Recombinant Saccharomyces Cerevisiae Strains Capable Of Cofermentation Of Glucose-xylose Engineered By Integrations At Different Chromosomal Positions

Shortage of crude oil reserve and environmental pollution from vehicle emissions make it urgent to develop alternatives such as biofuels. At present, fuel ethanol as a major biofuel is produced dominatedly from sugar-and starch-based feedstocks, which are not sustainable taking into account of increased population and consequent demand for food. Lignocellulosic biomass such as agricultural residues that are abundantly available has thus been acknowledged as sustainable biomass resource for fuel ethanol production, however, utilization of pentose sugars released during hydrolysis of hemicelluloses presents one of the bottlenecks.Xylose is the major pentose sugar in the hydrolysate of lignocellulosic biomass. In this thesis, the genes involved in xylose assimilation, which include xylose reductase gene PsXR and xylitol dehydrogenase gene PsXDH from Scheffersomyces stipites, as well as xylulose kinase gne ScXK and flocculin gene FLO1from Saccharomyces cerevisiae, were engineered into chromosomes of the industrial strains at different insertion sites by scattered and tandem integration strategies to investigate the position effect on mixed sugar assimilation, and in the meantime, site-directed mutation was employed with XR to modify the enzyme’s cofactor preference to address the intrinsic cofactor imbalance with the xylose-metabolic pathway. As a result,8recombinant yeast strains, in which2of them were engineered with the flocculating phenotype, were developed to assess expression of these genes and enzymatic activities as well as the impact on xylose and glucose assimilation. The results obtained were listed below:1. Using the industrial S. cerevisiae6525as the host strain, the genes PsXR, PsXDH and ScXK were ligated in head-to-tail way and integrated into the yeast chromosome XVI at the YPRCdeltal5position (tandem integration) to develop ZQ1(with wild type PsXR gene) and ZQ2(with mutated PsXR gene). On the other hand, PsXR, PsXDH and ScXK genes were incorporated into the chromosomes of the same host by scattered integration strategy to mimic their intrinsic distributions for xylose assimilation with PsXR and PsXDH inserted in the positions of YPRCdeltal5and YPRCtau3of the yeast chromosome XVI, and ScXK in the position YIRCdelta6of the chromosome IV to build ZQ3(with wild type PsXR gene) and ZQ4(with mutated PsXR gene). Both XR and XDH genes were fused to the strong expression promoter PGK1p, while ScXK gene was using the medium expression promoter ADH1p. All the3genes used CYC1, as terminator. Furthermore, the whole-genomic DNA transformation was applied to ZQ1and ZQ3by transforming genomic DNA of S. stipilis JCM10742to S. cerevisiae6525, and the hybrid strains displayed enhanced xylose assimilation ability compared to their parental strains.2. Semi RT-PCR, enzyme assays and sugar assimilation were experimented to assess these recombinant strains. ZQ2and ZQ4carrying mutated XR gene consumed xylose within fermentation time, in which the specific xylose uptake rate was both0.0035g/g CDW/h, and the specific xylose uptake rate of ZQ3was0.0033g/g CDW/h. Xylitol generated by ZQ4was reduced59%compared to that of ZQ2, while xylitol production of ZQ3was0.83g/g.0.9g/g xylitol was still produced by ZQl though xylose was only partially consumed in the medium. These results demonstrated that strains with the scattered chromosome integration strategy has better performance than strains with the tandem chromosome integration strategy in xylose assimilation, and the enzyme modification altered its cofactor preference in the meanwhile.3. The tandem gene cassette of PsXR, PsXDH and ScXK was also transferred into different industrial S. cerevisiae strains6525,4126and6508to develop ZQl, ZQ5and ZQ7to assess the influence of strain backgrounds. While ZQ5owned the highest levels of gene expression, enzyme activities, and consumed all sugars in the media. Enzyme activity of PsXR of ZQ5was4.1-fold of that of ZQ1and3.6-fold of that of ZQ7. The enzyme activity of PsXDH of ZQ5was6.2-fold and4.6-fold in contrast to that of ZQ1and ZQ7. The third enzyme demonstrated4.3-fold of ScXK in ZQ1and2.7-fold of ScXK in ZQ7. ZQ1had the lowest xylose utilization ability due to its inability to utilize xylose efficiently. Under20g/L xylose and40g/L glucose fermentation conditions, ZQ7was superior to ZQ1and ZQ5in that it could use80%xylose compared to70%xylose utilization of ZQ5in4days. It was demonstrated that xylose assimilation ability varied in yeast strains with different genetic backgrounds, demonstrating that xylose metabolism pathway was host-dependent.4. To improve tolerance ability of yeast strain, FLO1gene was incorporated into ZQ7at the sites of PHO13and HO to develop ZQ8and ZQ9. Cultivating ZQ8and ZQ9in YPD condition or plates containing high concentration of acetate acid/ethanol, it turned out that albeit the flocculation ability of ZQ8was stronger than that of ZQ9, but its tolerance to inhibitors was not improved. Preliminary confirmation revealed that the position effect of the FLO1gene integration in either position of HO or PHO13was responsible for the change of tolerance ability and flocculating ability of recombinant strains.