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The aim of this study was to develop a method to optimize expression levels of xylose-metabolizing enzymes to improve xylose utilization capacity of Saccharomyces cerevisiae. A xylose-utilizing recombinant S. cerevisiae strain YY2KL, able to express nicotinamide adenine dinucleotide phosphate, reduced (NADPH)-dependent xylose reductase (XR), nicotinamide adenine dinucleotide (NAD(+))-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK), showed a low ethanol yield and sugar consumption rate. To optimize xylose utilization by YY2KL, a recombinant expression plasmid containing the XR gene was transformed and integrated into the aur1 site of YY2KL. Two recombinant expression plasmids containing an nicotinamide adenine dinucleotide phosphate (NADP(+))-dependent XDH mutant and XK genes were dually transformed and integrated into the 5S ribosomal DNA (rDNA) sites of YY2KL. This procedure allowed systematic construction of an S. cerevisiae library with different ratios of genes for xylose-metabolizing enzymes, and well-grown colonies with different xylose fermentation capacities could be further selected in yeast protein extract (YPX) medium (1 % yeast extract, 2 % peptone, and 2 % xylose). We successfully isolated a recombinant strain with a superior xylose fermentation capacity and designated it as strain YY5A. The xylose consumption rate for strain YY5A was estimated to be 2.32 g/gDCW/h (g xylose/g dry cell weight/h), which was 2.34 times higher than that for the parent strain YY2KL (0.99 g/gDCW/h). The ethanol yield was also enhanced 1.83 times by this novel method. Optimal ratio and expression levels of xylose-metabolizing enzymes are important for efficient conversion of xylose to ethanol. This study provides a novel method that allows rapid and effective selection of ratio-optimized xylose-utilizing yeast strains. This method may be applicable to other multienzyme systems in yeast.

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