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 Petersson 1# , Jo˜ ao R. M. Almeida 2# , Tobias Modig 1 , Kaisa Karhumaa 2 , B¨ arbel Hahn-H¨ agerdal 2 , Marie F. Gorwa-Grauslund 2 and Gunnar Lid´ en 1 * 1 Department of Chemical Engineering, Lund University, PO Box 124, S-221 00 Lund, Sweden 2 Department of Applied Microbiology, Lund University, PO Box 124, S-221 00 Lund, Sweden *Correspondence to: Gunnar Lid´ en, Department of Chemical Engineering, Lund University, PO Box 124, S-221 00 Lund, Sweden. E-mail: Gunnar.Liden@chemeng.lth.se # 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 113- 5D. Strains overexpressing ADH6 had increased HMF conversion activity in cell- free 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 deriva- tives and aromatic compounds are released and formed (Larsson et al., 1999; Palmqvist and Hahn- H¨ agerdal, 2000). These compounds are inhibitory to the bioconversion/fermentation steps. One of the quantitatively most important inhibitors in ligno- cellulose hydrolysates is 5-hydroxymethyl furfural (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 Copyright  2006 John Wiley & Sons, Ltd.