[4S02e] 03. Fermentation Physiology/ Technology - Part 3

6:10 PM - 6:30 PM JST(9:10 AM - 9:30 AM UTC)

[4Ferm-14] Citrate exporter governs extracellular citric acid accumulation in koji fungi

○Taiki Futagami^1,2, Chihiro Kadooka³, Atsushi Nishitani⁴, Masatoshi Goto^2,5, Hisanori Tamaki^1,2 (1. Fac. Agric., Kagoshima Univ. (Japan), 2. United Grad. Sch. Agric. Sci., Kagoshima Univ. (Japan), 3. Fac. Biotechnol. Life Sci., Sojo Univ. (Japan), 4. Cent. Adv. Sci. Res. Promot., Kagoshima Univ. (Japan), 5. Fac. Agric., Saga Univ. (Japan))

Keywords:

Koji fungi,Citric acid,Exporter

The white koji fungus Aspergillus luchuensis mut. kawachii is used in the production of shochu, a traditional Japanese distilled spirit. A. kawachii plays a crucial role in the shochu production process by supplying amylolytic enzymes that convert starch present in primary ingredients such as rice, barley, buckwheat, and sweet potato into glucose, which is subsequently utilized by the yeast Saccharomyces cerevisiae to produce ethanol. In addition, A. kawachii secretes substantial amounts of citric acid, thereby lowering the pH of the shochu mash. This acidic environment prevents the growth of undesirable microorganisms, ensuring stable shochu production, particularly in the warmer regions of Japan. Our research focuses on understanding the mechanism of citric acid production in A. kawachii.
We investigated CexA (citrate exporter A) in A. kawachii, as previous studies have shown that CexA exports citric acid across the plasma membrane in Aspergillus niger, a close relative of A. kawachii, and plays a significant role in citric acid production. Disruption of AkcexA markedly decreased extracellular citric acid accumulation, whereas overexpression of AkcexA enhanced extracellular citric acid accumulation in A. kawachii. This result indicates that CexA-mediated citrate export is also important for citric acid production in A. kawachii.The yellow koji fungus Aspergillus oryzae, which is used in the production of miso, soy sauce, and sake, does not produce large amounts of citric acid. However, we identified two cexA homologs (AocexA and AocexB) encoded in the genome of A. oryzae. Transcriptomic data indicated that the expression levels of these two homologs are relatively low. Therefore, we evaluated the effects of overexpression of endogenous AocexA and AocexB genes as well as the AkcexA gene in A. oryzae under the control of the α-amylase gene promoter. Overexpression of AkcexA in A. oryzae significantly increased extracellular citric acid production, reaching levels comparable to that observed in A. kawachii. Overexpression of the two endogenous AocexA and AocexB also enhanced extracellular citric acid accumulation by A. oryzae, indicating that A. oryzae has the potential to produce a large amount of citric acid. These findings suggest that the expression level of the citrate exporter is one of the late limiting steps regulating citric acid production in koji fungi.