Antisense-mediated inhibition of acid trehalase (ATH1) gene expression promotes ethanol fermentation and tolerance in Saccharomyces cerevisiae
Young-Ji Jung & Heui-Dong Park*
Department of Life and Food Sciences, Kyungpook National University, 1370 Sangkyuk, Daegu, 702-701, Korea
*Author for correspondence (Fax: +82-53-950-6772; E-mail: [email protected])
Received 21 July 2005; Revisions requested 2 August 2005; Revisions received 20 September 2005; Accepted 23 September 2005
Key words: acid trehalase, anti-ATH1, ethanol fermentation, ethanol stress,S. cerevisiae
Abstract
Acid trehalase gene (ATH1) expression was decreased using the antisense-RNA technique inSaccharomyces cerevisiae. The 500 bp DNA fragments containing anti-ATH1gene between +1 and +500 were amplified using PCR and fused to yeastADH1,CYC1andATH1promoters. Yeast cells harboring the recombinant plasmids had a low activity of acid trehalase and promoted ethanol fermentation compared to the control yeast cells harboring the vector plasmid only. The recombinant yeast had a high viability with 8% (v/v) ethanol.
Introduction
Trehalose (a-D-glucopyranosyl-a-D-glucopyrano- side), a non-reducing disaccharide participating in the tolerance to various environmental stres- ses, occurs in many organisms including bacteria, fungi, insects and plants (Elbein et al. 2003). It can furnish endogenous carbon and energy to the cell and may act as a stabilizer of cellular mem- branes and proteins (Singer & Lindquist 1998).
Trehalose accumulation correlates with survival of Saccahromyces cerevisiae under adverse condi- tions (Estruch 2000). However, a high level inter- feres with the protein refolding by molecular chaperones both in vitro and in vivo (Singer &
Lindquist 1998).
Intracellular concentrations of trehalose are controlled by the balance between its synthesizing and hydrolyzing enzymes in S. cerevisiae (Kim et al. 1996). Among the trehalose hydrolyzing enzymes, there are two different types of trehalas- es: cytosolic neutral trehalases (NTH1p and NTH2p) and a vacuolar acid trehalase (ATH1p) (Estruch 2000). Among these three trehalases, a null mutation of ATH1 gene resulted in better
survival of the yeast under various environmental stresses including dehydration, freezing and ethanol shock (Kim et al. 1996, Parrou et al.
2005). However, the activity of trehalases showed the opposite roles in heat-shock recovery and heat- shock survival inS. cerevisiae(Weraet al.1999).
In this report, expression of the ATH1 gene was decreased in S. cerevisiaeusing the antisense RNA technique and its effects on ethanol fermentation were evaluated.
Materials and methods
Microorganisms and growth
E. coli DH5a used for transformation and plas- mid DNA isolation was grown at 37C in LB media (1% Bacto-tryptone, 0.5% yeast extract, and 0.5% NaCl) supplemented with 200lg ampi- cillin ml)1 when needed. S. cerevisiae TCY1 (a, lys2, ura3) was used for yeast transformation and acid trehalase activity assay (Park et al. 2001).
Plasmids YMCp31 (Park et al. 2001) and pLG669z (Kovari et al. 1990) were used as a
Japan) using a GENE cycler (BioRad Co., Richmond, USA) as described previously (Park et al.1999). Oligonucleotide primers for the PCR were synthesized by a commercial company (Bioneer Co., Chongwon, Korea). The PCR mix- ture consisted of 1lg template DNA, 100 pmol of each primer, 1 U Taq DNA polymerase, 0.25 mM each dNTP, 10 mM Tris–HCl (pH 8.3), 50 mM KCl and 2.5 mM MgCl2. The PCR cycle program for DNA amplification comprised one cycle of 94C (3 min), 30 cycles of 94 C (1 min), 45C (2 min), 72C (1.5 min), and finally one cycle of 74C (10 min). All the other methods including plasmid DNA isolation, restriction enzyme treatment, DNA ligation and E. coli transformation were carried out as described by Sambrooket al. (1989).
Yeast transformation
Transformation of the recombinant plasmids con- taining antisense ATH1 DNA fused to various yeast promoters intoS. cerevisiae TCY1 was per- formed using lithium acetate and dimethyl sulfox- ide solution by the method of Hillet al.(1991).
Enzyme assay
Acid trehalase activities were assayed using whole cells by the method of Alizadch & Klion- sky (1996). Cells, harvested from 5 ml culture broth, were resuspended in 0.9 ml 40 mM sodium citrate buffer (pH 4.5)/1 mM EDTA and were treated with 50ll toluene and 50 ll 0.1% SDS with vortexing for 20 s. The reaction mixture was composed of 400ll the toluene treated yeast
Ethanol fermentation and measurement of the fermentation ratio was carried out in 500 ml flasks containing 200 ml YPD media supple- mented with 10, 20 and 30% (w/v) glucose by the method of Kim & Seu (1988). A water trap apparatus containing conc. H2SO4 was attached to the top of each flask to trap water evaporated from the flask during the fermentation. The amount of CO2 produced was directly measured as the decrease in the weight of the whole flask.
The fermentation ratio was expressed as the per- centage of the amount of CO2 produced per the theoretical CO2 production from the glucose due to the ethanol fermentation.
Viable counts under the ethanol stress
After growth in YPD media to 3.1 mg dry cell wt/ml, yeast cells harboring the recombinant plasmids were harvested by centrifugation and resuspended in fresh YPD media with or without 8% (v/v) ethanol. During incubation of the resuspension at 30C with shaking (150 rpm), yeast viable counts were determined using YPD agar plates every 2 h.
Results and discussion
Construction of recombinant plasmids containing antisense ATH1 DNA fused to various yeast promoters
The ATH1 gene of S. cerevisiae was isolated. Its sequence is known (Destruelle et al. 1995). The
PCR primers were designed to amplify antisense ATH1 DNA fragments covering the translation initiation site (+1) to DNA position +500 bp of the yeastATH1 gene. The sequences of the prim- ers are shown in Figure 1. A KpnI site at 5¢-end and aBamHI site at 3¢-end of the PCR products were constructed using PCR primers for the cloning into the BamHI and KpnI sites down- stream of the yeast CYC1 promoter on the YMCp31 plasmid (Park et al. 2001). After PCR, using the primers anti-ATH1F and anti- ATH1R shown in the Figure 1 and the yeast chromosomal DNA as a template, the PCR products were resolved in a 1% agarose gel and confirmed as containing the amplified 500 bp DNA. The amplified DNA, digested withBamHI andKpnI, was fused to the yeast CYC1promoter
on the plasmid YMCp31 (Park et al. 2001) as shown in Figure 1 to produce a recombinant plasmid YJE12 containing antisense ATH1 DNA fused to yeast CYC1promoter.
A DNA fragment containing 1.7 kb yeast ADH1 promoter was amplified using primers pADH1F and pADH1R designed to generate SalI and BamHI sites. After digestion with SalI and BamHI, the PCR product containing yeast ADH1 promoter was cloned into the same sites of plasmid YJE12 to produce recombinant plas- mid YJE11 containing antisense ATH1 DNA fused to yeast ADH1 promoter. The DNA frag- ment containing the yeast ATH1 promoter was also amplified and cloned into the plasmid pLG669z in the same way described above. The DNA fragment containing antisense ATH1
Fig. 1.Construction of recombinant plasmids containing antisenseATH1DNA fused to several yeast promoters. AntisenseATH1 DNA as well asATH1andADH1promoters were amplified by PCR using primers shown in (a). The amplified 500 bp DNA frag- ment containing antisenseATH1 gene between +1 and +500 regions was fused toADH1andCYC1 promoters as well as to ATH1promoter as shown in (b).
DNA, isolated from the plasmid YJE11 digested BamHI and SacI, was then cloned in the down- stream side of the ATH1 promoter on the pLG669z-ATH1 plasmid digested with the same restriction enzymes to generate the recombinant plasmid YJE13 as shown in Figure 1.
Effects of antisense ATH1 DNA on the acid trehalase activity and ethanol fermentation
The recombinant yeast cells harboring antisense ATH1 DNA plasmids had decreased acid treha- lase activities. The highest decrease was in the re- combinant plasmid YJE11 containing ADH1 promoter-antisense ATH1 DNA (Figure 2). The recombinant yeasts had greater and faster ethanol production than in the control yeast harboring vector plasmid (Figure 2). Regardless of the glucose concentration, the antisenseATH1 DNA always resulted in increased ethanol pro- duction (Figure 3). With YPD media containing 30% (w/v) glucose, the time to reach the maxi- mal ethanol production by the recombinant yeast cells was shortened to 44 h from 88 h with the original strain.
Effects of antisense ATH1 DNA on the tolerance to ethanol stress
Tolerance of the recombinant yeast cells to ethanol stress was measured as the viability in
the presence of ethanol in YPD media. Without ethanol, yeast viable counts showed no big difference between the control yeast and the recombinant strains (Figure 4). In the presence of 8% ethanol, the recombinant yeast cells har- boring ADH1 promoter-antisense ATH1 DNA plasmid could grow faster than the control strain. After 8 h, the recombinant yeast showed about 1.5-fold viable counts compared to that of
ing various antisenseATH1plasmids. (b) Changes in the ethanol fermentation ratio by the yeast cells harboring YIp5 vector plas- mid only as a control (e), and recombinant plasmid YJE11 (n), YJE12 (s) and YJE13 (m). Ethanol fermentation was carried out at 30C in YPD media containing 20% (v/v) glucose. Fermentation ratio was expressed as the percentage of the amount of CO2
produced per the theoretical CO2production from the glucose due to the ethanol fermentation.
Fig. 3. Ethanol fermentation by the recombinant yeast cells in YPD liquid media containing high concentration of glucose. Changes in the ethanol fermentation ratio were determined every 4 h during the culture of the yeast cells harboring YIp5 vector plasmid only (open symbols) and recombinant plasmid YJE11 (closed symbols) in YPD liquid media containing 10 (e, r), 20 (h, n) and 30% (w/v) glu- cose (D,m).
the control strain. Therefore, it was thought that the expression of antisense ATH1 DNA resulted in the elevated tolerance to the ethanol stress.
Acknowledgements
This work was supported by Kyungpook National University Research Team Fund, 2003, and a grant from the Korea Research Founda- tion (2000-005-G00003).
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Fig. 4.Effect of ethanol concentration on the viability ofS. cerevisiaeTCY1 harboring the recombinant plasmid YJE11 containing ADH1promoter-antisenseATH1DNA. After grown in YPD liquid media to 3.1 mg dry cell weight/ml, the yeast cells were har- vested and resuspended in the same volume of fresh YPD media with (b) or without 8% (v/v) ethanol (a). During incubation of the resuspension at 30C, viable counts of the yeast cells harboring YIp5 vector plasmid only (h) and recombinant plasmid YJE11 (n) were assayed on YPD agar plates.
