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Applied and Environmental Microbiology, July 2008, p. 4222-4225, Vol. 74, No. 13
0099-2240/08/$08.00+0     doi:10.1128/AEM.02874-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Discovery of a Modified Transcription Factor Endowing Yeasts with Organic-Solvent Tolerance and Reconstruction of an Organic-Solvent-Tolerant Saccharomyces cerevisiae Strain

Ken Matsui, Shinya Teranishi, Shohei Kamon, Kouichi Kuroda, and Mitsuyoshi Ueda^*

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received 20 December 2007/ Accepted 27 April 2008

Top ABSTRACT INTRODUCTION REFERENCES

 
Organic-solvent tolerance in Saccharomyces cerevisiae strain KK-211, which was first isolated as an organic-solvent-tolerant strain, depends on point mutation (R821S) of the transcription factor Pdr1p. The integration of the PDR1 R821S mutation into wild-type yeast results in organic-solvent tolerance, and the PDR1 R821S mutant can reduce carbonyl compounds in organic solvents.

Top ABSTRACT INTRODUCTION REFERENCES

 
Living microorganisms are widely used in complex chemical conversions in industry, and the term "white biotechnology" has become popular. In general, chemical syntheses of hydrophobic compounds are carried out in organic solvents, which are highly toxic to living microorganisms. The accumulation of toxic organic solvents in the membrane increases membrane fluidity and membrane swelling and disrupts the normal functions of membrane-associated proteins (18, 22, 23). The incorporation of organic solvents leads to membrane structure disruption, loss of membrane functions, and, ultimately, to cell death.

Despite the lethal toxicity of organic solvents, organic-solvent-tolerant Saccharomyces cerevisiae strain KK-211 was first isolated among eukaryotic cells through the serial culture of dry yeast in a medium containing isooctane (8). The KK-211 strain can grow normally in medium containing isooctane. It was reported that the fatty acid composition of the cell membrane of this strain grown with isooctane also changed (8). Furthermore, the transcriptional levels were analyzed on a genome-wide scale using the KK-211 strain and the parent strain, DY-1 (12, 13). Some genes determined by DNA microarray analysis to be activated in the organic-solvent-tolerant KK-211 strain (12) contain pleiotropic drug response elements (PDREs) in their upstream sequences (Table 1) (7). Genes containing PDREs are controlled by the transcription factor Pdr1p, which is a master regulator involved in recruiting other zinc cluster proteins to PDREs (4). Furthermore, the transcription pattern of the KK-211 strain is similar to those of the PDR1 mutant strains (Table 1). Therefore, it was possible that the KK-211 strain had mutations in PDR1. To examine the possibility of mutations, the amino acid sequences of Pdr1p derived from the DY-1 and KK-211 strains were compared (Table 2). These strains are considered to be diploid. Predictably, Pdr1p of the KK-211 strain had some mutations. It is striking that Pdr1p of the KK-211 strain consisted of Ser821.

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TABLE 1. Comparison of transcription patterns by microarray analysis in PDR1 mutants

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TABLE 2. Mutations and polymorphisms in Pdr1p

To confirm that the PDR1 R821S point mutation was the factor that contributed to organic-solvent tolerance, the MT8-1 mutant (PDR1 A820T, R821S) strain was constructed by site-directed mutagenesis and homologous recombination. Compared with the DY-1 and KK-211 strains, the MT8-1 strain contained Ala820 (Table 2). To ascertain the requirement for the PDR1 R821S mutation for organic-solvent tolerance in the KK-211 strain, Ala820 in the MT8-1 strain was replaced by threonine. The growth of DY-1, KK-211, wild-type MT8-1, and the MT8-1 mutant (PDR1 A820T, R821S) strains in yeast extract-peptone-dextrose (YPD) medium with and without organic solvents, such as isooctane and n-nonane, was monitored (Fig. 1). Although the MT8-1 mutant (PDR1 A820T, R821S) showed slower growth than the wild-type MT8-1 strain in YPD medium without organic solvents, the MT8-1 mutant could grow in YPD medium with organic solvents. Compared with the KK-211 strain, the MT8-1 mutant (PDR1 A820T, R821S) showed smaller cell populations in YPD media with and without organic solvents. Differences in ploidy and strains might cause the difference seen between KK-211 and the MT8-1 (PDR1 A820T, R821S) mutant strains. In this study, the two point mutations (A820T and R821S) were applied to the wild-type MT8-1 strain. Considering that other wild-type strains that have threonine at amino acid position 820 showed no organic-solvent tolerance, one point mutation (R821S) was sufficient for organic-solvent tolerance.

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FIG. 1. Growth curves of PDR1 R821S mutant strains in organic solvents. The DY-1 and KK-211 strains (A) or wild-type (WT) MT8-1 and MT8-1 mutant (PDR1 R821S) strains (B) were precultured exponentially in YPD medium and transferred into YPD medium or YPD medium overlaid with isooctane or n-nonane. The initial OD600 was 0.1. The results of three independent trials are shown, and the error bars indicate standard deviations.

Some ABC transporters regulated by Pdr1p export toxic drugs (3, 11). The organic-solvent-tolerant strain KK-211 showed high transcriptional levels of some ABC transporter-encoding genes (12). To test drug resistance, the PDR1 R821S mutants, including KK-211 strains, were spotted with serial dilutions on YPD agarose medium containing cycloheximide. The PDR1 R821S mutants showed drug resistance (Fig. 2). Although the organic-solvent tolerance of the MT8-1 mutant (PDR1 R821S) strain was lower than that of the KK-211 strain, the drug resistance of the MT8-1 mutant (PDR1 R821S) strain was higher than that of the KK-211 strain. This result demonstrates that the mechanism underlying organic-solvent tolerance is not the same as that underlying drug resistance.

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FIG. 2. Resistance of organic-solvent-tolerant strains to cycloheximide. Fivefold serial dilutions (left to right) of exponential-phase cells (OD600, 5.0) were spotted onto YPD agarose medium with or without cycloheximide (1.0 mg/liter). The KK-211, parent DY-1, wild-type MT8-1, and MT8-1 mutant (PDR1 R821S) strains were used.

Living cells are essential for application of yeast metabolism to biocatalytic reactions. To confirm the catalytic activity of the PDR1 R821S mutant strain in organic solvents, the reduction of butyl 3-oxobutanoate to butyl 3-hydroxybutanoate in isooctane was performed in an aqueous/organic-solvent two-phase system. Preincubated yeasts were transferred to fresh YPD medium additionally containing 10% (wt/vol) glucose overlaid with isooctane containing butyl 3-oxobutanoate to obtain a final optical density at 600 nm (OD₆₀₀) of 4.0, and butyl 3-oxobutanoate reduction was carried out with shaking at 30°C. To calculate the conversion rate, the concentration of residual butyl 3-oxobutanoate was determined by high-performance liquid chromatography (Fig. 3). Despite the toxicity, the MT8-1 (PDR1 R821S) strain reduced most of the substrate after 30 h. This result showed the great potential for the PDR1 R821S mutant strain to be applied to various biocatalytic reactions in aqueous/organic-solvent two-phase systems.

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FIG. 3. Microbial reduction of butyl 3-oxobutanoate in isooctane. Wild-type (WT) MT8-1 and MT8-1 (PDR1 R821S) mutant strains were precultured exponentially in YPD medium and transferred into aqueous/organic-solvent two-phase systems containing 20 mM butyl 3-oxobutanoate. The initial OD600 was 4.0. The ordinate axis shows the residual amount of butyl 3-oxobutanoate in isooctane. The results of three independent trials are shown, and the error bars indicate standard deviations.

Our study is the first to show that mutation of a transcription factor is mainly related to organic-solvent tolerance and that an organic-solvent-tolerant microorganism can be artificially reconstructed by genetic methods based on our results. Many organic-solvent-tolerant bacteria have been isolated, and the elucidation of their tolerance mechanisms is in progress (10, 15-17). However, there is insufficient knowledge concerning organic-solvent-tolerant eukaryotic cells (26). On the basis of DNA microarray analysis, we found that the organic-solvent tolerance of the KK-211 strain depended on an R821S mutation in the transcription factor Pdr1p. The reconstructed MT8-1 mutant (PDR1 R821S) strain can grow in a medium containing organic solvents. As an approach to bioconversion, the MT8-1 mutant (PDR1 R821S) strain was able to reduce butyl 3-oxobutanoate to butyl 3-hydroxybutanoate in an aqueous/organic-solvent two-phase system. This result indicates that yeast endowed with organic-solvent tolerance by the PDR1 R821S mutation has catalytic activity even in organic solvents, such as isooctane, and this suggests novel methods of bioproduction using eukaryotic cells in organic solvents, including n-nonane. Some organic-solvent-tolerant bacteria also acquired organic-solvent tolerance induced by the overproduction of one or a few proteins, such as transporters (1, 14). Although organic-solvent-tolerant bacteria are useful for bioproduction, organic-solvent-tolerant yeasts, which are eukaryotic cells, are more useful for industries in relation to "white biotechnology" because of their greater abilities as biocatalysts. Our findings enable the reconstruction of organic-solvent-tolerant yeast and might provide us with a strategy for elucidating the mechanisms underlying organic-solvent tolerance in eukaryotic cells.

 
* Corresponding author. Mailing address: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Phone: 81-75-7536110. Fax: 81-75-7536112. E-mail: miueda{at}kais.kyoto-u.ac.jp

Published ahead of print on 9 May 2008.

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Applied and Environmental Microbiology, July 2008, p. 4222-4225, Vol. 74, No. 13
0099-2240/08/$08.00+0     doi:10.1128/AEM.02874-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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