Effects of a novel variant of the yeast \u03b3-glutamyl kinase Pro1 on its enzymatic activity and sake brewing

Naoyuki Murakami,Kengo Matsumura,Keiko Ashida,Shota Isogai,Akira Nishimura,Atsushi Kotaka,Hiroki Ishida,Hiroshi Takagi,Yoji Hata

doi:10.1007/s10295-020-02297-1

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.

Highlights

Sake is a traditional Japanese alcoholic beverage made from steamed rice by multiple parallel fermentations of the fungus Aspergillus oryzae and the yeast Saccharomyces cerevisiae, which produce saccharification enzymes and ethanol from glucose, respectively
Many proline-accumulating mutant strains [7] were confirmed to have mutations in the PRO1 gene encoding glutamyl kinase (GK), which is a key enzyme in proline biosynthesis in S. cerevisiae [5, 10, 15, 24]
The results showed that the sequence of the PRO1 gene in Kyokai no. 9 (K-9) was identical to that in strain Kyokai no. 7 (K-7)

Summary

Introduction

Sake is a traditional Japanese alcoholic beverage made from steamed rice by multiple parallel fermentations of the fungus Aspergillus oryzae and the yeast Saccharomyces cerevisiae, which produce saccharification enzymes and ethanol from glucose, respectively. The taste of sake is determined by a combination of many compounds such as sugars, organic acids, amino acids, nucleotides, and inorganic salts. Amino acids and other taste components originate mainly from S. cerevisiae cells, and a variety of yeast strains have been constructed to develop differentiated sake products [6]. From strain K-9, many mutants including strain K-9-AZC were isolated from minimal agar medium containing 500 mg/l of toxic proline analogue azetidine2-carboxylic acid (AZC) by spontaneous mutagenesis [7]. Microorganisms that overproduce various amino acids have been obtained by isolating mutants resistant to analogues of corresponding amino acids. Some AZC-resistant mutants were found to accumulate larger amounts of intracellular proline than the parent strain. The majority of proline-accumulating strains have been obtained by expressing GK variants, such as Asp154Asn (D154N) and Ile150Thr (I150T), which are less sensitive to feedback inhibition by proline [10, 15]

Methods

Results

Conclusion