950-19.