Construction of a xylose-fermenting Saccharomyces cerevisiae strain by combined approaches of genetic engineering, chemical mutagenesis and evolutionary adaptation

Abstract

Recombinant Saccharomyces cerevisiae strains constructed by metabolic engineering approaches can ferment xylose but with low efficiency. We constructed a S. cerevisiae strain via combined approaches of recombinant DNA technology, chemical mutagenesis and evolutionary adaptation for an efficient xylose utilization and ethanol fermentation. A haploid derivative of an industrial ethanol-fermenting S. cerevisiae strain was first engineered to express the XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK). This recombinant strain, LEK122, was then subjected to EMS mutagenesis followed by adaptive evolution, resulting in a single isolate, LEK513, which displayed significantly improved xylose-utilizing property. The specific growth rate of the LEK513 strain was 0.225 h−1 under aerobic condition (0.205 h−1 under oxygen-limited condition) with xylose as the sole carbon source, while that of the LEK122 was 0.055 h−1. During 100 h batch cultivation, the optical density of LEK513 reached 60, while LEK122 only grew to 7.5. In the same time period, LEK513 consumed 95% of the xylose in the medium, while LEK122 only consumed 20% of that. The LEK513 strain produced 11% more ethanol in oxygen-limited fermentation than it did in aerobic fermentation.