Effects of a novel variant of the yeast γ-glutamyl kinase Pro1 on its enzymatic activity and sake brewing

Abstract

Sake is a traditional Japanese alcoholic beverage brewed with the yeast Saccharomyces cerevisiae. Sake taste is affected by sugars, organic acids, and amino acids. We previously isolated mutants resistant to the proline analogue azetidine-2-carboxylate derived from a diploid sake yeast strain. Some of the mutants produced a greater amount of proline in the brewed sake. One of them (strain K-9-AZC) carried a novel mutation in the PRO1 gene encoding the Gln79His variant of the γ-glutamyl kinase Pro1, a key enzyme in proline biosynthesis in S. cerevisiae. This mutation resulted in extreme desensitization to feedback inhibition by proline, leading to proline overproduction. Interestingly, sake brewed with K-9-AZC contained 3.7-fold more proline, but only 25% less succinate than sake brewed with the parent strain. Metabolome analysis suggests that the decrease in succinate was attributable to a lower level of 2-oxoglutarate, which is converted into glutamate. The approach here could be a practical method for breeding of yeast strains involved in the diversity of sake taste.

High-Level Production of Isoleucine and Fusel Alcohol by Expression of the Feedback Inhibition-Insensitive Threonine Deaminase in Saccharomyces cerevisiae

“…Sake brewing test was carried out according to the previous report ( 41 ) with modification. Briefly, sake mash contained 29 g of pre-gelatinized rice with a polishing ratio of 70%, 7 g of dry koji ( A. oryzae was grown on steamed rice), 150 μl of 9% (vol/vol) lactic acid, 71 ml of water and 1 × 10 9 sake yeast cells.…”

Isolation and analysis of a sake yeast mutant with phenylalanine accumulation

“…After fermentation, the sake mash was centrifuged. The general components of the resulting sake were analyzed by standard methods established by the National Tax Administration Agency (Murakami et al, 2020 ).…”