Influence of individual HXT transporters in xylose fermentation by recombinant Saccharomyces cerevisiae strains

Abstract

Background Lignocellulosic biomass is an attractive raw material for bioethanol production since it is an abundant and renew-able feedstock that does not compete with food and feed production [1]. Xylose is the most abundant pentose pre-sent on these feedstocks, and although S. cerevisiae can-not readily ferment this sugar, the overexpression of the genes for xylose reductase (XR) and xylitol dehidrogenase (XDH) from P. stiptis and xylulokinase (XK) from S. cere-visiae allows the utilization of xylose [2]. However S. cere-visiae also lacks specific transporters for this sugar and thus the uptake of xylose is carried out by native hexose transporters encoded by the HXT1-HXT7 genes [3]. In the present report we analyzed the impact of individual HXT transporters on xylose fermentation by recombinant S. cerevisiae yeast strains overexpressing the genes for XR, XDH and XK [4]. Methods Cultivations were perfomed in rich (YP) or synthetic complete (SC) medium containing the required sugars and when necessary, 2% Bacto agar, 0.5 mg/l aureobasi-din A and 200 mg/l Geneticin were added to the med-ium. The chromosome-integrative plasmid pAUR-XKXDHXR [4] containing PGK promoters for overex-pression of XR, XDH and XK was digested with BsiWI and then chromosomally integrated into the AUR1 locus of the yeast strains. HXT1, HXT2, HXT5 and HXT7 genes were obtained by PCR from S288c S. cerevisiae genomic DNA and cloned individually into a pPGK multicopy plasmid [5], and these plasmids were transformed into the strains lacking all HXT genes or individual HXT genes, respectively. Anaerobic batch fer-mentations were performed at 30°C in closed 50-ml bot-tles with a magnetic stir bar and 100 rpm. Assays with 2-6% of glucose, xylose or both sugars were performed. During fermentation cell growth was monitored and samples were removed for further analysis. Glucose, xylose, ethanol, xylitol, glycerol, and acetic acid were determined by HPLC as previously described [4]. Results and conclusion The deletion of individual HXT genes had no detectable effect on glucose fermentations, but these knockout strains ferment xylose poorly, even under glucose plus xylose conditions. The low-affinity HXT1 permease allowed the maximal consumption of sugars and ethanol production rates during xylose plus glucose co-fermen-tation, but was incapable to allow xylose consumption when this sugar was the only carbon source. The high-affinity HXT7permease allowed efficient xylose fermen-tation, but during xylose plus glucose co-fermentation this permease showed a clear preference for glucose. While the HXT5 permease performed bad with glucose and did not allow xylose utilization, the moderately high-affinity HXT2 permease was a transporter that allowed xylose consumption with the same rates as glu-cose, even under co-fermentation conditions, but had the drawback of producing stuck fermentations. Thus, our results indicate that new approaches to engineer selected HXT transporters to increase their affinity towards pentoses, or to avoid their sugar-induced degra-dation, are promising strategies to improve second gen-eration bioethanol production by xylose-fermenting yeasts.