Construction of an efficient xylose-fermenting diploid Saccharomyces cerevisiae strain through mating of two engineered haploid strains capable of xylose assimilation

► Classic yeast mating method was employed for improving xylose-fermenting capabilities of engineered Saccharomyces cerevisiae. ► Complementation of sub-optimal xylose-assimilating pathways was responsible for improved phenotypes of the heterozygous diploid strain. ► Activities of the xylose-assimilating enzymes could determine efficiency of xylose fermentation of engineered S. cerevisiae. Saccharomyces cerevisiae can be engineered for xylose fermentation through introduction of wild type or mutant genes (XYL1/XYL1 (R276H), XYL2, and XYL3) coding for xylose metabolic enzymes from Scheffersomyces stipitis. The resulting engineered strains, however, often yielded undesirable phenotypes such as slow xylose assimilation and xylitol accumulation. In this study, we performed the mating of two engineered strains that exhibit suboptimal xylose-fermenting phenotypes in order to develop an improved xylose-fermenting diploid strain. Specifically, we obtained two engineered haploid strains (YSX3 and SX3). The YSX3 strain consumed xylose rapidly and produced a lot of xylitol. On the contrary, the SX3 strain consumed xylose slowly with little xylitol production. After converting the mating type of SX3 from alpha to a, the resulting strain (SX3-2) was mated with YSX3 to construct a heterozygous diploid strain (KSM). The KSM strain assimilated xylose (0.25gxyloseh−1gcells−1) as fast as YSX3 and accumulated a small amount of xylitol (0.03ggxylose−1) as low as SX3, resulting in an improved ethanol yield (0.27ggxylose−1). We found that the improvement in xylose fermentation by the KSM strain was not because of heterozygosity or genome duplication but because of the complementation of the two xylose-metabolic pathways. This result suggested that mating of suboptimal haploid strains is a promising strategy to develop engineered yeast strains with improved xylose fermenting capability.