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  • APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2009, p. 3765–3776 Vol. 75, No. 11 0099-2240/09/$08.00�0 doi:10.1128/AEM.02594-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

    Comparative Proteomic Analysis of Tolerance and Adaptation of Ethanologenic Saccharomyces cerevisiae to Furfural, a

    Lignocellulosic Inhibitory Compound�† Feng-Ming Lin, Bin Qiao, and Ying-Jin Yuan*

    Key Laboratory of Systems Bioengineering, Ministry of Education and Department of Pharmaceutical Engineering, School of Chemical Engineering & Technology, Tianjin University, P.O. Box 6888, Tianjin 300072, People’s Republic of China

    Received 13 November 2008/Accepted 1 April 2009

    The molecular mechanism involved in tolerance and adaptation of ethanologenic Saccharomyces cerevisiae to inhibitors (such as furfural, acetic acid, and phenol) represented in lignocellulosic hydrolysate is still unclear. Here, 18O-labeling-aided shotgun comparative proteome analysis was applied to study the global protein expression profiles of S. cerevisiae under conditions of treatment of furfural compared with furfural-free fermentation profiles. Proteins involved in glucose fermentation and/or the tricarboxylic acid cycle were upregulated in cells treated with furfural compared with the control cells, while proteins involved in glycerol biosynthesis were downregulated. Differential levels of expression of alcohol dehydrogenases were observed. On the other hand, the levels of NADH, NAD�, and NADH/NAD� were reduced whereas the levels of ATP and ADP were increased. These observations indicate that central carbon metabolism, levels of alcohol dehydrogenases, and the redox balance may be related to tolerance of ethanologenic yeast for and adaptation to furfural. Furthermore, proteins involved in stress response, including the unfolded protein response, oxidative stress, osmotic and salt stress, DNA damage and nutrient starvation, were differentially expressed, a finding that was validated by quantitative real-time reverse transcription-PCR to further confirm that the general stress responses are essential for cellular defense against furfural. These insights into the response of yeast to the presence of furfural will benefit the design and development of inhibitor-tolerant ethanologenic yeast by metabolic engineering or synthetic biology.

    Bioethanol produced from renewable resources such as lignocelluloses is considered to be an attractive alternative to fossil fuels, for it is renewable, can make use of fast-rotation plants, produces fewer emissions, and generates no net carbon dioxide. Nevertheless, there are some barriers in the lignocel- lulosic-to-ethanol conversion process, including inhibitor tol- erance, ethanol tolerance, and utilization of xylose (62). Inhib- itors formed by acid-catalyzed hydrolysis of lignocelluloses, which include furan derivatives, weak acids, and phenolic com- pounds, reduce both the growth rate and fermentation of eth- anologenic Saccharomyces cerevisiae (2). The mechanisms of inhibition acting upon yeast during fermentation of lignocel- lulosic hydrolysate have been studied intensively, but mainly with traditional methods such as metabolite analysis, enzyme activity analysis, metabolic flux analysis, and kinetic analysis (50). Furfural is one of the major inhibitors for lignocellulosic hydrolysates. Previous studies have shown that in most cases, furfural can be converted by yeast to furfural alcohol (12, 30). Sometimes furoic acid (64), furoin and furil (47), and acyloin products (28, 61) can also be detected in the medium under different sets of cultivation conditions. The genetic mecha-

    nisms involved in furfural tolerance have been investigated by screening an S. cerevisiae disruption library to find potential relative genes (18). Through gene cloning and enzyme activity study, Liu et al. found that the conversion of furfural is cata- lyzed by multiple aldehyde reductases (40).

    The traditional methods described above can analyze only one or a few metabolites, proteins, or genes and are unable to globally assess the inhibition issue, which is complex and sys- tematic. Moreover, previous work mainly focused on extracel- lular metabolites and the activity of some key enzymes, whereas what happens inside yeast cells in response to inhib- itors remains a “black box” to us. Integration of different “om- ics” tools, including those of transcriptomics, proteomics, and metabolomics, into the study of systems biology is a potentially powerful approach to address these challenges (61, 68). Many proteomic, transcriptomic, and/or metabolomic studies of S. cerevisiae have provided us with an increasingly rich under- standing of the response of this organism to various environ- mental perturbations. Investigation of genomic expression profiles of the ethanologenic yeast S. cerevisiae to HMF (5- hydroxymethylfurfural) stress conditions showed that up to several hundred genes were differentially expressed signifi- cantly in response to HMF treatment (41, 42, 58). Comparative lipidomics analysis has been applied to study the ethanologenic yeast response to different inhibitors, such as furfural, acetic acid, and phenol (69). The results of comparative proteome analysis (8, 23) and small-molecule metabolite profiling of eth- anologenic yeast during industrial fermentation (13) have been previously reported, enhancing the molecular understanding of physiological adaptation of industrial strains for optimizing the

    * Corresponding author. Mailing address: Key Laboratory of Sys- tems Bioengineering, Ministry of Education and Department of Phar- maceutical Engineering, School of Chemical Engineering & Technol- ogy, Tianjin University, P.O. Box 6888, Tianjin 300072, People’s Republic of China. Phone: 86-22-87401546. Fax: 86-22-27403888. E-mail: yjyuan@tju.edu.cn.

    † Supplemental material for this article may be found at http://aem .asm.org/.

    � Published ahead of print on 10 April 2009.

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  • performance of industrial bioethanol fermentation. However, the specifics of global protein expression in response to the presence of biomass conversion inhibitors have not yet been quantitatively measured for ethanologenic yeast.

    Quantitative proteomics, i.e., quantifying protein expression levels in different sets of complex biological samples on a large scale, is critical for our understanding of biological systems and pathways as a whole. It is considered likely to be a potential cornerstone of systems biology in the near future (56). Current quantitative proteomic methods fall into three categories: the traditional two-dimensional (2D) electrophoresis, stable iso- tope labeling, and nonlabeling methods (45). Each of the dif- ferent labeling methods undergoing development has its ad- vantages and disadvantages (see reference 45 for reviews). Among quantitative proteomic methods, 18O stable isotope labeling is convenient to use, low in cost, highly specific in terms of specific 18O C-terminal modifications, and capable in theory of labeling proteins globally. The 18O-labeling method has demonstrated its applicability in differential comparative proteomics with biological applications performed using Por- phyromonas gingivalis strain W50 (4), the human plasma pro- teome (55), breast cancer cells (5), and the low-molecular- weight serum proteome (26). 18O labeling is becoming a powerful labeling strategy for quantitative proteomics ap- plication.

    To give insights into the tolerance and adaptation of eth- anologenic yeast to biomass conversion inhibitors at the pro- tein level, comparative shotgun proteomic investigations com- bining 18O labeling with 2D liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) were performed here to systematically identify proteins by the use of an industrial strain of S. cerevisiae and to quantify cells treated with furfural compared with control cells under aerobic batch culture con- ditions. Quantitative real-time reverse transcription-PCR (RT- PCR) and metabolite analysis were utilized to provide orthog- onal evidence for the comparative proteome results.

    MATERIALS AND METHODS

    Yeast strain. An industrial strain of S. cerevisiae, purchased from Angel Yeast Co., Ltd. (Hubei, People’s Republic of China), in the form of alcohol instant active dry yeast, was utilized in this study. This industrial strain has the advan- tages of thermal resistance (38 to 42°C), low-acid tolerance (pH 2.5), and high glucose tolerance (60%) and can tolerate 13% (vol/vol) ethanol.

    Cultivation conditions. After recovery from a lyophilized form, S. cerevisiae was maintained on agar slants containing YEPD medium (2% glucose, 2% yeast extract, 1% peptone, and 2% agar). S. cerevisiae was initially grown in 250-ml conical flasks containing 50 ml of YEPD medium (2% glucose, 2% yeast extract, and 1% peptone) on a rotary shaker at 30°C and 160 rpm for 12 h. Subsequently, the 50-ml seed cultures were transferred into 2-liter conical flasks containing 450 ml of YEPD medium on a rotary shaker at 30°C and 90 rpm for approximately 12 h and grown to an optical density (OD) of about 3. Cells for the control experiment and the furfural treatment experiment were harvested from the same inoculation culture. An initial OD of 0.35 was used for aerobic bath cultures performed at 30°C in 2-liter conical flasks containing 450 ml of medium, with a stirrer speed of 90 rpm. The aerobic bath culture medium was composed of 10% glucose, 2% yeast extract, and 1% peptone. During exponential growth in the respiratory-fermentative phase, when the OD was approximately 3 to 4, 50-ml volumes of aerobic bath culture media con