Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e4, 2014 www.elsevier.com/locate/jbiosc

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Trehalose accumulation enhances tolerance of Saccharomyces cerevisiae to acetic acid Yoko Yoshiyama,1 Koichi Tanaka,1 Kohei Yoshiyama,2 Makoto Hibi,3 Jun Ogawa,4 and Jun Shima1, * Research Division of Microbial Sciences, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-ku, Kyoto 606-8502, Japan,1 River Basin Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan,2 Industrial Microbiology, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-ku, Kyoto 606-8502, Japan,3 and Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-ku, Kyoto 606-8502, Japan4 Received 27 December 2013; accepted 27 June 2014 Available online xxx

Trehalose confers protection against various environmental stresses on yeast cells. In this study, trehalase gene deletion mutants that accumulate trehalose at high levels showed significant stress tolerance to acetic acid. The enhancement of trehalose accumulation can thus be considered a target in the breeding of acetic acid-tolerant yeast strains. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Stress tolerance; Trehalose accumulation; Acetic acids; Propionic acid; Saccharomyces cerevisiae; Bioethanol production]

Tolerance to organic acids in yeast has significant advantages for bioprocesses such as bioethanol production. It is known that acetic acid is generated from acetylated hemicellulose and strongly inhibits the fermentation ability of Saccharomyces cerevisiae during ethanol production from lignocellulose, which is considered a suitable feedstock in second-generation bioethanol production (1). We consider that the utilization of S. cerevisiae with high acetic acid tolerance can prevent stuck fermentation even in the presence of acetic acid. In addition, we have proposed a bioprocess combining organic acids as a bacteriostatic agent and a yeast strain that is tolerant to organic acids (2,3). One of the advantages of this process is that it eliminates the need for antibiotics to prevent bacterial contamination. This is crucial, since antibiotics such as virginiamycin are still being used in commercial bioethanol production today (4), despite their undesirability from an ecological viewpoint. Unlike many other wastes generated during bioethanol production, antibiotics cannot be recycled as useful byproducts like forage or fertilizer (3). Propionic acid, in contrast, is usually used as a food preservative (5,6). In S. cerevisiae, the uptake of organic acids occurs both by facilitated diffusion via the Fps1 channel (7) and by passive diffusion. At natural cytosolic pH, however, the dissociation of the acids leads to the release of protons and the respective anions, which induce the intracellular acidification and inhibition of fermentation ability (8). Haa1, a transcriptional activator, is considered to be responsible for acetic acid responses (5,6). Approximately 80% of the genes induced by acetic acid addition are regulated by Haa1 (6). Haa1 binds to an acetic acid-responsive element (ACRE), activating the expression of * Corresponding author at: Present address: Faculty of Law, Ryukoku University, Fukakusatsukamoto-cho, Fushimi-ku, Kyoto 612-5662, Japan. Tel.: þ81 75 645 5662; fax: þ81 75 645 2096. E-mail address: [email protected] (J. Shima).

several targets, including membrane transporter genes TPO2 and TPO3 (6). Recently, we reported that S. cerevisiae overexpressing HAA1 acquired a higher level of acetic acid tolerance (9). Trehalose (a-D-glucopyranosyl a-D-glucopyranoside), which is a non-reducing disaccharide of glucose, is considered one of the most important molecules protecting against environmental stresses in S. cerevisiae. The most significant function of trehalose is to protect proteins and lipids included in the membrane structure against different kinds of stress conditions, such as heat and freezeethaw (10). Cellular levels of trehalose are controlled by an enzymatic balance between its synthesis enzymes (Tps1 and Tps2) and degradation enzymes (Nth1 and Nth2) (11,12). Nth1 activity is regulated by the Ras/adenylate cyclase signal-transduction pathway, which converts the inactive form into the phosphorylated active form (10,13). It has been reported that the deletion mutants of NTH1 accumulate higher levels of trehalose relative to their respective parent strains and tolerate environmental stresses such as high temperature, freezeethaw, and high osmolality (10,13,14). As far as we know, detailed analyses of the correlation between trehalose and organic acid tolerance have not been performed. In this study, we show that there is a strong correlation between the trehalose accumulation level and organic acid tolerance, and we demonstrate that trehalose accumulation fully compensates for acetic acid sensitivity due to the deletion of HAA1. The S. cerevisiae strains used in this study are listed in Table S1. Because differences in auxotrophy could affect the estimation of organic acid tolerance in S. cerevisiae, we used yeast strains that have the same auxotrophy. Yeast cells were cultivated in liquid YPD medium [2% glucose, 1% yeast extract (Difco Laboratories, Detroit, MI, USA) and 2% peptone (Difco)] or YPD agar medium containing  2% agar at 30 C, unless otherwise stated. For the fermentation evaluation (Fig. 2), YP5D medium (5% glucose, 1% yeast extract and

1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.06.021

Please cite this article in press as: Yoshiyama, Y., et al., Trehalose accumulation enhances tolerance of Saccharomyces cerevisiae to acetic acid, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.06.021

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FIG. 1. Organic acid stress tolerance of the mutants of trehalose metabolisms from a wild-type background (A, B) and the mutants of trehalose metabolisms from a haa1 background (C). Panel A shows the results of an assay using 0.45% and 0.5% acetic acid, and panel B, 0.40% and 0.45% propionic acid, to determine the level of tolerance to organic acids between trehalose accumulation mutants and the wild-type strain (BY4741). Panel C shows the results of an assay using 0.40% acetic acid and 0.40% propionic acid to compare the level of tolerance to organic acids between haa1D and haa1D with trehalose accumulation mutations, and the wild-type strain (BY4741). Approximately 105 cells and serial dilutions of 101 to 104 (from left to right) of each strain were spotted on the plates shown in each panel and incubated at 30  C.

2% peptone) was used. In the trehalose-loading experiment (Fig. S1B), SD medium (2% glucose, 0.17% yeast nitrogen base w/o amino acids, 0.5% ammonium sulfate, 0.5% casamino acids and 0.13% amino acid dropout mixture) was exclusively used because we wanted to avoid media containing yeast extract, which generally includes trehalose. In this experiment, casamino acids and amino acid dropout mixture were used in order to enhance the growth rate. To determine the correlation between the trehalose accumulation level and organic acid tolerance, we employed strains nth1D, nth2D, tps1D, and tps2D (Table S1). The trehalose levels of such strains in the exponential phase (i.e., optical density at 600 nm [OD600] ¼ 1.0) were estimated by a method described previously (14). Both nth1D and nth2D accumulated higher levels of trehalose than tps1D, tps2D, and wild-type (Fig. S1A). The spot assays for the estimation of acetic acid and propionic acid in YPD medium containing 0.45% and 0.50% acetic acid (pH 4.3; Fig. 1A) or YPD medium containing 0.40% and 0.45% propionic acid (pH 4.5; Fig. 1B) were performed as described previously (5,9). As shown in Fig. 1A and B, nth1D and nth2D were more tolerant to both acetic and propionic

acids than the wild-type strain. In contrast, tps1D and tps2D were hypersensitive to such organic acids, the levels of which were determined in a medium containing lower levels of acetic (0.45%) or propionic (0.40%) acid (Fig. 1A and B). Based on these results we constructed an nth1D nth2D double-knockout mutant using the hphMX6 gene as a selective marker and evaluated its acetic and propionic acid stress tolerance (Fig. 1A and B). The nth1D nth2D strain showed much higher acetic and propionic acid stress tolerance depending on trehalose accumulation levels (Fig. 1A, B and Fig. S1A), compared with either single-knockout mutant. To the best of our knowledge, these are the first data that show a significant correlation between organic acid stress tolerances and trehalose accumulation. Our results strongly suggest that intercellular trehalose contributed to tolerance to organic acids stresses in S. cerevisiae. We focused on acetic acid in the following analyses because the cellular response systems of yeast cells to acetic acid have been investigated in detail (5e7). To gain insights into the correlation between the Haa1 acetic acid response system and trehalose accumulation, double deletion mutants (haa1D tps2D and haa1D nth1D) were constructed by the

Please cite this article in press as: Yoshiyama, Y., et al., Trehalose accumulation enhances tolerance of Saccharomyces cerevisiae to acetic acid, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.06.021

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FIG. 2. Ethanol fermentation profiles of the wild-type strain BY4741 and the mutants of trehalose metabolisms (A, B), and those of BY4741 with or without trehalose loading (C, D). (A, C) The ethanol fermentation profiles in YP5D without acetic acid addition are shown as controls. (B, D) Fermentation profiles in YP5D with 0.5% acetic acid are shown. The cells were inoculated into 40 ml YP5D medium at a final OD600 ¼ 0.1, and incubated at 30 C for 3 days with shaking at 10 rpm. Fermentation was continuously monitored as the CO2 evolution with a Fermograph II apparatus (Atto, Tokyo, Japan). (A, C) Yeast cells were pre-culture in YPD medium for 24 h until the stationary phase. (C, D) Wild-type cells were precultured in SD medium (SD) or in SD medium containing 0.5 mol/L of trehalose (SD þ Tre) for 24 h until the stationary phase.

conventional molecular biological method using the primers listed in Table S2 and antibiotic-resistant genes (kanMX4 or hphMX6) as selection markers (15). The acetic acid tolerance of haa1D and the double deletion mutants was assayed (Fig. 1C). In accordance with previous reports (5,6), haa1D showed hypersensitivity to acetic acid. Interestingly, the deletion of NTH1 fully compensated for the acetic acid sensitivity caused by the deletion of HAA1. In contrast, haa1D tps1D and haa1D tps2D showed more severe acetic acid sensitivity than haa1D. These results suggest that trehalose accumulation protects yeast cells from acetic acid stress even under haa1 deletion. To determine whether acetic acid stress induced trehalose accumulation, we measured changes in intracellular trehalose levels after acetic acid challenge to wild-type cells in the exponential phase (OD600 ¼ 1.0). Although we employed several experimental conditions (i.e., acetic acid concentrations of 0.3e0.5% and 0e2 h cultivation periods after acetic acid challenge), we did not observe a rapid increase in trehalose content (

Trehalose accumulation enhances tolerance of Saccharomyces cerevisiae to acetic acid.

Trehalose confers protection against various environmental stresses on yeast cells. In this study, trehalase gene deletion mutants that accumulate tre...
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