Elucidation of the stationary-phase specific amino acid catabolic
pathway in budding yeast and its application to cell longevity
Akira MATSUURA
Department of Biology, Graduate School of Science, Chiba University
Research objective
The budding yeast Saccharomyces cerevisiae, which has been used in the field of brewing forthousands of years, has been recognized as a model organism to study the basic mechanismsof biological phenomena. The process by which budding yeast cells in liquid culture lose theirviability after the stationary phase is called chronological senescence, and has been analyzed
as a model for the aging of non-proliferating cells. Extensive studies have shown that thesenescence process involves internal factors, such as the production of reactive oxygen species
and the storage of carbohydrates during the stationary phase, as well as external factors, suchas the amount of carbon sources and amino acids and changes in the pH of the culture medium.
In most aging studies of budding yeast, synthetic defined (SD) medium, a minimal synthetic
medium containing ammonium sulfate as a single nitrogen source, has been used. Whenbudding yeast is cultured in this medium, the pH of the medium, which is approximately 4.5,
drops rapidly to approximately 2.2 the next day. When synthetic complete (SC) medium1) withan additional 14 amino acids was used, the decrease in pH was suppressed and the pH wasmaintained at approximately 2.8. We have shown that the amino acid-mediated suppression ofmedium acidification prolongs the lifespan of yeast2). The purpose of this study was to analyze
the mechanism of delayed aging induced by the addition of amino acids, focusing on metabolicchanges in yeast cells. This study is expected to lead to the elucidation of new adaptation
pathways to physiological stresses and to novel strategies for extending the lifespan ofindustrial yeasts.
Methods
Yeast: S. cerevisiae strain YOM36 (MATa wild-type)2)
Medium: SD medium (2% glucose, 0.17% Bacto-yeast nitrogen base without amino acids andammonium sulfate, and 0.5% ammonium sulfate) and AYD medium (Patent 6429627, Kohjin
Life Science Co., Ltd.).
Measurements of the medium composition: The culture medium was centrifuged at 3,000 rpm
for 2 min, and the supernatant was used for various measurements: pH was measured with apH electrode (Mettler Toledo), ammonia concentration with an ammonium electrode (TokoChemical Laboratories Co., Ltd.), and serine concentration with a DL-Serine assay kit (SigmaAldrich).
Results(1) Determination of amino acids that inhibit acidification of the culture medium
We prepared a medium with only one of the amino acids present in the SC medium at thesame molar concentration and measured the pH of the medium during the stationary phase (9days after culturing). The results showed that L-glutamic acid and L-serine had inhibitory
effects on medium acidification, and L-serine had a remarkable inhibitory effect.
We found that the pH of the medium increased during the stationary phase when cultured
in the AYD medium containing high concentrations of amino acids, and the increase in the pH
was not observed when L-serine was removed from the medium. This observation confirmedthat L-serine had the greatest effect on inhibiting acidification of the medium environment.
This effect was not observed when D-serine was used, suggesting that serine uptake andmetabolism in yeast cells is involved in this phenomenon.
(2) Time course of serine consumption in the medium
In the medium supplemented with L-serine, the pH increase occurred after more than one
week in the stationary phase. The amount of serine in the medium decreased at a constant ratefrom the beginning of the culture. In addition, cultivation in an L-serine-supplemented
medium suppressed the consumption of ammonium ions in the medium.
(3) Involvement of the metabolic pathway induced by serine supplementation
Among the genes induced by L-serine addition, CHA1 encodes serine threonine
dehydratase3). This enzyme has the ability to degrade serine into ammonium and pyruvate. In
fact, the Cha1 protein, which is rarely expressed under normal culture conditions, was induced
from the logarithmic growth phase when L-serine was added to the culture.
In the CHA1-deficient strain, the suppression of ammonium ion consumption by serine
addition was no longer observed, and the pH of the culture medium was greatly reduced, aswas observed when the wild-type strain was cultured in serine-free medium. This result
indicates that the ammonium produced by the degradation of serine by the Cha1 protein isutilized as a nitrogen source during the logarithmic growth phase and thereafter.
(4) Function of serine as a carbon source
Although S. cerevisiae can utilize a single amino acid as the sole nitrogen source, it cannot
grow in a medium containing amino acids as a carbon source4). This means that thismicroorganism typically grows in environments with limited nitrogen sources.
Cha1, which plays an important function in the growth of yeast populations after thestationary phase, has the ability to degrade threonine in addition to serine, but the addition ofthreonine did not reproduce the effect of serine. This suggests that pyruvate, rather thanammonium ions, is responsible for the observed effect. The fact that the maximum yield ofcells increases in serine-supplemented cultures suggests that serine is also used as an energy
source under certain conditions. Moreover, the fact that the pH of the medium increased earlyin the culture using the medium fraction recovered from the late stationary phase suggests thata change in the composition of the medium promotes an increase in pH.
Conclusion
In the process of analyzing the medium conditions that delay chronological senescence, weobserved that the survival rate increased when a certain amount of a specific amino acid waspresent in the medium, and that amino acid utilization continued during the stationary phasewhen growth had stopped. Although it is known that budding yeast cannot grow using freeamino acids as a sole carbon source, our results indicate that metabolic reactions using specificamino acids as a source occur in stationary phase cells with limited carbon sources, and thatthese reactions contribute to the maintenance of viability.
Elucidation of the function of amino acid metabolism in S. cerevisiae under extreme
conditions will lead to the elucidation of the ability of this yeast to utilize amino acids, which
has not been observed under normal conditions. Based on the results of this study, we proposethat the use of yeast with constitutively activated amino acid catabolism to avoid the problem
of medium acidification in fermentation using a carbon source, and the suppression ofsenescence and cell death caused by acidification of the culture medium. In the future, we
hope to genetically modify the metabolism of microorganisms to increase their longevity andcontribute to the improvement of material production capacity in the fermentation industry
using S. cerevisiae and other microorganisms.
References
1) Sherman, F. (1991) Getting started with yeast. Meth. Enzymol. 194: 3-21.
2) Maruyama, Y., Ito, T., Kodama, H., and Matsuura, A. (2016) Availability of amino acids
extends chronological lifespan by suppressing hyper-acidification of the environment in
Saccharomyces cerevisiae. PLoS ONE 11: e0151894.
3) Lee, J. C. Y., Tsoi, A., Kornfeld, G. D., and Dawes, I. W. (2013) Cellular responses to Lserine in Saccharomyces cerevisiae: roles of general amino acid control,compartmentalization, and aspartate synthesis. FEMS Yeast Res. 13: 618-634.
4) Large, P. J. (1986) Degradation of organic nitrogen compounds by yeasts. Yeast 2: 1-34.