Direct enzyme assay evidence confirms aldehyde

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Yeast Yeast 2015; 32: 399–407. Published online 26 February 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/yea.3067

Research Article

Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae Jaewoong Moon# and Z. Lewis Liu* BioEnergy Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, IL USA

*Correspondence to: Z. L. Liu, US Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, 1815 N University Street, Peoria, IL 61604, USA. E-mail: [email protected] #

Present address: Xyleco Inc., 271 Salem Street, Woburn, MA 01801, USA

Received: 16 September 2014 Accepted: 28 January 2015

Abstract The aldehyde reductase gene ARI1 is a recently characterized member of an intermediate subfamily within the short-chain dehydrogenase/reductase (SDR) superfamily that clarified mechanisms of in situ detoxification of 2-furaldehyde and 5-hydroxymethyl-2furaldehyde by Saccharomyces cerevisiae. Uncharacterized open reading frames (ORFs) are common among tolerant candidate genes identified for lignocellulose-toadvanced biofuels conversion. This study presents partially purified proteins of two ORFs, YDR541C and YGL039W, and direct enzyme assay evidence against aldehydeinhibitory compounds commonly encountered during lignocellulosic biomass fermentation processes. Each of the partially purified proteins encoded by these ORFs showed a molecular mass of approximately 38 kDa, similar to Ari1p, a protein encoded by aldehyde reductase gene. Both proteins demonstrated strong aldehyde reduction activities toward 14 aldehyde substrates, with high levels of reduction activity for Ydr541cp toward both aromatic and aliphatic aldehydes. While Ydr541cp was observed to have a significantly higher specific enzyme activity at 20 U/mg using co-factor NADPH, Ygl039wp displayed a NADH preference at 25 U/mg in reduction of butylaldehyde. Amino acid sequence analysis identified a characteristic catalytic triad, Ser, Tyr and Lys; a conserved catalytic motif of Tyr–X–X–X–Lys; and a cofactor-binding sequence motif, Gly–X–X–Gly–X–X–Ala, near the N-terminus that are shared by Ydr541cp, Ygl039wp, Yol151wp/GRE2 and Ari1p. Findings of aldehyde reductase genes contribute to the yeast gene annotation and aids development of the next-generation biocatalyst for advanced biofuels production. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: aldehyde reductase family; direct enzyme assay; gene annotation; in situ detoxification; Saccharomyces cerevisiae

Introduction For advanced biofuels production from lignocellulosic biomass, including agricultural and industrial processing residues, a pretreatment of the biomass is needed in order to release fermentable sugars for microbial use. Depolymerization of cellulose and hemicellulose materials typically produces toxic by-products that inhibit microbial growth and fermentation. Inhibitory compounds liberated from lignocellulose pretreatment are well known (Klinke et al., 2004; Larsson et al., 1999; Liu and Copyright © 2015 John Wiley & Sons, Ltd.

Blaschek, 2010; Palmqvist and Hahn-Hägerdal, 2000). Remediation of inhibitory compounds by additional physical and chemical means appears too expensive and economically impractical (Liu and Blaschek, 2010). Using tolerant ethanologenic strains of Saccharomyces cerevisiae to detoxify lignocellulosic inhibitors, such as 2furaldehyde (2-furancarbaldehyde, or furfural) and 5-(hydroxymethyl)-2-furaldehyde (5-hydroxythyl2-furancarbaldehyde, or HMF) in situ while producing ethanol was successful (Liu and Moon, 2009; Liu et al., 2009). It was concluded that

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multiple gene-mediated NAD(P)H-dependent aldehyde reduction is one of the most significant mechanisms of the in situ detoxification for the tolerant yeast (Heer et al., 2009; Liu et al., 2008). New pathways and reprogrammed metabolic pathways were discovered for an improved tolerant industrial yeast strain to maintain cofactor regeneration balance in S. cerevisiae (Liu et al., 2009). Key regulatory elements and molecular mechanisms were identified at the genome level for yeast tolerance and in situ detoxification (Ma and Liu, 2010; Yang et al., 2012). However, with a large number of genes involved in yeast tolerance and detoxification, many genes and open reading frames (ORFs), including tolerance candidate genes, in the S. cerevisiae genome remain uncharacterized. We recently characterized a novel NADPHdependent aldehyde reductase gene, ARI1, from S. cerevisiae, as a member of a subclass intermediate within the short-chain dehydrogenase/reductase (SDR) superfamily (Liu and Moon, 2009; http:// www.yeastgenome.org/). The enzyme kinetics of this reductase have been characterized and its stereochemistry in furan aldehyde reduction established (Bowman et al., 2010; Jordan et al., 2011). Several ORFs, such as YDR541C, YGL039W and YOL151W (GRE2), were observed to have similar enhanced gene expression response to furfural or HMF (Liu, 2006; Liu and Slininger, 2006; Liu and Moon, 2009; Moon and Liu, 2012). These ORFs encode similar amino acid sequences; however, evidence of direct enzyme assay is not available to establish a valid gene annotation. Gene annotation by computation and high-throughput data is commonly used to predict the functions of unknown genes. However, such predicted functions are often inconsistent with low confidence, such as on annotation of the previously unknown ORF YGL157W/ARI1 (Baxter et al., 2004; Joshi et al., 2004; Liu and Moon, 2009; Pir et al., 2006). Gene function inferred by direct assay is more reliable and is preferred as a functional annotation method. In this study, we isolated proteins and evaluated aldehyde reduction activities for two previously identified target ORFs, YDR541C and YGL039W, toward 14 aldehyde compounds related to lignocellulose hydrolysates. Evidence of direct enzyme assay aids identification and characterization of functions of unknown genes. Copyright © 2015 John Wiley & Sons, Ltd.

J. Moon and Z. L. Liu

Materials and methods Yeast and bacterial strains, plasmids, media, and culture conditions S. cerevisiae NRRL Y-12632, obtained from Agricultural Research Service Culture Collection (Peoria, IL, USA), was used for this study. Cultures were maintained and cultured on a synthetic complete medium, as previously described (Liu et al., 2004). Amino acids and all chemicals were provided by Sigma-Aldrich (St. Louis, MO, USA). For cloning procedures, the E. coli Top10 strain from Invitrogen (Carlsbad, CA, USA) was used. The genomic DNA from wildtype S. cerevisiae NRRL Y-12632 was used to amplify fragments of ORFs YDR541C and YGL039W.

DNA construction and gene overexpression The E. coli–yeast shuttle vector pYes2/NT (Invitrogen), carrying a URA3 selection marker and a galactose-inducible promoter of GAL1, was used to clone and overexpress the selected target genes YDR541C and YGL039W in the fast-growing diploid yeast strain INVSc1 (Invitrogen). The E. coli strain was grown at 37°C in LB medium supplemented with 50 μg/ml ampicillin for selection of desired plasmid constructs. Yeast cells were grown at 30°C on minimal medium without uracil, supplemented with 2% glucose for initial yeast growth. For overexpression evaluation, cells of the gene clone were grown on minimum medium without uracil and containing 2% glucose at 30°C for initial growth for 24 h. The yeast cells were harvested and introduced into an induction medium containing varied concentrations of butyl aldehyde, furfural or HMF in the range 10–30 mM. The induction medium consisted of minimum medium containing 2% galactose and 1% raffinose without any solvate. A transformant without a gene insert, grown under the same conditions, served as a control. Cell growth was monitored over time and measured by absorbance at OD600. Three replicated experiments were carried out for each treatment. Cells harvested 15 h after incubation on induction medium were used for the examination of protein expression. Yeast 2015; 32: 399–407. DOI: 10.1002/yea

Aldehyde reductase function of Ydr541cp and Ygl039wp

Protein purification Yeast cells were harvested and disintegrated by Y-PER® Plus reagent (Pierce, Rockford IL, USA). Cell pellets were resuspended in the Y-PER® Plus reagent solution and incubated at 25°C with shaking for 20 min. The homogenate was centrifuged at 20 000 × g for 30 min and the pellet was discarded. The supernatant fraction was applied to a column (1.5 × 1.0 cm) of Ni-NTA equilibrated with binding buffer (50 mM sodium chloride, pH 7, with 10 mM imidazole). A washing buffer was prepared with a binding buffer plus 20 mM imidazole and applied for the protein sample cleaning. Then, the expressed protein was eluted by using as 10 ml step-wise gradient of 250–500 mM imidazole in binding buffer. The molecular weight of the partially purified proteins was estimated using SDS–PAGE gel electrophoresis, following standard procedures (Sambrook and Russell, 2001).

Enzyme assay Crude cell extract or purified protein was assayed for activity using a Genesys 10UV spectrophotometer, as previously described (Liu et al., 2008), with modifications. Activity was monitored by recording a decrease in absorbance at 340 nm using NADH or NADPH. Assays were carried out in 500 μl volumes at 25°C or 37°C and lasted for 1 min. The reaction mixture consisted of a final concentration of 10 mM HMF or furfural or other aldehyde substrates and 100 μM cofactor in 100 mM sodium phosphate buffer, pH 6.5. All reagents were maintained in a 25°C or 37°C water bath prior to use. Cell lysates were kept on ice until use. Crude cell extract or purified protein was added to initiate a reaction. The spectrophotometer was blanked before each assay, using the reaction mixture as stated above without enzyme. Duplicated experiments were carried out for all assays.

Enzyme kinetics A typical reaction was performed in a sodium phosphate (100 mM, pH 6.5) buffer containing 200 μM NADH or NADPH, aldehyde substrate (2-furaldehyde or cinnamic aldehyde) at each optimized temperature of 37°C or 25°C for YDR541C and YGL039W, respectively. The consumption of Copyright © 2015 John Wiley & Sons, Ltd.

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NADH or NADPH was determined by measuring the decrease of absorbance at 340 nm with a spectrophotometer for up to 1 min. The kinetic parameters were measured with varied concentrations of a defined aldehyde compound (0.2–75 mM). A reaction with a cofactor of NADH or NADPH but without enzyme addition was used as a control measurement. Duplicated experiments were carried out for each assay. Enzyme activities and kinetic parameters were calculated using Grafit data analysis software (Erithacus Software, Surrey, UK).

Results Cell growth response to selective aldehyde inhibitors Gene constructs of pYes2NT/C–YDR541C and pYes2NT/C–YGL039W were transformed into yeast strain INVSc1 for test on cell growth response to selective inhibitors. Initial cultures with a cell density in the range OD600 0.064–0.099 were used to evaluate cell growth response. Cells of each gene overexpressing yeast strains showed more resistance to butyl aldehyde, furfural and HMF at varied concentrations (Figure 1). Both strains were able to recover with a shortened lag phase against inhibitor challenge compared with a control without a target gene insert.

Protein expression and purification A partially purified protein from each gene clone was obtained for each with a net molecular weight of approximately 38 kDa. To facilitate protein purification using Ni-NTA column chromatography, we added a polyhistidine-tag with 65 extra amino acid residues, accounting for about 7 kDa in molecular weight, to each of the above overexpressed clones. Thus, on SDS–PAGE gel, both partially purified proteins showed a molecular weight of approximately 45 kDa (Figure 2) with the artificial polyhistidine-tag.

Cofactor preference and enzyme kinetics Using butylaldehyde as a substrate, Ydr541cp protein showed a significantly higher specific enzyme activity toward reduction of the substrate with cofactor NADPH than NADH (Table 1). On the Yeast 2015; 32: 399–407. DOI: 10.1002/yea

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J. Moon and Z. L. Liu 1.2

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Figure 1. Comparison of cell growth in the (a) absence or (b) presence of 10 mM butyl aldehyde, (c) 30 mM HMF and (d) 20 mM furfural in a synthetic minimum medium for clone YDR541C (filled square) and YGL039W (filled triangle) from Saccharomyces cerevisiae, and a control without a target gene insert (open circle)

other hand, Ygl039wp protein displayed a significantly higher enzyme activity with cofactor NADH than NADPH in reduction of butylaldehyde. Ydr51cp showed a stronger Km of 8.66 mM toward furfural and Ygl039wp had a Vmax (s–1) of 19.9 and 8.87 toward furfural and cinnamic aldehyde, respectively (Table 2). Due to the instability of the enzyme, active concentrations of Vmax for Ydr541cp were not available.

Optimal temperature and pH

Figure 2. An SDS-PAGE gel showing molecular weight markers (MM, in kDa) and partially purified proteins encoded by (a) YDR541C and (b) YGL039W with a 7 kDa polyhistidine tag, using Ni-NTA affinity chromatography. The Ydr541cp– and Ygl039wp–histidine complexes each showed a molecular mass of 45 kDa and a net molecular mass of Ydr541cp and Ygl039wp proteins of approximately 38 kDa each Copyright © 2015 John Wiley & Sons, Ltd.

The highest enzyme activity was observed at pH 6.5 for Ydr541cp with pH values tested in the range 5–8.5 (Figure 3a). In the same range, Ygl039wp appeared less sensitive to a pH and showed similar levels of enzyme activity pH 6–7 (Figure 3b). Enzyme assay for Gre2p was commonly performed at pH 7.2 (Larroy et al., 2002a; Moon and Liu, 2012). The optimal pH of all these enzymes appeared similar, toward a neutral pH condition. Similar to Ari1p and Gre2p, Ygl039wp displayed a lower temperature preference for its Yeast 2015; 32: 399–407. DOI: 10.1002/yea

Aldehyde reductase function of Ydr541cp and Ygl039wp

Table 1. Cofactor preference of enzyme activity for Ydr541cp and Ygl039wp toward reduction of butylaldehyde Gene

Cofactor

Specific activity (mU/mg)

NADH NADPH NADH NADPH

306.5 ± 39.4 21054.9 ± 1959.0 25560.8 ± 2556.0 337.6 ± 36.7

Ydr541cp Ygl039wp

maximum enzyme activity (Figure 3d). In contrast, Ydr541cp showed its highest enzyme activity at 45°C (Figure 3c), a much higher temperature than the other proteins of Ygl039wp, Gre2p and Ari1p.

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Lys171 and Tyr170–X–X–X–Lys174 for Ydr541p and Ygl039cp, respectively. For Yol151wp/GRE2, this site was located at Tyr165–X–X–X–Lys169. These sequences were found to share an indispensible catalytic triad of Ser, Tyr and Lys (Figure 5), a common characteristic for SDR enzymes. Another typical characteristic of intermediate subfamily enzymes is a conserved cofactor-binding motif, Gly–X–X–Gly–X–X– Ala. It was shared with all the four proteins and located at a starting position of Gly11, Gly12, Gly7 and Gly9, near the N-terminus for ARI1, Ygl039wp, Yol151wp/GRE2 and Ydr541cp, respectively (Figure 5).

Substrate specificity Both Ydr541cp and Ygl039wp showed specific reduction activity to a wide range of aldehyde substrates. Among 14 aldehydes associated with lignocellulosic hydrolysate tested in this study, Ydr541cp was highly active toward both aromatic and aliphatic aldehydes (Figure 4). The highest specific activity in reduction of butyaldehyde reached almost 20 U/mg. On the other hand, Ygl039wp showed a stronger enzyme activity toward aliphatic aldehydes but a relatively weaker activity toward aromatic aldehydes. Its highest specific activity toward butylaldehyde was recorded at 25 U/mg.

Amino acid sequence analysis The amino acid sequences of proteins encoded by YDR541C, YGL039W and YOL151W/GRE2 are highly similar to those of YGL157W, a recently defined aldehyde reductase gene, ARI1 (Figure 5). A typical conserved catalytic site of Tyr169–X–X–X– Lys173 in ARI1was observed at Tyr167–X–X–X– Table 2. Parameters of enzyme kinetics for a partially purified protein of Ydr541cp and Ygl039wp of Saccharomyces cerevisiae Ydr541cp

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Ygl039wp

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n.a. n.a.

28.4 ± 4.7 18.6 ± 2.5

19.9 ± 1.38 8.87 ± 0.42

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Active concentration of Ydr541cp was not available (n.a.), due to instability of the enzyme.

Copyright © 2015 John Wiley & Sons, Ltd.

Discussion Functions of aldehyde reductase activity in S. cerevisiae have not been realized until the recent comprehensive characterization of a novel gene, ARI1, and its protein by sequence analysis, gene expression assay, direct enzyme assay, protein characterization, enzyme kinetics and stereochemistry (Bowman et al., 2010; Jordan et al., 2011; Liu and Moon, 2009). The functions of Ari1p were strongly supported by yeast in situ detoxification of toxic compounds associated with lignocellulosic hydrolysate. Ari1p was categorized as a member of an intermediate subfamily belonging to the SDR superfamily, based on its distinct amino acid sequence structure (Kavanagh et al., 2008; Liu and Moon, 2009). Recognition of an aldehyde reduction function in the yeast is particularly significant for biomass-to-advanced biofuels conversion, since many aldehydeinhibitory compounds are commonly encountered during the biomass fermentation process. This study provided the first evidence of direct enzyme assay for Ydr541cp and Ygl039wp toward 14 aldehyde substrates associated with lignocellulosic hydrolysates. Both proteins showed a similar molecular weight of 38 kDa, similar to the previously identified aldehyde reductase Ari1p (Liu and Moon, 2009). We confirmed a catalytic triad, Ser, Tyr and Lys; a conserved catalytic motif, Tyr–X–X–X–Lys; and a conserved cofactorbinding sequence motif, Gly–X–X–Gly–X–X–Ala, near the N-terminus shared by these proteins. Based on their characteristic sequence and specific enzyme Yeast 2015; 32: 399–407. DOI: 10.1002/yea

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Figure 3. Specific enzyme activity performance of a partially purified protein at (a, b) a varied range of pH and (c, d) temperatures for (a, c) Ydr541cp and (b, d) Ygl039wp. Enzyme assay was performed at the optimized condition for each, using the corresponding cofactor of NADH or NADPH. Optimal pH was tested at 37°C and temperature tested at pH 6.5 for ydr541cp. For ygl039w, the optimal pH evaluation was performed at 25°C and the optimal temperature was tested at pH 6.5 in a sodium phosphate buffer

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Figure 4. Specific enzyme activity of a partially purified protein of Ydr541cp (open bar) and Ygl039wp (grey shaded bar) toward 14 aldehyde substrates at 100 mM each, using cofactor NADPH or NADH, respectively, in a sodium phosphate buffer Copyright © 2015 John Wiley & Sons, Ltd.

Yeast 2015; 32: 399–407. DOI: 10.1002/yea

Aldehyde reductase function of Ydr541cp and Ygl039wp

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Figure 5. A comparison of amino acid sequence for four closely related proteins encoded by ORFs or genes of YGL157W/ ARI1, YOL151W/GRE2, YDR541C and YGL039W from Saccharomyces cerevisiae. Conserved consensus sequences are highlighted in dark grey and relatively conserved and similar sequences are shaded in medium and light grey, respectively. The conserved catalytic triad sites are marked by an arrow. A shared typical cofactor-binding motif region, Gly–X–X– Gly–X–X–Ala, is boxed near the N-terminus, and a conserved catalytic site, Tyr170–X–X–X–Lys174, commonly presented for all four genes, is underlined

activity toward a wide range of aldehyde substrates, our results suggest YDR541 and YGL039W to be members of the aldehyde reductase gene family. YDR541C is an uncharacterized ORF and its biological functions are unknown. Information on its protein product is not available. Based on sequence or structural similarity, it was suggested as a putative dihydrokaempferol 4-reductase (http://www.yeastgenome.org/). Similar to Ari1p, Ydr541cp showed stereoselective reduction of bicyclic diketone bicyclo [2.2.2] octane-2,6-dione and anti-2 stereoselective reduction (Kaluzna et al., 2005; Katz et al., 2003). A YDR541Cdeletion mutation strain also showed approximate 80% reduced reductase activity toward a model carbonyl compound ethyl acetoacetate. The ORF Copyright © 2015 John Wiley & Sons, Ltd.

YGL039W is an oxidoreductase showing reduction activity of carbonyl compounds to chiral alcohols (Kaluzna et al., 2005). It has been used to reduce α-chloro-β-keto ester (Feske et al., 2005) and effectively catalysed the reduction of α-azidoacetophenone (Ankati et al., 2008). Its reduction activity toward phenylacetaldehyde to produce 2-phenylethanol has also been observed previously (Hwang et al., 2009). Isolation of these proteins and evidence of direct enzyme assay presented in this study suggested a function of aldehyde reduction of these proteins. Due to a close relationship of sequence similarity with GRE2, ORFs YGL039W, YDR541C and YGL157W/ARI1 were previously reported collectively as the GRE2 group. Originally, GRE2 was Yeast 2015; 32: 399–407. DOI: 10.1002/yea

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identified as a stress-responsive gene (genes de respuesta a estres in Spanish; Garay-Arroyo and Covarrubias, 1999) and it did not indicate a specific enzymatic function. Consequently, GRE3 was similarly identified. Both GRE2 and GRE3 were strongly induced by challenges of aldehyde inhibitors, such as furfural and HMF, and were identified as candidate tolerant genes (Liu et al., 2008; Ma and Liu, 2010). Multiple functions of a gene are commonly observed. New functions of aldehyde reduction were discovered for numerous genes that have been characterized previously. For example, alcohol dehydrogenase VI and VII (ADH6 and ADH7) were found to be significantly enhanced, expressed by challenges of furfural and HMF, and their aldehyde reductase activity is significantly higher than their originally reported alcohol dehydrogenase activity (Larroy et al., 2002a, 2002b; Liu et al., 2008; Petersson et al., 2006). Enhanced expressions of these genes were also commonly observed to be associated with stress conditions for yeast, including challenges by furfural and HMF (Garay-Arroyo and Covarrubias, 1999; Rep et al., 2001; Krantz et al., 2004; Liu and Slininger, 2006; Liu et al., 2009; Ma and Liu, 2010). A gene is more appropriately characterized and named after its major enzymatic function. Our results suggest the existence of an aldehyde reductase gene family in S. cerevisiae represented by the newly characterized ARI1 and its closely related ORFs. The recently reported Ari1p was identified to have high enzyme activity using NADPH as a cofactor in its aldehyde reduction reactions (Liu and Moon, 2009). In this study, Ydr541cp showed a significantly stronger activity using NADPH than NADH. On the other hand, Ygl039wp demonstrated the highest enzyme activity using cofactor NADH, although it was active using NADPH as well. In general, proteins of GRE2 displayed a strong aldehyde reductase activity against furfural and HMF using NADH (Liu and Moon, 2009; Liu et al., 2008). Gre2p is also able to use the additional cofactor NADPH through protein-engineering efforts. Evidence of site-directed mutagenesis confirmed that this function was a result of amino acid substitution of Val285 to Asp285 for Gre2p (Moon and Liu, 2012). Efficient reductions of aldehydes by other genes were observed to use either NADH or NADPH (Liu et al., 2008). No critical cofactor preference appeared to be found for this group of enzymes. Copyright © 2015 John Wiley & Sons, Ltd.

J. Moon and Z. L. Liu

Overcoming toxic inhibitory compounds liberated from lignocellulosic biomass pretreatment is a significant challenge for the development of next-generation biocatalysts. Aldehyde inhibitors are potent and representative inhibitors commonly encountered for biomass-to-biofuel conversions. The discovery and identification of functions of genes able to specifically degrade these toxic compounds are necessary for tolerant strain development. Key tolerance genes, including those aldehyde reduction genes, were highly activated in the reprogrammed tolerant yeast metabolic pathways (Liu et al., 2009). Recent genetic engineering efforts using furfural reduction genes to enhance bacterial tolerance for biofuels production are encouraging. Our results in identification of members of an aldehyde reduction gene family not only contribute to the annotation of yeast genes and genomes but also aid tolerant strain development of the next-generation biocatalyst for advanced biofuels production from lignocellulosic materials.

Acknowledgements This work was supported in part by the National Research Initiative award of the USDA National Institute of Food and Agriculture (Project No. 2006-35504-17359). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer.

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Yeast 2015; 32: 399–407. DOI: 10.1002/yea