Abstract
We previously reported that intracellular proline (Pro) confers tolerance to ethanol on the yeast Saccharomyces cerevisiae. In this study, to improve the ethanol productivity of sake, a traditional Japanese alcoholic beverage, we successfully isolated several Pro-accumulating mutants derived from diploid sake yeast of S. cerevisiae by a conventional mutagenesis. Interestingly, one of them (strain A902-4) produced more than 10-fold greater amounts of ornithine (Orn) and Pro compared to the parent strain (K901). Orn is a non-proteinogenic amino acid and a precursor of both arginine (Arg) and Pro. It has some physiological functions, such as amelioration of negative states such as lassitude and improvement of sleep quality. We also identified a homo-allelic mutation in the ARG5,6 gene encoding the Thr340Ile variant N-acetylglutamate kinase (NAGK) in strain A902-4. The NAGK activity of the Thr340Ile variant was extremely insensitive to feedback inhibition by Arg, leading to intracellular Orn accumulation. This is the first report of the removal of feedback inhibition of NAGK activity in the industrial yeast, leading to high levels of intracellular Orn. Moreover, sake and sake cake brewed with strain A902-4 contained 4–5 times more Orn than those brewed with strain K901. The approach described here could be a practical method for the development of industrial yeast strains with overproduction of Orn.
Highlights
During the fermentation of sake, a traditional Japanese alcoholic beverage, the yeast Saccharomyces cerevisiae is exposed to high concentrations of ethanol which inhibits the growth, viability and fermentation of yeast cells (Kodama, 1993)
We identified a homo-allelic mutation in the ARG5,6 gene encoding the Thr340Ile variant N-acetylglutamate kinase (NAGK) in strain A902-4
There might be the other genomic mutations which contribute to the Orn overproduction in strain A902-4, these results indicate that the amino acid replacement of Thr to Ile at position 340 in N -Acetyl glutamate kinase (NAGK) confers Pro and
Summary
During the fermentation of sake, a traditional Japanese alcoholic beverage, the yeast Saccharomyces cerevisiae is exposed to high concentrations of ethanol which inhibits the growth, viability and fermentation of yeast cells (Kodama, 1993). Increased tolerance to ethanol in sake yeast strains could improve ethanol productivity and reduce fermentation time. Ethanol tolerance would simplify the production process of bioethanol. Intracellular proline (Pro) can be expected to alleviate the ethanol toxicity (Takagi et al, 2016). S. cerevisiae cells synthesize Pro from glutamate (Fig. 1). Γ-Glutamyl kinase (GK) is the rate-limiting enzyme of. S. cerevisiae has an alternative pathway for Pro synthesis from arginine (Arg) via ornithine (Orn) (Fig. 1). N -Acetyl glutamate kinase (NAGK) is one of the rate-limiting enzymesin Arg biosynthesis, and its activity is subject to feedback inhibition by Arg
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