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Involvement of ergosterol in tolerance to vanillin, a potential inhibitor of bioethanol fermentation, in Saccharomyces cerevisiae

Ayako Endo, Toshihide Nakamura, Jun Shima
DOI: http://dx.doi.org/10.1111/j.1574-6968.2009.01733.x 95-99 First published online: 1 October 2009

Abstract

A vanillin-tolerant strain of Saccharomyces cerevisiae was screened and its intracellular ergosterol levels were compared with several laboratory yeast strains to study the potential relationship between ergosterol content and vanillin tolerance. Saccharomyces cerevisiae NBRC1950 was selected as a vanillin-tolerant strain. Its ergosterol content was higher than those of the laboratory strains. The results of DNA microarray and quantitative reverse transcriptase-polymerase chain reaction analysis showed that five genes involved in ergosterol biosynthesis (ERG28, HMG1, MCR1, ERG5, and ERG7) were upregulated in NBRC 1950 compared with strain X2180, suggesting that high expression of genes involved in ergosterol biosynthesis may cause high ergosterol content in strain NBRC 1950. The S. cerevisiae HX strain, which was a high-ergosterol-containing strain derived from X2180, was more tolerant to vanillin than the parental strain, suggesting that high ergosterol content may, in part, be responsible for vanillin tolerance. These findings provide a biotechnological basis for the molecular engineering of S. cerevisiae with increased tolerance to vanillin.

Keywords
  • vanillin
  • bioethanol
  • Saccharomyces cerevisiae
  • fermentation inhibitor
  • ergosterol

Introduction

Lignocellulosic materials such as crop residues and wood chips are among the most important potential sources for bioethanol production (Gray et al., 2006; Hahn-Hagerdal et al., 2006). However, due to the close association of cellulose and hemicellulose with lignin in the plant cell wall, pretreatment is necessary to render the carbohydrates available for enzymatic hydrolysis and fermentation (McMillan, 1994). For economic reasons, dilute acid hydrolysis is commonly used to prepare lignocelluloses for enzymatic saccharification and fermentation (Galbe & Zacchi, 2002).

Numerous byproducts, including furan derivatives, weak acids, and phenolic compounds, are generated during pretreatment. It has been suggested that most of these compounds inhibit the growth and fermentation of yeasts (Olsson & Hahn-Hagerdal, 1996; Palmqvist & Hahn-Hagerdal, 2000; Saha, 2003). In particular, vanillin, a major phenolic compound, has been suggested to be a stronger inhibitor of growth and bioethanol fermentation than other inhibitors because vanillin acts at low concentrations (Klinke et al., 2004). Although the bioconversion mechanism of vanillin in the culture medium of Saccharomyces cerevisiae was demonstrated previously (Fitzgerald et al., 2003), there have been no reports describing vanillin tolerance in S. cerevisiae. Saccharomyces cerevisiae is the preferred microorganism for industrial ethanol production due to its high fermentation capacity, high ethanol yield, and accepted generally regarded as safe status (Knox et al., 2004). Previously, we showed the genes required for tolerance to vanillin using the complete deletion strain collection (Endo et al., 2008). We suggested that the genes involved in ergosterol biosynthesis are required for vanillin tolerance, which in turn suggested that ergosterol is a key component of vanillin tolerance (Endo et al., 2008). Ergosterol is a ubiquitous component of cellular membranes in yeast and is required for the correct fluidity and functioning of the cellular membrane (Daum et al., 1998).

To analyze the function of cellular ergosterol levels in the mechanism of vanillin tolerance, we examined a vanillin-tolerant strain of S. cerevisiae and compared the intracellular ergosterol levels with other yeast strains. Moreover, as strains derived from S288C carry a defective Ty1 element inserted into the 3′ region of the HAP1 ORF, the expression levels of ergosterol-related genes and cellular ergosterol levels are reduced in strain X2180 relative to yeast containing a functional HAP1 gene (Kwast et al., 1998; Gaisne et al., 1999; Tamura et al., 2004). As a result, we compared the vanillin tolerance of strain X2180 with that of strain HX, which is a derivative of strain X2180 containing a wild-type HAP1 gene originating from the sake yeast strain Kyokai no. 7 (Tamura et al., 2004).

Materials and methods

Yeast strains and media

The Saccharomyces cerevisiae HX strain was kindly provided by Dr H. Shimoi of the National Research Institute of Brewing (Hiroshima, Japan). Saccharomyces cerevisiae (383 strains) used for the screening of vanillin-tolerant strains were obtained from the Microbiology Bank of the National Food Research Institute (NFRI). These included strains isolated from fermented foods, strains used in the production of bread and alcoholic beverages, and strains purchased from other culture collections. Saccharomyces cerevisiae laboratory strains X2180, S288C, and W303 were used as control strains. Yeast cells were grown at 30 °C on a solid and in a liquid yeast extract peptone and dextrose (YPD) medium consisting of 10 g of yeast extract (Difco), 20 g of peptone (Difco) and 20 g of glucose (per liter).

Screening of vanillin-tolerant S. cerevisiae

Yeast strains from the NFRI Microbiology Bank were inoculated into 100 μL of YPD medium in microtiter plates (Corning Inc., Corning, NY) and incubated at 30 °C for 48 h (preculture). Portions (1.2 μL) of the preculture were transferred into 100 μL of YPD medium containing 15 mM vanillin in microtiter plates at 30 °C for 24 h. The OD630 nm was then measured using a microtiter plate reader (Elx800, BioTek Instruments, Winooski, VT).

Measurement of ergosterol content

Yeast cells were cultivated in YPD medium at 30 °C with shaking at 150 r.p.m. and harvested at the log phase (OD600 nm=1.0). Extraction of total sterols was carried out as reported previously (Tamura et al., 2004). The ergosterol concentrations were measured using an HPLC system containing a UV detector (Prominence UFLC, Shimadzu, Kyoto, Japan) equipped with a reverse-phase column (TSKgel ODS80Ts, Tosoh, Tokyo, Japan).

DNA microarray analysis

Yeast cells were cultivated in YPD medium at 30 °C with shaking at 150 r.p.m. and harvested at the log phase (OD600 nm=1.0). The experimental procedures of RNA extraction and DNA microarray analysis have been described previously (Tanaka-Tsuno et al., 2007). Affymetrix Yeast Genome 2.0 arrays (Affymetrix, Santa Clara, CA) were used as the DNA microarrays. Statistical analysis after data acquisition and normalization of expression data were performed using genespring ver. 7.3.1 (Agilent Technologies, Palo Alto, CA) based on the gene expression data from two independent experiments. After data transformation to genespring, perchip normalization to the 50th percentile was performed, and pergene normalization to the specific samples (X2180 samples) was applied to the perchip normalized data. Quality control was performed based on experimental confidence levels (each strain in which all samples were present or marginal) and on statistical confidence levels (the condition in which the P-value of t-test comparisons between X2180 and NBRC 1950 was <0.05). Microarray data from the present study have been deposited in the Gene Expression Omnibus repository at the National Center for Biotechnology Information (NCBI) under series accession no. GSE13755.

Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis

Quantitative RT-PCR (LightCycler Instrument, Roche, Mannheim, Germany) was performed using a hot-start PCR kit (LightCycler FastStart DNA MasterPLUS SYBRGreen I, Roche). Gene-specific primers (ACT1 forward 5′-AGCCTTCTACGTTTCCATCC-3′, reverse 5′-CTTTCAGCAGTGGTGGAGAA-3′; ERG28 forward 5′-TGTACTTGAATGAACCACAC-3′, reverse 5′-GTGGTTGAGACAACCAATG-3′; HMG1 forward 5′-GTACCATCGGTGGTGGTACTG-3′, reverse 5′-CTGCTAGGGCAGCACATAAGG-3′; MCR1 forward 5′-GATGACCAAGACTTTGATGG-3′, reverse 5′-GATCAATTCACCTTGGTCC-3′; ERG5 forward 5′-CTGATGAGTTCATCCCTG-3′, reverse 5′-GTCACTGTATGATGGAAATCA-3′; ERG7 forward 5′-CTAATTGCACTTCTTTTCGCTG-3′, reverse 5′-ACTTGGGTATTCAATTGCAC-3′) were used for cDNA amplification. The reaction mixture was prepared according to the kit protocol. The PCR conditions were as follows: one cycle of 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 5 s at 55 °C (except HMG1) or at 63 °C (for HMG1), and 10 s at 72 °C. A melting curve analysis program showed that the correct product was amplified. Standard curves were produced by serially diluting the RNA sample. The concentration of each gene was calculated by reference to the respective standard curve with the aid of lightcycler software. Relative gene expression was expressed as the ratio of the target gene concentration to the ACT1 gene concentration, based on the data from two independent experiments.

Results and discussion

Screening of vanillin-tolerant S. cerevisiae

To obtain vanillin-tolerant S. cerevisiae, we monitored the growth of 383 yeast strains in YPD medium containing 15 mM vanillin. We found that only S. cerevisiae NBRC 1950 could grow in the vanillin-containing medium, and its turbidity exceeded OD630 nm=0.3 on 24-h cultivation (data not shown). To verify the result of the screening, we compared the growth of this strain with that of S. cerevisiae strains (X2180, S288C, W303, and HX) in YPD media containing 0 or 15 mM vanillin with shaking at 150 r.p.m. As shown in Fig. 1, NBRC 1950 could grow in a medium containing 15 mM vanillin, but the growth of other strains was drastically inhibited by the addition of 15 mM vanillin.

Figure 1

Growth of vanillin-tolerant (NBRC 1950) and laboratory strains (X2180, S288C, W303, and HX) of Saccharomyces cerevisiae. NBRC 1950 (closed circles), X2180 (open circles), S288C (closed triangles), W303 (open triangles), and HX (closed squares) strains were cultivated by shaking YPD media containing 0 mM (a) or 15 mM (b) of vanillin. All values are means±SDs of three independent experiments.

Measurement of ergosterol content

To clarify the relationship between ergosterol content and vanillin tolerance, we measured the total ergosterol content in NBRC 1950 and other strains. As shown in Fig. 2, the total ergosterol content in NBRC 1950 was higher than that in other strains. These results suggested that a high ergosterol content may be responsible for the vanillin tolerance observed in NBRC 1950.

Figure 2

Ergosterol contents of vanillin-tolerant (NBRC 1950) and laboratory strains (X2180, S288C, W303, and HX) of Saccharomyces cerevisiae. All values are means±SDs of three independent experiments.

Expression analysis of genes involved in ergosterol biosynthesis of NBRC 1950

To analyze the mechanisms of the higher ergosterol content of NBRC 1950 in detail, we compared the expression patterns of genes involved in ergosterol biosynthesis between NBRC 1950 and X2180. We found that the five genes involved in ergosterol biosynthesis (ERG28, HMG1, MCR1, ERG5, and ERG7) were upregulated twofold or more in strain NBRC 1950 compared with strain X2180 (data not shown). To confirm the microarray data, we performed quantitative RT-PCR for the upregulation of the five genes. As shown in Table 1, the expression levels of the five genes in NBRC 1950 were higher than those in strain X2180 in YPD medium. In particular, ERG28 and HMG1 were quite highly expressed in NBRC 1950. Erg28p is a transmembrane protein localized in the endoplasmic reticulum (ER) that tethers the sterol C-4 demethylation enzyme complex (Erg25p, Erg26p, and Erg27p) to the ER and also interacts with Erg6p (Mo et al., 2002, 2004). Hmg1p, 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, which catalyzes the conversion of HMG-CoA to mevalonate (Basson et al., 1986). This step is considered to be rate limiting in sterol biosynthesis (Polakowski et al., 1998). Our results suggest that high expressions of the genes involved in ergosterol biosynthesis such as ERG28 and HMG1 may be responsible for the high ergosterol content in strain NBRC 1950.

View this table:
Table 1

Expression levels of ergosterol synthesis genes in NBRC 1950 and X2180

GeneRatioFunction
ERG286.62ER membrane protein, may facilitate protein–protein interactions between the Erg26p dehydrogenase and the Erg27p 3-ketoreductase and/or tether these enzymes to the ER, also interacts with Erg6p
HMG13.90One of two isozymes of HMG-CoA reductase that catalyzes the conversion of HMG-CoA to mevalonate, which is a rate-limiting step in sterol biosynthesis; localizes to the nuclear envelope; overproduction induces the formation of karmellae
MCR13.79Mitochondrial NADH-cytochrome b5 reductase, involved in ergosterol biosynthesis
ERG52.32C-22 sterol desaturase, a cytochrome P450 enzyme that catalyzes the formation of the C-22(23) double bond in the sterol side chain in ergosterol biosynthesis; may be a target of azole antifungal drugs
ERG72.38Lanosterol synthase, an essential enzyme that catalyzes the cyclization of squalene 2,3-epoxide, a step in ergosterol biosynthesis
  • * The values indicate the relative mRNA levels normalized to strain X2180. All values are means of two independent experiments.

Comparison of vanillin tolerance in HX and X2180 strains

To further verify the involvement of ergosterol in vanillin tolerance, we compared the growth of X2180 with that of the HX strain in YPD media containing 0 or 12 mM vanillin. It was shown that the HX strain was a high-ergosterol-containing strain derived from X2180 (Tamura et al., 2004 and Fig. 2). As shown in Fig. 3, the HX strain grew with a shorter lag phase than X2180 in medium containing 12 mM vanillin. These results suggest that the changes in the amount of ergosterol affected the tolerance to vanillin in S. cerevisiae. A lag phase of an extended delay of cell growth for the vanillin-sensitive strain (e.g. X2180) was observed within a narrow range of vanillin concentrations (10–15 mM). However, at these concentrations, growth retardation of the vanillin-sensitive strain was dependent on the vanillin concentrations, suggesting a low margin of capacity for the degradation or sequestration of vanillin.

Figure 3

Comparison of growth of X2180 with that of HX in a vanillin-containing medium. X2180 (closed circles), HX (open circles), and NBRC 1950 (closed triangles) were cultivated by shaking YPD media containing 12 mM vanillin. All values are means±SDs of three independent experiments.

In this study, we successfully isolated a vanillin-tolerant S. cerevisiae strain, NBRC 1950. Saccharomyces cerevisiae is the preferred microorganism for industrial ethanol production due to its high fermentation ability. We believe that NBRC 1950 may be a useful material for the breeding of commercial strains of yeast for bioethanol production. In NBRC 1950, the total amount of ergosterol was higher than that in the S. cerevisiae laboratory strains. The results of DNA microarray and quantitative RT-PCR analysis showed that five genes related to ergosterol biosynthesis (ERG28, HMG1, MCR1, ERG5, and ERG7) were upregulated in NBRC 1950. Ergosterol is considered to be one of the most important components of cellular membranes in yeasts and is required for the correct fluidity and functioning of the cellular membrane (Daum et al., 1998). Aguilera et al. (2006) inferred that the more ethanol-tolerant strains have the ability to decrease membrane fluidity and offset the membrane-fluidizing effect of ethanol, because a high ergosterol concentration in S. cerevisiae is typically associated with low membrane fluidity (Zinser et al., 1991; Alexandre et al., 1994). Although it is unknown whether the inhibitory mechanisms of vanillin were similar to those of ethanol, we speculated that the high ergosterol content in NBRC 1950 was one of the reasons for its vanillin tolerance. As shown in Figs 1 and 3, NBRC 1950 showed considerable tolerance to vanillin, suggesting the existence of other mechanisms for vanillin tolerance. We also found that mitochondrial function was upregulated in NBRC 1950 using DNA microarray analysis (data not shown). In particular, the expressions of the Mn-SOD gene and genes involved in complex III in the mitochondrial respiratory chain (RIP1, QCR6, QCR10, CYC1, CYT1, and QCR2) were upregulated. Thus, we speculate that not only ergosterol content but also mitochondrial functions, especially Mn-SOD and complex III in the mitochondrial respiratory chain, were potentially important for vanillin tolerance. Finally, we showed that the HX strain was more tolerant to vanillin than the X2180 strain. The HX strain, which was a derivative of X2180, had higher ergosterol contents than strain X2180. These results suggest that the construction of yeast strains containing higher amounts of ergosterol may bring about an increase of vanillin tolerance. The data obtained in this study might be useful for the molecular engineering of S. cerevisiae strains with increased tolerance to vanillin.

Acknowledgements

We would like to thank Dr Shimoi for his generous gift of the HX strain. This work was supported by the Project for the Development of Biomass Utilization Technologies for Revitalizing Rural Areas of the Ministry of Agriculture, Forestry and Fisheries of Japan.

Footnotes

  • Editor: Derek Jamieson

References

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