Nishitani A., Hiramatsu K., Kadooka C., Hiroshima K., Sawada K., Okutsu K., Yoshizaki Y., Takamine K., Goto M., Tamaki H., Futagami T. . Overexpression of the DHA1 family, ChlH and ChlK, leads2024 · 記述言語： 日本語 出版者・発行元： Journal of Bioscience and Bioengineering The white koji fungus Aspergillus luchuensis mut. kawachii secretes substantial amounts of citric acid through the expression of the citric acid exporter CexA, a member of the DHA1 family. In this study, we aimed to characterize 11 CexA homologs (Chl proteins) encoded in the genome of A. luchuensis mut. kawachii to identify novel transporters useful for organic acid production. We constructed overexpression strains of chl genes using a cexA disruptant of the A. luchuensis mut. kawachii as the host strain, which prevented excessive secretion of citric acid into the culture supernatant. Subsequently, we evaluated the effects of overexpression of chl on producing organic acids by analyzing the culture supernatant. All overexpression strains did not exhibit significant citric acid accumulation in the culture supernatant, indicating that Chl proteins are not responsible for citric acid export. Furthermore, the ChlH overexpression strain displayed an accumulation of 2-oxoglutaric and fumaric acids in the culture supernatant, while the ChlK overexpression strain exhibited the accumulation of 2-oxoglutaric, malic and succinic acids. Notably, the ChlH and ChlK overexpression led to a substantial increase in the production of 2-oxoglutaric acid, reaching approximately 25 mM and 50 mM, respectively. Furthermore, ChlH and ChlK overexpression also significantly increased the secretory production of dicarboxylic acids, including 2-oxoglutaric acid, in the yellow koji fungus, Aspergillus oryzae. Our study demonstrates that overexpression of DHA1 family gene results in enhanced secretion of organic acids in koji fungi of the genus Aspergillus. DOI： 10.1016/j.jbiosc.2024.01.010 Scopus PubMed
Kobashi Y., Yoshizaki Y., Okutsu K., Futagami T., Tamaki H., Takamine K. . THI3 contributes to isoamyl alcohol biosynthesis through thiamine diphosphate homeostasis . Journal of Biosci2023 · 記述言語： 日本語 出版者・発行元： Journal of Bioscience and Bioengineering Isoamyl alcohol is a precursor of isoamyl acetate, an aromatic compound that imparts the ginjo aroma to sake. The isoamyl alcohol biosynthesis pathway in yeasts involves the genes PDC1, PDC5, PDC6, ARO10, and THI3 encoding enzymes that decarboxylate α-ketoisocaproic acid to isovaleraldehyde. Among these genes, THI3 is the main gene involved in isoamyl alcohol biosynthesis. Decreased production of isoamyl alcohol has been reported in yeast strains with disrupted THI3 (Δthi3). However, it has also been reported that high THI3 expression did not enhance decarboxylase activity. Therefore, the involvement of THI3 in isoamyl alcohol biosynthesis remains unclear. In this study, we investigated the role of THI3 in isoamyl alcohol biosynthesis. While reproducing previous reports of reduced isoamyl alcohol production by the Δthi3 strain, we observed that the decrease in isoamyl alcohol production occurred only at low yeast nitrogen base concentrations in the medium. Upon investigating individual yeast nitrogen base components, we found that the isoamyl alcohol production by the Δthi3 strain reduced when thiamine concentrations in the medium were low. Under low-thiamine conditions, both thiamine and thiamine diphosphate (TPP) levels decreased in Δthi3 cells. We also found that the decarboxylase activity of cell-free extracts of the Δthi3 strain cultured in a low-thiamine medium was lower than that of the wild-type strain, but was restored to the level of the wild-type strain when TPP was added. These results indicate that the loss of THI3 lowers the supply of TPP, a cofactor for decarboxylases, resulting in decreased isoamyl alcohol production. DOI： 10.1016/j.jbiosc.2023.11.006 Scopus PubMed
Kobashi Y., Nakayama E., Fukumori N., Shimojima A., Tabira M., Nishimura Y., Mukae M., Muto A., Nakashima N., Okutsu K., Yoshizaki Y., Futagami T., Takamine K., Tamaki H. . Homozygous gene dis2023 · 記述言語： 日本語 出版者・発行元： Journal of Bioscience and Bioengineering As industrial shochu yeast is a diploid strain, obtaining a strain with mutations in both allelic genes was considered difficult. We investigated a method for disrupting two copies of a homozygous gene with a single transformation. We designed a disruption cassette containing an intact LYS5 flanked by nonfunctional ura3 gene fragments divided into the 5′- and 3′-regions. These fragments had overlapping sequences that enabled LYS5 removal as well as URA3 regeneration through loop-out. Furthermore, both ends of the disruption cassette had an additional repeat sequence that allowed the cassette to be removed from the chromosome through loop-out. First, 45 bases of 5′- and 3′-regions of target gene sequences were added on both ends of this cassette using polymerase chain reaction; the resultant disruption cassette was introduced into a shochu yeast strain (ura3/ura3 lys5/lys5); then, single allele disrupted strains were selected on Lys drop-out plates; and after cultivation in YPD medium, double-disrupted strains, in which replacement of another allelic gene with disruption cassette by loss of heterozygosity and regeneration of URA3 in one of the cassettes by loop-out, were obtained by selection on Ura and Lys drop-out plates. The disruption cassettes were removed from the double-disrupted strain via loop-out between repeat sequences in the disruption cassette. The strains that lost either URA3 or LYS5 were counter-selected on 5-fluoroorotic acid or α-amino adipic acid plates, respectively. Using this method, we obtained leu2/leu2 and leu2/leu2 his3/his3 strains in shochu yeast, demonstrating the effectiveness and repeatability of this gene disruption technique in diploid yeast Saccharomyces cerevisiae. DOI： 10.1016/j.jbiosc.2023.11.003 Scopus PubMed
