APPLIED MICROBIAL AND CELL PHYSIOLOGY Carbon fluxes of xylose-consuming Saccharomyces cerevisiae strains are affected differently by NADH and NADPH usage in HMF reduction João R. M. Almeida & Magnus Bertilsson & Bärbel Hahn-Hägerdal & Gunnar Lidén & Marie-F. Gorwa-Grauslund Received: 3 April 2009 / Revised: 20 May 2009 / Accepted: 21 May 2009 / Published online: 9 June 2009 # Springer-Verlag 2009 Abstract Industrial Saccharomyces cerevisiae strains able to utilize xylose have been constructed by overexpression of XYL1 and XYL2 genes encoding the NADPH-preferring xylose reductase (XR) and the NAD + -dependent xylitol dehydrogenase (XDH), respectively, from Pichia stipitis. However, the use of different co-factors by XR and XDH leads to NAD + deficiency followed by xylitol excretion and reduced product yield. The furaldehydes 5-hydroxymethyl- furfural (HMF) and furfural inhibit yeast metabolism, prolong the lag phase, and reduce the ethanol productivity. Recently, genes encoding furaldehyde reductases were identified and their overexpression was shown to improve S. cerevisiae growth and fermentation rate in HMF containing media and in lignocellulosic hydrolysate. In the current study, we constructed a xylose-consuming S. cerevisiae strain using the XR/XDH pathway from P. stipitis. Then, the genes encoding the NADH- and the NADPH-dependent HMF reductases, ADH1-S110P-Y295C and ADH6, respectively, were individually overexpressed in this background. The performance of these strains, which differed in their co-factor usage for HMF reduction, was evaluated under anaerobic conditions in batch fermentation in absence or in presence of HMF. In anaerobic continuous culture, carbon fluxes were obtained for simultaneous xylose consumption and HMF reduction. Our results show that the co-factor used for HMF reduction primarily influenced formation of products other than ethanol, and that NADH-dependent HMF reduction influenced product formation more than NADPH-dependent HMF reduction. In particular , NADH-dependent HMF reduc- tion contributed to carbon conservation so that biomass was produced at the expense of xylitol and glycerol formation. Keywords ADH1 . ADH6 . Furaldehyde reduction . HMF . Xylose . Lignocellulosic hydrolysate Introduction The use of lignocellulosic biomass for sustainable produc- tion of fuels and chemicals with bioconversion is an attractive alternative to fossil raw material (Farrell et al. 2006; Hahn-Hägerdal et al. 2006) and baker ’ s yeast Saccharomyces cerevisiae is the preferred organism in the fermentation industry. However, commercial bioconversion of lignocellulose requires yeast strains capable to utilize both hexose and pentose sugars (Hahn-Hägerdal et al. 2007a, b; Jeffries 2006), and to do so in presence of metabolic inhibitors produced during lignocellulose pretreatment and hydrolysis (Almeida et al. 2007; Hahn-Hägerdal et al. 2007a; Klinke et al. 2004). For S. cerevisiae to be able to utilize the pentose sugar xylose, the second most abundant sugar in nature, heterologous xylose isomerase or xylose reductase/ xylitol dehydrogenase (XR/XDH) pathways have been introduced in recombinant strains (Chu and Lee 2007; Hahn-Hägerdal et al. 2007a; van Maris et al. 2007). Concurrently, a number of genetic engineering strategies to reduce the influence of lignocellulose derived inhibitors have also been developed, including construction of S. cerevisiae strains expressing laccase (Larsson et al. 2001a), phenyl- Appl Microbiol Biotechnol (2009) 84:751–761 DOI 10.1007/s00253-009-2053-1 J. R. M. Almeida : B. Hahn-Hägerdal : M.-F. Gorwa-Grauslund (*) Department of Applied Microbiology, Lund University, P.O. Box 124, S-221 00 Lund, Sweden e-mail: Marie-Francoise.Gorwa@tmb.lth.se M. Bertilsson : G. Lidén Department of Chemical Engineering, Lund University, P.O. Box 124, S-221 00 Lund, Sweden