Vanillin causes the activation of Yap1 and mitochondrial fragmentation in Saccharomyces cerevisiae Trinh Thi My Nguyen, 1 Aya Iwaki, 1 Yoshikazu Ohya, 2 and Shingo Izawa 1 , * Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Kyoto 606-8585, Japan 1 and Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan 2 Received 8 May 2013; accepted 10 June 2013 Available online xxx Vanillin and furfural are derived from lignocellulosic biomass and inhibit yeast growth and fermentation as biomass conversion inhibitors. Furfural has been shown to induce oxidative stress in Saccharomyces cerevisiae. Since there has been no report on the relationship between vanillin and oxidative stress, we investigated whether vanillin caused oxidative stress in yeast cells. We showed that vanillin caused the nuclear accumulation of Yap1, an oxidative stress responsive transcription factor, and subsequent transcriptional activation of Yap1-target genes. The growth of the null mutant of the YAP1 gene (yap1D) was delayed in the presence of vanillin, which indicated that Yap1 plays a role in the acquisition of tolerance to vanillin. We also demonstrated that vanillin facilitated the fragmentation of mitochondria. These findings suggest that the toxicity of vanillin involves damage induced by oxidative stress. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Vanillin; Furfural; Yap1; Lignocellulosic biomass; Mitochondrial fragmentation; Saccharomyces cerevisiae] Lignocellulosic biomass, such as wood and agricultural residues, is a potential source for biofuel production because it is abundant, regenerable, and does not compete with food resources. Lignocel- lulosic biomass is commonly pretreated with acid or hot-com- pressed water for fermentable sugar production (1,2). Fermentation inhibitors such as furfural, 5-hydroxymethylfurfural (HMF), glyco- laldehyde, and vanillin are also generated as by-products under the extreme conditions of this pretreatment step (1,3e6). They have been shown to inhibit yeast growth and subsequent fermentation in a dose-dependent manner (5,7). Vanillin is one of the major phenolic aldehyde compounds produced by the degradation of lignin, and has been shown to be a more potent inhibitor of fermentation than other by-products (5). Vanillin is also one of the most important flavor compounds, originally obtained from the pods of Vanilla planifolia. De novo synthesis of vanillin from glucose was recently implemented in yeast cells, and offers a sustainable means of vanillin production (8). However, the adverse effects of vanillin have prevented the efficient production of vanillin and bioethanol using yeast cells. Therefore, breeding vanillin-tolerant yeast is an important pre- requisite for efficient cell factories of bioethanol production from lignocellulosic biomass and vanillin synthesis. More information regarding the physiological effects of vanillin on yeast cells is required to improve their tolerance against vanillin. A genome-wide analysis using 4718 non-essential deletion mutants identified 76 vanillin-sensitive mutants that were classified into the functional categories chromatin remodeling, vesicle transport, and ergosterol biosynthesis process, which suggests the importance of these functions for vanillin tolerance (9). Other genome-wide screenings identified various genes that conferred resistance to vanillin (10,11). Additionally, we very recently reported that vanillin repressed bulk translational activity and induced the formation of processing bodies and stress granules (12). These findings clearly indicate that various intracellular processes are involved in the yeast response to vanillin. Yeast cells can reduce furfural to its less toxic alcohol derivative, furan methanol, in a NAD(P)H-dependent manner (13,14). The pentose phosphate pathway (PPP), in which NADPH is generated, plays an essential role in tolerance to furfural, and deficient mu- tants in the PPP such as the zwf1D mutant were shown to be hy- persensitive to furfural (15). Since the PPP is also necessary to protect cells against oxidative stress (16,17), the toxicity of furfural was also considered to involve damage induced by oxidative stress. Allen et al. verified that furfural induced the accumulation of TABLE 1. List of primer pairs for quantitative real time-PCR. Gene Orientation Primer sequence GPX2 Forward 5 0 -GGCGAATCTTTTAAGTTTGA-3 0 GPX2 Reverse 5 0 -TCCATTTGATACCCTTGAAA-3 0 TRX2 Forward 5 0 -TGGATGTTGATAAGTCTCAGATG-3 0 TRX2 Reverse 5 0 -CCTCCTTACCGCCCTTGTAG-3 0 ACT1 Forward 5 0 -TTGGATTCCGGTGATGGTGTTACT-3 0 ACT1 Reverse 5 0 -TGAAGAAGATTGAGCAGCGGTTTG-3 0 * Corresponding author at: Laboratory of Microbial Technology, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Kyoto 606- 8585, Japan. Tel./fax: þ81 75 724 7764. E-mail address: thioredoxin@kit.ac.jp (S. Izawa). www.elsevier.com/locate/jbiosc Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2013 1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.06.008 Please cite this article in press as: Nguyen, T. T. M., et al., Vanillin causes the activation of Yap1 and mitochondrial fragmentation in Saccha- romyces cerevisiae, J. Biosci. Bioeng., (2013), http://dx.doi.org/10.1016/j.jbiosc.2013.06.008