Characterization of an .ALPHA.-Ketoglutarate-Resistant Sake Yeast Mutant with High Organic Acid Productivity

Abstract

A yeast with high organic acid productivity was isolated from α-ketoglutarate-resistant mutants obtained by mutagenizing a sake yeast, Kyokai no. 701 (K-701). The new strain, 20G-R39, produces about twice as much malate and succinate as the parental strain. DNA microarray analyses revealed that the transcriptional levels of genes involved in the TCA cycle, oxidative phosphorylation, and respiration were higher in strain 20G-R39 than in strain K-701. Expression of these genes is regulated by the Hap2/3/4/5p complex, and especially by expression of the HAP4 gene. In a Northern blot analysis, the transcriptional level of the HAP4 gene was higher in strain 20G-R39 than in strain K-701. We constructed a plasmid that expresses the HAP4 gene constitutively and introduced it into strain K-701. The HAP4 -overexpression-strain produced more malate and succinate than strain K-701 both in YPD medium and in a sake brewing test. These results indicate that strain 20G-R39 produces more organic acids than strain K-701 because strain 20G-R39 has a higher level of expression of the HAP4 gene than strain K-701.