A 5-hydroxymethyl furfural reducing enzyme ... - Wiley Online Library

Yeast Yeast 2006; 23: 455–464. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1370

Research Article

A 5-hydroxymethyl furfural reducing enzyme encoded by the Saccharomyces cerevisiae ADH6 gene conveys HMF tolerance Anneli Petersson1# , João R. M. Almeida2# , Tobias Modig1 , Kaisa Karhumaa2 , Bärbel Hahn-Hägerdal2 , Marie F. Gorwa-Grauslund2 and Gunnar Lidén1 * 1 Department 2 Department

of Chemical Engineering, Lund University, PO Box 124, S-221 00 Lund, Sweden of Applied Microbiology, Lund University, PO Box 124, S-221 00 Lund, Sweden

*Correspondence to: Gunnar Lidén, Department of Chemical Engineering, Lund University, PO Box 124, S-221 00 Lund, Sweden. E-mail: [email protected] # These authors contributed equally to this study.

Received: 1 November 2005 Accepted: 23 February 2006

Abstract The fermentation of lignocellulose hydrolysates by Saccharomyces cerevisiae for fuel ethanol production is inhibited by 5-hydroxymethyl furfural (HMF), a furan derivative which is formed during the hydrolysis of lignocellulosic materials. The inhibition can be avoided if the yeast strain used in the fermentation has the ability to reduce HMF to 5-hydroxymethylfurfuryl alcohol. To enable the identification of enzyme(s) responsible for HMF conversion in S. cerevisiae, microarray analyses of two strains with different abilities to convert HMF were performed. Based on the expression data, a subset of 15 reductase genes was chosen to be further examined using an overexpression strain collection. Three candidate genes were cloned from two different strains, TMB3000 and the laboratory strain CEN.PK 113-5D, and overexpressed using a strong promoter in the strain CEN.PK 1135D. Strains overexpressing ADH6 had increased HMF conversion activity in cellfree crude extracts with both NADPH and NADH as co-factors. In vitro activities were recorded of 8 mU/mg with NADH as co-factor and as high as 1200 mU/mg for the NADPH-coupled reduction. Yeast strains overexpressing ADH6 also had a substantially higher in vivo conversion rate of HMF in both aerobic and anaerobic cultures, showing that the overexpression indeed conveyed the desired increased reduction capacity. Copyright  2006 John Wiley & Sons, Ltd. Keywords: 5-hydroxymethyl furfural; ADH6; genome-wide analysis; lignocellulose hydrolysates; Saccharomyces cerevisiae

Introduction The utilization of lignocellulosic biomass for the production of liquid fuels and chemicals involves acidic pretreatment of the raw material prior to hydrolysis and bioconversion/fermentation (Galbe and Zacchi, 2002). In the pretreatment step, low molecular weight fatty acids, furan derivatives and aromatic compounds are released and formed (Larsson et al., 1999; Palmqvist and HahnHägerdal, 2000). These compounds are inhibitory to the bioconversion/fermentation steps. One of the quantitatively most important inhibitors in lignocellulose hydrolysates is 5-hydroxymethyl furfural Copyright  2006 John Wiley & Sons, Ltd.

(HMF), which is formed as a result of hexose degradation (Ulbricht et al., 1984). HMF has been reported to reduce both cell growth and ethanol production for baker’s yeast Saccharomyces cerevisiae, most commonly used for industrial ethanol production (Delgenes et al., 1996; Liu et al., 2004; Taherzadeh et al., 2000). The mechanism of HMF inhibition is, however, only partially understood. HMF has been shown to cause accumulation of lipids and decrease the protein content in yeast cells (Banerjee and Viswanathan, 1976). The lag-phase observed in ethanolic fermentation in the presence of HMF has been ascribed to inhibition of triose-phosphate

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dehydrogenase and alcohol dehydrogenase (Sanchez and Bautista, 1988). In addition to inhibition of alcohol dehydrogenase, HMF has also been shown to inhibit aldehyde dehydrogenase and pyruvate dehydrogenase (Modig et al., 2002). HMF inhibition is overcome in strains of S. cerevisiae that are able to reduce HMF to 5hydroxymethylfurfuryl alcohol (Liu et al., 2004; Taherzadeh et al., 1999). The reduction of HMF has been found to be both NADPH- and NADHcoupled, the ratio of the activities being straindependent (Wahlbom and Hahn-Hägerdal, 2002; Nilsson et al., 2005). In the present investigation we compared two strains of S. cerevisiae; TMB3000, known to be inhibitor-tolerant (Lindén et al., 1992; Mart´ın and Jönsson, 2003; Brandberg et al., 2004), and a laboratory strain, CBS8066, using genome-wide transcription analysis of known yeast reductase and dehydrogenase genes (Stewart et al., 2001; Katz et al., 2003). Selected genes from the transcription analysis were evaluated using the Ex-Clone overexpression collection. Candidate genes were finally cloned from TMB3000 and from a control laboratory strain and overexpressed in a CEN.PK strain, where HMF reducing activity was further verified.

Materials and methods Strains Saccharomyces cerevisiae CBS8066, a widely used diploid laboratory strain (Verduyn et al., 1990), and TMB3000 (ATCC 96 581), isolated from a spent sulphite liquor fermentation plant (Lindén et al., 1992; denoted ‘isolate 3 in the reference) were used for genome-wide transcription analysis. The Ex-Clone overexpression collection (Resgen, Invitrogen Corp., UK) was used to verify

the genome-wide transcription analysis. S. cerevisiae CEN.PK 113-5D (MAT a ura3)(van Dijken et al., 2000) was used to overexpress candidate genes. Escherichia coli strain DH5α [F endA1 hsdR17 (rK − mK + ) glnV44 thi1 recA1 gyrA (Nalr ) relA1(lacIZYA-argF )U169 deoR (φ80dlac (lacZ )M15] was used routinely for plasmid amplification. All strains were stored at −80 ◦ C or maintained on YPD agar plates (1% yeast extract, 2% soy peptone, 2% glucose and 2% agar in distilled water).

Genetic constructs Standard molecular biology techniques were used (Sambrook et al., 1989). Yeast strains were grown in YPD medium or defined mineral medium (Verduyn et al., 1992), supplemented with 20 g/l glucose, and an Escherichia coli strain in LB medium (0.5% yeast extract, 1% peptone and 1% NaCl) supplied with appropriated antibiotic. Yeast chromosomal DNA was extracted with Easy-DNA Kit (Invitrogen, Groningen, The Netherlands). Alcohol dehydrogenase II (ADH2), alcohol dehydrogenase VI (ADH6 ) and formaldehyde dehydrogenase (SFA1) genes from S. cerevisiae TMB3000 and CEN.PK 113-5D were amplified using the primers summarized in Table 1. The 5 region of the forward and reverse primers contained around 30 and 33 nucleotides, respectively, corresponding to the sequence of the HXT7 truncated promoter (Hauf et al., 2000) and PGK1 terminator, respectively. After amplification, the PCR products were analysed by electrophoresis in agarose gels and purified using QIAquick PCR Purification kit (Qiagen). The vector YEplacHXT (Karhumaa et al., 2005) was linearized using the restriction endonuclease BamHI. A mix containing the linear vector, the ADH2, ADH6 or SFA1

Table 1. Primers for ADH2, ADH6 and SFA1 amplification Sequence (5 to 3 )

Size (bp)

TTAATTTTAATCAAAAAAGGATCCCCGGGCTGCAatgtcttatcctgagaaatttgaagg CACCACCAGTAGAGACATGGGAGATCTAGAATTCctagtctgaaaattctttgtcgtagc TTTAATTTTAATCAAAAAAGGATCCCCGGGCTGCAatgtccgccgctactgttggtaaac CCACCACCAGTAGAGACATGGGAGATCTAGAATTCctattttatttcatcagacttcaagacg AATTTTAATCAAAAAAGGATCCCCGGGCTGCAatgtctattccagaaactcaaaaagcc ACCAGTAGAGACATGGGAGATCTAGAATTCttatttagaagtgtcaacaacgtatctac

60 60 60 63 59 59

Primer ADH6-FOR ADH6-REV SFA1-FOR SFA1-REV ADH2-FOR ADH2-REV ∗ Upper-case

letters: homologous sequences for HXT7 truncated promoter and PGK terminator in forward (XXX-FOR) and reverse (XXX-REV) primers, respectively.

Copyright  2006 John Wiley & Sons, Ltd.

Yeast 2006; 23: 455–464.

5-Hydroxymethyl furfural reducing enzyme in S. cerevisiae

PCR product from TMB3000 (referred to as T/ADH2, T/ADH6 and T/SFA, respectively) or from CEN.PK 113-5D (referred to as C/ADH2, C/ADH6 and C/SFA1, respectively) were used to transform S. cerevisiae CEN.PK 113-5D using the lithium acetate method (Gietz et al., 1995). The strains were given the numbers TMB3282, TMB3286, TMB3291, TMB3281, TMB3285 and TMB3290, respectively. A yeast control strain (TMB3280) was also constructed by transformation with the vector YEplacHXT without the structural genes. Transformant yeast strains were selected by colony PCR, using the primers shown in Table 1, and assayed for ethanol oxidation. Plasmids from positive transformants (ADH2, ADH6 and SFA1) were recovered, amplified in E. coli DH5α (Dagert and Ehrlich, 1979) and submitted to automatic sequencing using the Abi-Prism BigDye cycle sequencing kit (Applied Biosystems, Weiterstadt, Germany).

Continuous cultures Cells of CBS8066 and TMB3000 to be used for genome-wide transcription analysis were grown in continuous cultures in the absence and presence of 0.5 g/l HMF. Inoculum cultures were grown in 300 ml cotton-plugged unbaffled shake-flasks with 100 ml synthetic media, using 15 g/l glucose as carbon and energy source. The synthetic medium contained (per litre distilled water): 7.5 g (NH4 )SO4 , 3.5 g KH2 PO4 , 0.75 g MgSO4 · 7H2 O, 30 mg EDTA, 9 mg CaCl2 · 2H2 O, 9 mg ZnSO4 · 7H2 O, 6 mg FeSO4 · 7H2 O, 2 mg H3 BO3 , 1.6 mg MnCl2 · 2H2 O, 0.8 mg Na2 MoO4 · 2H2 O, 0.6 mg CoCl2 · 2H2 O, 0.6 mg CuSO4 · 5H2 O, 0.2 mg KI, 50 µg D-biotin, 0.2 mg p-aminobenzoic acid, 1 mg nicotinic acid, 1 mg calcium pantothenate, 1 mg pyridoxine HCl, 1 mg thiamine HCl, 25 mg m-inositol, 10 mg ergosterol, and 420 mg Tween 80. Inoculum cultures were grown for 24 h at 30 ◦ C and 150 r.p.m. 20 ml inoculum was used to start a batch cultivation. A Belach BR 0.5 bioreactor (Belach Bioteknik AB, Solna, Sweden) with a working volume of 500 ml was used. The concentration of the synthetic medium was increased by 33% relative to the inoculum medium and the glucose concentration was 20 g/l. The feed was started at a dilution rate of 0.1/h when glucose had been consumed. The reactor was sparged with 300 ml/min nitrogen. pH was maintained at 5.0, Copyright  2006 John Wiley & Sons, Ltd.

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using 0.75 M NaOH. The temperature was 30 ◦ C and the stirrer speed was set to 500 r.p.m. Samples for genome-wide transcription analysis were collected at steady state following five residence times after start of feed or change in feed media.

mRNA preparation and microarray analysis Samples from the reactor were spun at 0 ◦ C at 3000 r.p.m. for 1 min and thereafter frozen in liquid nitrogen and stored at −80 ◦ C until mRNA isolation. mRNA was isolated using the Fast RNA kit (Q-biogene, USA). mRNA was then purified, and cDNA was synthesized, in vitro transcribed and fragmented as described by Affymetrix (Santa Clara, CA, USA). Hybridization, washing, staining and scanning of microarray chips (Yeast Genome S98 Arrays) were performed in an Affymetrix Gene Chip Oven 640, a Fluidics Station 400 and a GeneArray Scanner (Affymetrix). The array signals were normalized to give the same total signal intensity in order to compensate for any difference in loading. The results were processed with Affymetrix Microarray Suite (MAS 5.0) and sorted in Microsoft Excel.

Shake-flask experiments Ex-Clone strains were grown in 300 ml shake flasks containing 100 ml SD — Ura omission medium and 40 g/l glucose, as described by the supplier. In addition, 80 µM Cu2+ were added when the shake flasks were inoculated. Samples for enzyme activity measurements were collected after 16 h of growth at 30 ◦ C and 150 r.p.m. Anaerobic and aerobic batch cultivations were carried out in 300 ml shake flasks placed in a shaker bath running at 170 r.p.m. and at a temperature of 30 ◦ C. The medium volume was 200 ml (medium composition identical to the inoculum medium used for the continuous cultures) containing 13 g glucose at initial pH 5.5. In the anaerobic cultivations each shake flask was equipped with a loop-trap containing sterile glycerol. Nitrogen gas was sparged through the flasks, initially for 5 min to establish and, while sampling, to maintain anaerobic conditions. The aerobic shake flasks were fully aerated by sparging with air during the cultivation. All cultivations were grown until a biomass concentration of 0.8 g/l was reached before 1.5 g/l Yeast 2006; 23: 455–464.

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HMF was added to the medium. pH was then readjusted to 5.5 with 2 M NaOH and the cultivations were followed until complete uptake of glucose.

Biomass and metabolite analyses Cell concentrations were determined from absorbance measurements at 610 nm on samples diluted to give an optical density (OD) of less than 0.5. In this range the absorbance values were linearly related to cell dry weight. The OD was calibrated against dry-weight measurements from duplicate 5 ml samples, which were centrifuged, washed with distilled water and dried for 24 h at 105 ◦ C. Metabolite samples were immediately centrifuged and stored at −20 ◦ C until analysis. The amounts of glucose, ethanol, glycerol and HMF were determined by liquid chromatography. The HPLC set-up consisted of an Aminex HPX-87H (Bio-Rad) column and a refractive index detector (Waters 410, Millipore, Milford, USA) eluted with 5 mM H2 SO4 prepared in ultra-pure water.

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photometrically at 340 nm in 1.0 cm path-length cuvettes at 30 ◦ C (Bergmeyer, 1974). The reaction mixture contained 5.0 mM NAD+ and the reaction was started by adding 1.7 M ethanol in 100 mM glycine buffer, pH 9.0. Furfural and HMF reducing activity was measured according to Wahlbom and Hahn-Hägerdal (2002). 5–10 µl cell-free extract (using different dilutions) was diluted in 1 ml 100 mM phosphate buffer (50 mM KH2 PO4 and 50 mM K2 HPO4 ) and NAD(P)H was added to a concentration of 100 µM. The samples were heated to 30 ◦ C and thereafter the reaction was started by addition of HMF or furfural to a concentration of 10 mM. The oxidation of NAD(P)H was followed as the change in absorbance at 340 nm. Dihydroxyacetone phosphate (DHAP) reducing activity was determined the same way as furfural and HMF with 0.7 mM of substrate. The molar absorption coefficient, ε340 , was 6.22 mM−1 cm−1 for NADH and NADPH.

Results

Enzyme activity

Comparative expression analysis

Crude cell extracts were prepared using Y-PER reagent (Pierce, Rockford, IL, USA). The protein content in the cell-free preparation was determined using Micro BCA Protein Assay Kit (Pierce). Dehydrogenase/reductase activity with ethanol, furfural, HMF and dihydroxyacetone phosphate (DHAP) as substrate was measured in cell extract samples. For ethanol, the rate of oxidation was determined by monitoring the reduction of NAD+

Cells of TMB3000 and CBS8066 were collected from anaerobic steady-state continuous cultures, where cells were grown in the absence and in the presence of HMF, and mRNA was extracted for microarray transcriptome analyses using Affymetrix chips. The obtained biomass yields for the two strains were 0.115 ± 0.01 g/g and 0.104 ± 0.006 g/g for TMB3000 and CBS8066, respectively. The resulting dataset was analysed for

Figure 1. Results from microarray analyses for selected reductase/dehydrogenase genes. Black bars, expression levels for TMB3000. Striped bars, expression levels for TMB3000 grown on synthetic media supplemented with 0.5 g/l HMF. White bars, expression levels for CBS8066. Grey bars, expression levels for CBS8066 grown on synthetic media supplemented with 0.5 g/l HMF Copyright  2006 John Wiley & Sons, Ltd.

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known reductase and dehydrogenase genes (Katz et al., 2003, Stewart et al., 2001). AAD15, ADH2, ADH5, ADH6, ADH7, ALD6, ERG27, GDH3, GND2, HMG1, IDP3, LAT1, LYS5, MDH2, OYE3, SER3, SFA1 and SPS19 (Figure 1) had at least two-fold higher expression levels in TMB3000 in comparison with CBS8066, in either the absence or presence of HMF. In particular, ADH2 was highly overexpressed as well as induced by HMF in TMB3000. In contrast, GND2, MDH2 or SER3 were not induced by HMF in either strain.

HMF activity in strains overexpressing candidate genes First, Ex-Clone strains overexpressing genes identified in the mRNA analysis (Figure 1) were grown in synthetic medium containing 40 g/l glucose in shake flasks. NADH- and NADPH-dependent HMF reductase activity was measured in crude cell extracts (Figure 2). Strains overexpressing SER3, GND2 or MDH2 were not included, since these genes were not found to be induced by HMF in the array analysis (Figure 1). Ex-Clone strains overexpressing ADH6, ADH7 and SFA1 showed increased HMF reduction activity compared to the control strain carrying the vector without the structural gene (Figure 2). The increase was particularly pronounced in the Ex-Clone strain overexpressing ADH6, with a co-factor preference for NADPH, for which the activity was 3.9 times higher than for the control strain. Also the ADH7 Ex-Clone overexpressing strain showed a 1.8-fold increased NADPH-coupled activity.

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However, the SFA1 overexpressing strain showed 1.9-fold higher reduction activity than the control strain with NADH as co-factor. Contrary to the array analysis, which showed ADH2 to be the most highly expressed and induced gene, the ExClone strain overexpressing ADH2 displayed only a very modestly increased HMF reducing activity with NADPH as co-factor (Figure 2). Next, candidate genes ADH2, ADH6 and SFA1 were overexpressed using the high-copy vector YEplacHXT (Karhumaa et al., 2005) carrying the strong truncated HXT7 promotor (Hauf et al., 2000). ADH2 was included because of its outstanding expression level in strain TMB3000 (Figure 1). Using the same reasoning, ADH7 was excluded because of the low mRNA expression level (Figure 1). The candidate genes were cloned in CEN.PK 113-5D by recombination from both CEN.PK 113-5D and TMB3000, in order to identify possible mutations. Yeast strains carrying plasmids YEplacHXT–ADH2 (named C/ADH2 and T/ADH2 when carrying the ADH2 gene from CEN.PK 113-5D and TMB3000, respectively), YEplacHXT–ADH6 and YEplacHXT–SFA1 (same nomenclature used for ADH2 ) were successfully selected by colony PCR. Among these strains, strains with efficient expression were selected based on their ethanol oxidation capacity (Figure 3).

In vivo and in vitro HMF reduction capacity In vivo HMF conversion capacity of the ADH2, ADH6 and SFA1 overexpressing strains was

Figure 2. NADH (black bars)- and NADPH (grey bars)-dependent HMF reduction in crude cell extracts of Ex-Clone strains overexpressing different reductase/dehydrogenase genes. The activity is reported as relative activity compared to the activity for the control strains of the Ex-Clone collection containing an empty vector taken as 100%. The activities for the control strain were 12 mU/mg and 72 mU/mg for NADH and NADPH-coupled activity, respectively Copyright  2006 John Wiley & Sons, Ltd.

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Figure 3. HMF reduction activities in crude cell extracts from the control strain (CEN.PK 113-5D) and CEN.PK 113-5D overexpressing ADH2, ADH6 or SFA1 genes originating from TMB3000 (T) or CEN.PK 113-5D (C). The activities were measured using (A) NADH or (B) NADPH as co-factor

compared with the CEN.PK 113-5D control strain and TMB3000 in minimal medium under both anaerobic (see Figure 4) and aerobic (i.e. respirofermentative) conditions and the metabolite profiles were compared. For all strains, the specific growth rate decreased in the presence of HMF during both anaerobic and respirofermentative conditions. None of the modified strains had a significantly higher specific growth rate than the reference strain (Tables 2 and 3). The ADH6 overexpressing strains showed three-fold higher specific HMF uptake rates than the reference strain under anaerobic conditions. The biomass yields were not significantly affected, whereas there was a slight increase in glycerol yields (Table 2, Figure 4). Under aerobic conditions, there was an Copyright  2006 John Wiley & Sons, Ltd.

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Figure 4. Uptake of HMF (ž, right hand scale), and profiles of glycerol ( ), ethanol () and glucose (◊) concentrations (left hand scale) in anaerobic batch fermentations with CEN.PK 113-5D (A) and CEN.PK 113-5D over expressing ADH6 from TMB 3000 (B). Glucose (65 g/l) was used as carbon and energy source. 1.5 g/l HMF was added at time 0

°

even stronger increase in HMF uptake rates (fourfold) for the ADH6 overexpressing strains than the reference strain, compared under anaerobic conditions. The biomass yields were somewhat lower, whereas the glycerol yields were about 70% higher than that of the reference strain. The specific uptake rate of HMF was somewhat lower for strains overexpressing ADH2 and, to some extent, for strains overexpressing SFA1 (Table 3). Yields and specific growth rates were almost unaffected by overexpression of ADH2 and SFA1 compared to the reference strain under both anaerobic and aerobic conditions. Since overexpression of ADH6 resulted in increased glycerol yield in vivo (Tables 2 and 3), dihydroxyacetone phosphate (DHAP) reducing activity was determined for strains C-ADH6-2 and Yeast 2006; 23: 455–464.


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Table 2. Results from pulse addition of 1.5 g/l HMF in anaerobic batch cultivations of CEN. PK 113-5D, TMB3000 and CEN.PK 113-5D overexpressing ADH2, ADH6 and SFA1. 65 g/l glucose was used as carbon and energy sourcea

Strain CEN.PK 113-5D TMB3000 C/ADH2 T/ADH2 C/ADH6 T/ADH6 C/SFA1 T/SFA1

Specific growth rate (h−1 ) before addition of HMF

Specific growth rate (h−1 ) after addition of 1.5 g/l HMFb

Specific uptake rate of HMF (g/g/h)c

Glycerol yield (g/g)d

Biomass yield (g/g)d

0.38 ± 0.01 0.35 ± 0.01 0.36 ± 0.01 0.38 ± 0.02 0.34 ± 0.02 0.34 ± 0.01 0.39 ± 0.03 0.33 ± 0.02

0.23 ± 0.01 0.27 ± 0.03 0.23 ± 0.01 0.24 ± 0.04 0.21 ± 0.001 0.23 ± 0.01 0.23 ± 0.001 0.21 ± 0.01

0.10 ± 0.02 0.39 ± 0.13 0.10 ± 0.05 0.09 ± 0.01 0.35 ± 0.08 0.42 ± 0.002 0.10 ± 0.01 0.11 ± 0.004

0.069 ± 0.004 0.090 ± 0.006 0.071 ± 0.003 0.074 ± 0.003 0.089 ± 0.017 0.096 ± 0.002 0.081 ± 0.012 0.078 ± 0.005

0.057 ± 0.002 0.073 ± 0.001 0.059 ± 0.006 0.063 ± 0.001 0.068 ± 0.006 0.061 ± 0.008 0.061 ± 0.002 0.070 ± 0.002

Presented values are the mean value of two independent experiments ± SD. was added in the exponential growth phase when the biomass concentration reached 0.8 g/l. c Specific uptake rate of HMF (g/g/h) was calculated from the first 2.0–2.5 h after HMF addition. d Yields (g/g glucose) were calculated after glucose depletion. a

b HMF

Table 3. Results from pulse addition of 1.5 g/l HMF in aerobic batch cultivations of CEN. PK 113-5D, TMB3000 and CEN.PK 113-5D overexpressing ADH2, ADH6 and SFA1. 65 g/l glucose was used as carbon and energy source

Strain CEN.PK 113-5D TMB3000 C/ADH2 T/ADH2 C/ADH6 T/ADH6 C/SFA1 T/SFA1

Specific growth rate (h−1 ) before addition of HMF

Specific growth rate (h−1 ) after addition of 1.5 g/l HMFa

Specific uptake rate of HMF (g/g/h)b

Glycerol yield (g/g)c

Biomass yield (g/g)c

0.43 0.44 0.40 0.41 0.35 0.37 0.37 0.40

0.29 0.33 0.29 0.29 0.32 0.33 0.31 0.31

0.19 0.27 0.15 0.08 0.80 0.71 0.14 0.19

0.049 0.057 0.047 0.043 0.083 0.085 0.051 0.047

0.099 0.092 0.095 0.093 0.078 0.077 0.087 0.093

a

HMF was added in the exponential growth phase when the biomass concentration reached 0.8 g/l. uptake rate of HMF (g/g/h) was calculated from the first 2.0–2.5 h after HMF addition. c Yields (g/g glucose) were calculated only in the respirofermentative phase after HMF addition. b Specific

T-ADH6-2. No increase in DHAP reduction activity was found with either of the co-factors (data not shown), which would suggest that ADH6p does not have a directed catalytic role of in the glycerol pathway. However, further assays may be necessary to completely rule out this possibility. The 5-hydroxymethyl-furfural (HMF) reducing activity in crude extracts from ADH2, ADH6 and SFA1 overexpressing strains was determined using NADH and NADPH as co-factors (Figure 3). The highest HMF reducing activity was found with NADPH for strains overexpressing ADH6, where the specific activity was several orders of magnitudes higher than in any of the other strains. Copyright  2006 John Wiley & Sons, Ltd.

A low NADH-coupled HMF reduction activity was found for ADH6 and SFA1 overexpressing strains, whereas essentially no activity was found for ADH2 overexpressing strains, using either NADH or NADPH as co-factor. The specific HMF reducing activity was the same irrespective of the origin of the ADH6 gene (Figure 3), which suggested that there was no important difference in the structure and activity of the ADH6p protein in the CEN.PK 133-5D and TMB3000 strains, respectively. Indeed, a comparison of ADH6 gene sequences showed only a minor change — a substitution of G-203 in C-ADH6 for E-203 in T-ADH6 (data not shown). Yeast 2006; 23: 455–464.

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Figure 5. Furfural reduction activities in crude cell extracts from the control strain (CEN.PK 113-5D) and CEN.PK 113-5D overexpressing ADH6 gene originating from TMB3000 (T) or CEN.PK 113-5D (C). The activities were measured using (A) NADH or (B) NADPH as co-factor

The ability of the ADH6p protein to reduce the other main furan usually present in lignocellulose hydrolysates, furfural, was also investigated. With NADH as a co-factor, no increased furfural reduction was found for strains overexpressing ADH6 (Figure 5A). However, with NADPH furfural reduction was increased (Figure 5B), which agrees with reports that the ADH6 gene encodes an NADPH-dependent reductase able to reduce cinnamaldehyde, veratraldehyde and furfural (Larroy et al., 2002).

Discussion In the present study, we used genome-wide transcription analysis to identify the ADH6 gene from S. cerevisiae to encode NADPH-dependent HMF Copyright  2006 John Wiley & Sons, Ltd.

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reduction activity. HMF is a prominent inhibitor in lignocellulose hydrolysates and is therefore of significant interest. To the best of our knowledge, this is the first report in which genome-wide transcription analysis has been used for yeast, to successfully identify a yeast gene and to subsequently verify its function. Two strains of S. cerevisiae were compared, CBS8066, a laboratory strain (Verduyn et al., 1990), and TMB3000 (Lindén et al., 1992), known to tolerate inhibiting lignocellulose hydrolysates. The difference in HMF reducing activity of theses two strains has recently been correlated to their fermentation capacity in lignocellulose hydrolysates (Nilsson et al., 2005). Among known reductase/dehydrogenase genes (Katz et al., 2003; Stewart et al., 2001) the transcriptome analysis identified 18 candidate genes, which were upregulated in TMB3000 compared to CBS8066 and/or were induced by HMF. When the NADHand NADPH-dependent HMF reducing activity of the products of these candidate genes was further analysed in the Ex-Clone overexpression collection (Resgen, Invitrogen Corp., UK), the number of candidate genes could be reduced to three, ADH6, ADH7 and SFA1. Of these, ADH6 and SFA1 were cloned from the laboratory strain CEN.PK and from TMB3000 and overexpressed in CEN.PK. In addition ADH2 was also cloned and overexpressed because if its high expression in TMB3000, whereas ADH7 was not further investigated because of its low expression level. In vivo and in vitro HMF reduction by the CEN.PK strains overexpressing these three selected candidate genes unequivocally showed that HMF is reduced by the gene product of ADH6 using NADPH as co-factor. ADH6 encodes a strictly NADPH-dependent alcohol dehydrogenase with a high specificity for a variety of long-chain aliphatic and bulky substrates (Larroy et al., 2003). In addition, it has been reported to reduce cinnamaldehyde, veratraldehyde and furfural (Larroy et al., 2002), of which furfural reduction was confirmed in the present investigation. Overexpression of ADH6 from the two strains CEN.PK and TMB3000 resulted in almost identical HMF-reducing activity, suggesting that the high reduction capacity of TMB3000 was not due to mutations in the coding region of ADH6. This was further confirmed by analysis of the gene sequences. The high expression level in TMB3000 may be associated with mutations in the regulator sequences. Yeast 2006; 23: 455–464.


Both the genome-wide transcription analysis and the Ex-Clones analysis indicated that SFA1 encoded HMF-reducing activity. SFA1 encodes an enzyme that catalyses the final step of amino acid catabolism where its converts aldehydes to long-chain alcohols (Dickinson et al., 2003). Its product has also been shown to convey formaldehyde resistance (Wehner et al., 1993). The ExClone strain overexpressing SFA1 indeed showed an increased HMF reduction activity using NADH as co-factor, and overexpression of SFA1 from TMB3000 or CEN.PK 113-5D confirmed increased NADH-dependent HMF-reducing activity. However, this activity did not result in increased HMF uptake rate. ADH2 was cloned and overexpressed in CEN.PK because of its high expression level in strain TMB3000. ADH2 encodes an ethanol dehydrogenase, which is usually repressed by glucose (Lee and DaSilva, 2005). The enzyme is believed to oxidize ethanol to acetaldehyde during aerobic growth on ethanol (Leskovac et al., 2002). The product of ADH2 has also been shown to accept other primary unbranched aliphatic alcohols as substrate (Dickinson et al., 2003). However, the present investigation gave no evidence for a direct HMF-reducing activity of ADH2p with either NADH or NADPH. In vivo, ADH6 overexpression increased the glycerol yield (under both anaerobic and aerobic conditions) and decreased the biomass yield (aerobic conditions) in HMF-containing media in comparison with the control strain. Whereas the reduced biomass yield most likely is a consequence of NADPH being rerouted towards HMF reduction at the expense of biosynthetic reactions, the increased glycerol yield is more complicated to rationalize. The role of anaerobic glycerol production is to regenerate NAD+ required in anabolic reactions (Nordström, 1966). Since HMF reduction was shown to be strictly NADPH-dependent, the increased glycerol yield was unexpected and could not be explained by an increased anabolic NADH production. A direct reduction of DHAP by ADH6p with either NADH or NADPH was furthermore not supported by measurements. Therefore, further work is needed to understand ADH6-related glycerol formation. In conclusion, the identification of ADH6p to be responsible for NADPH-dependent HMF reduction could be accomplished by genome-wide transcription analysis of cells from two yeast strains Copyright  2006 John Wiley & Sons, Ltd.

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differing substantially in HMF reduction capacity. The verification of the transcription results required screening of the Ex-Clone collection in combination with cloning and overexpression of a small number of selected genes. Furthermore, verification of the function of candidate genes also included in vivo and in vitro characterization of strains in which these genes were overexpressed. The current investigation proves the power of genomewide transcription analysis, while it demonstrates the importance of biochemical and physiological methods to provide useful data from such analysis.

Acknowledgements The work was sponsored by the Swedish Energy Agency and the Swedish Foundation for international cooperation in Research and Higher Education (STINT).

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