Xylose Fermentation By Recombinant Saccharomyces Cerevisiae For Ethanol Production

D-Xylose is the second most abundant carbohydrate in nature, which is considered to be of great economic and environmental significance for future biofuels. While Saccharomyces cerevisiae has natural advantage in ethanolic fermentation of hexose, it is incapable of xylose utilization.In the first part of the work, we describe the construction of 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 strain KAM-2 was first engineered to functionally 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). The resulting recombinant strain LEK122, which has acquired basic xylose-utilizing ability, was then subjected to EMS mutagenesis followed by adaptive evolution, resulting a single isolate, LEK513, that displayed significantly improved xylose-utilizing property. The specific growth rate of the LEK513 strain was 0.225h-1 under aerobic condition with xylose as the sole carbon source, while that of the LEK122 was 0.055h-1. When cultured under oxygen-limited condition, the specific growth rate of LEK513 was 0.205h-1 comparable to that under aerobic condition. During 100 hour 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 the xylose in the medium. The LEK513 strain produced 11% more ethanol in oxygen-limited fermentation than it did in aerobic fermentation. The significantly improved xylose-utilizing property demonstrated the feasibility of the combination of rational and random approaches in construction of efficient xylose-utilizing, ethanol fermenting S. cerevisiae strains for industrial application.The flux through the nonoxidative PPP in S. cerevisiae is insufficient, so the four genes involved in nonoxidative PPP (TAL1, TKL1, RKI1, RPE1) were overexpressed by the PGK1 promoter and the GRE3 gene encoding endogenous xylose (aldose) reductase which mediates unwanted production of xylitol was deleted to construct a original strain. In consideration of Western blot results when xylose as sole carbon source, the relatively strong promoter TPI1 promoter and HXT7 truncated promoter were chosed to introduce the xylose assimilation pathway into the yeast. The new xylose assimilating cassette with ADH1 promoter replaced by TPI1 promoter and PGK1 promoter replaced by HXT7 truncated promoter was inserted into genome of original strain to generate the strain LEK631, compared with strain LEK630 in which using the ADH1 & PGK1 promoter to express xylose assimilating pathway, the fermentation results as follows: both of them were taken to inoculate YPX(50g/L) with an initial cell concentration of OD600=0.6, in 216h batch cultivation, the final OD600 of LEK630 was 7.0, whereas LEK631 was 31.2; LEK630 consumed 11.9% of total xylose, whereas LEK631 consumed 96.8%; and the highest ethanol concentration during fermentation period in LEK630 was 1.052g/L, whereas in LEK631 that was 7.575g/L. From the results, the changing of the promoters significantly improved growth ability, xylose-utilizing and ethanol-yielding property. The Piromyces xylose isomerase gene xylA that synthesized by fusion PCR and endogenous XKS1 gene were placed under the control of HXT7 truncated promoter. Both of them were inserted into original strain genome, the resulting strain could grow on the medium that xylose as sole carbon source, but no ethanol was detected in fermentation broth.