Molecular mechanism for the aldehyde tolerance of S. cerevisiae
and its application in the fermentation industry
Tomoyuki NAKAGAWA
Faculty of Applied Biological sciences, Gifu University
Research aims
Acetaldehyde is a volatile organic chemical compound, which serves as an importantintermediate product in several intracellular metabolic pathways, contributing to amino acidmetabolism, ethanol detoxification, and ethanol fermentation. However, acetaldehyde readily
forms adducts with both protein and DNA, inhibiting cell growth and interfering with a wide
range of cellular metabolic activities. Therefore, despite a central role in many criticalpathways, acetaldehyde also exhibits high toxicity and has irritant properties, mutability, andmay be carcinogenic to living organisms.
The budding yeast Saccharomyces cerevisiae is likely to have evolved an especially welldeveloped acetaldehyde tolerance system owing to the fact the species produces
acetaldehyde as the predominant intermediate during anaerobic metabolism and thegeneration of ethanol.
In this study, we demonstrated the molecular mechanisms for acetaldehyde tolerance of S.
cerevisiae, and apply an understanding of these mechanisms to molecular breeding ofindustrial yeast strains.
Methods
S. cerevisiae strain BY4741 was used as the wild type strain. The gene disrupted strains of
BY4741 used in this study were purchased from Invitrogen Co. (Carlsbad, CA., USA).
Cell-free extracts from yeast strains were prepared using a method outlined in Oku et al.1).
Intracellular levels of NADP+/NADPH were identified using a Prominence nano HPLC
(Shimadzu)-4000QTRAP Mass spectrograph system (AB SCIEX) (1).

Results
1. Physiological role and functions of the intracellular redox regulation system inacetaldehyde tolerance of S. cerevisiae.
Under acetaldehyde stress, the intracellular NADP+/NADPH ratio in the S. cerevisiae strain
was increased. In strain zwf1Δ, which shows particularly high acetaldehyde sensitivity2), theNADP+/NADPH ratio in the cell was significantly increased under acetaldehyde stress. Thisindicates that the main function of the pentose phosphate pathway in acetaldehyde tolerance
is to supply NADPH to the acetaldehyde tolerance system.
Additionally, under acetaldehyde stress, the intracellular levels of NADP+/NADPH
concentrations in S. cerevisiae increased. This demonstrates that S. cerevisiae not only
controls the NADP+/NADPH ratio, but also the production of NADP+/NADPH underacetaldehyde stress. We observed specific phenotypes in the NADP+/NADPH kinasegene-disrupted strains. Strain pos5Δ, in which the mitochondrial NADP+/NADPH kinase
gene is deleted, showed severe acetaldehyde sensitivity. Strain yef1Δutr1Δ, in whichcytosolic NADP+/NADPH kinase genes were deleted, also demonstrated acetaldehyde
sensitivity. Furthermore, among the NADP+/NADPH kinase genes, YEF1 was found to beacetaldehyde-inducible.
2. Physiological roles and functions of the glutathione degradation pathway inacetaldehyde tolerance of S. cerevisiae.
S. cerevisiae has at least three acetaldehyde tolerance pathways, each requiring NADPH for
their activities (2). These acetaldehyde tolerance pathways include the glutathione (GSH)
synthesis and reduction pathways2). We have already demonstrated that GSH forms adductswith acetaldehyde3), briefly reducing acetaldehyde toxicity3). However, GSH alone cannot
explain the whole mechanism of acetaldehyde tolerance. On the other hand, Anni et al.
reported that Cys-Gly, a decomposition product from GSH, has an ability to form adductswith acetaldehyde4). We have therefore observed a possible effect that the GSH degrading
pathway has on the acetaldehyde tolerance system. Expression of Dug2/Dug3 was up
regulated by acetaldehyde stress, and strain dug2Δ showed slightly elevated acetaldehyde
sensitivity. However, intracellular Cys-Gly level was maintained despite the presence orabsence of acetaldehyde.
From these results, it seems that S. cerevisiae uses both GSH and Cys-Gly as anacetaldehyde scavenger in the cell, although it is possible that GSH is the primary scavenger,rather than Cys-Gly.
Conclusions
In this study, we have shown that PPP plays an important role in the acetaldehyde tolerance
system of S. cerevisiae by supplying NADPH. NAD+/NADH kinases also contribute byelevating NADPH levels in the cell. Pos5, a NAD+/NADH kinase localized in mitochondria,
also has an important function in acetaldehyde tolerance. Mitochondria possess the synthesis
pathway for oleic acid, a key component for acetaldehyde tolerance (2) and are therefore key
organelles in acetaldehyde tolerance.

GSH, which requires NADPH for reduction, is also involved in the acetaldehyde tolerance ofS. cerevisiae (3)and may be more important for this function than Cys-Gly.
These findings can be applied to improve molecular breeding of industrial budding yeasts.
References(1) Oku, M., Hozeki, J., Ichiki, Y. and Sakai, Y. (2013). A fluorescence resonance energy
transfer (FRET)-based redox sensor reveals physiological role of thioredoxin in theyeast Saccharomyces cerevisiae. FEBS Lett. 587: 793-798.
(2) Matsufuji, Y., Fujimura, S., Ito, T., Nishizawa, M., Miyaji, T., Nakagawa, J., Ohyama, T.,Tomizuka, N. and Nakagawa, T. (2008). Acetaldehyde tolerance in Saccharomycescerevisiae involves the pentose phosphate pathway and oleic acid biosynthesis. Yeast.
25: 825-833.
(3) Matsufuji, Y., Yamamoto, K., Yamauchi, K., Mitsunaga, T., Hayakawa, T. and Nakagawa,T. (2013). The novel physiological roles for glutathione in sequestering acetaldehyde toconfer acetaldehyde tolerance in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol.
97: 297-303.
(4) Anni, H., Pristatsky, P. and Israel, Y. (2003). Binding of acetaldehyde to a glutathione
metabolite: mass spectrometric characterization of an acetaldehyde-cysteinylglycineconjugate. Alcohol Clin. Exp. Res. 27: 1613-1621.