Construction Of Recombinant S. Cerevisiae Strains With High Ethanol Fermentation Efficiency And Functional Study Of Sfi1p In Sporulation And Karyogamy

This thesis consists of two parts: In the first part, a recombinant S. cerevisiae strain with significantly improved ethanol fermentation performance was constructed via combined approaches of recombinant DNA technology,chemical mutagenesis and sexual recombination; in the second part, the function of Sfi1p, a new component of yeast SPB, in sporulation and karyogamy was studied.Due to the increasing demands for energy and the decreasing reserves of fosil fuels and the concern over the issue of environment protection, bioethanol production and the development of its related technology become more and more important. Currently bioethanol is mainly produced through fermentation of starch- or sugar-based feedstocks by Saccharomyces cerevisiae. Increasing sugar-ethanol conversion rate for efficient substrate utilization and reducing process cost are two major problems to be solved. In this study, a combination of the recombinant DNA technology, chemical mutagenesis and sexual recombination was used for construction of Saccharomyces cerevisiae strains with improved fermentation performance.The fermentation performance of the recombinant industrial yeast strain S812 carrying deletions in the two functional redundant gene, GPD1 and GPD2, encoding the NAD+-dependent glycerol-3-phosphate dehydrogenase and expressing the PGK1 promoter-driven GLT1, encoding the NAD+-dependent glutamate synthase was investigated. Results showed that, in YPD medium containing 2% glucose, the S812 strain produced 17% more ethanol than its parental strain did. In media with higher glucose concentration, however, the S812 strain grew and metabolized slowly compared to the control strain due to the defect in producing glycerol. Addition of 30 mM glycerol to the medium released the S812 strain from high osmolarity to some extent. However, the strain was still unable to adapt to the high osmotic pressure imposed by high glucose concentration in industrial media.To facilitate glycerol transport in to the cells and enhance cell's resistance to high osmolarity, the STL1 gene encoding a glycerol/H+ symporter and the GUP1 gene proposed to be involved in glycerol transport and it's close homolog, the GUP2 gene, were each overexpressed in the S812 strain, generating strain LE17U(STL1 overexpressed), LE18U(GUP1 overexpressed)and LE19U(GUP2 overexpressed), respectively. Fermentation test demonstrated that, in the presence of 30 mM glycerol and with glucose concentration below 20%, strain LE17U performed better in terms of growth and metabolism and produced 3.4% more ethanol than its parental strain S812U did. However, no significantly improved fermentation behavior was observed for strain LE18U and LE19U compared to the control strain under same conditions. When glucose concentration was increased to 30%, the advantages of the LE17U strain over its parental strain S812U were abolished.For further improvement of strain's performance in high gravity fermentation, we developed a new method for sexual recombination. The STE2 gene encodingα-factor receptor was deleted in the diploid strain LE17D, generating strain LE17DS2. After EMS mutagenesis, LE17DS2 was then subjected to sexual recombination-mediated genome shuffling. Due to the lack of a functional STE2, haploid cells resulted from genome shuffling cannot mate to reform dipoilds, and therefore, greatly simplified the process of selection and made the method a high-throughput one. Strain LS75 was obtained by this approach and, during fermentation in a medium containing 30% glucose, this strain exhibited 10.88% increased ethanol yield and the fermentation period of this strain was shortened by 10 hours compared to the LE17U strain. In addition, ethanol and high osmolarity tolerance of the LS75 strain were also significantly improved. These results verified the feasibility of the strain improvement strategy developed in this work and demonstrated that it is an efficient method for development of S. cerevisiae strains with improved ethanol fermentation performance.As the microtubule organizing center of the yeast S. cerevisiae, the spindle pole body (SPB) plays an essential role in cell division and maintenance of genome stability, and therefore has attracted a great deal of attention and enormous research efforts in elucidating its structure feature and function in the field of cell biology. Sfi1p is a newly identified SPB component of Saccharomyces cerevisiae, which localizes at Half-Bridge and spans its full length. Structural characterization and functional analysis of Sfi1p will not only shed new light on our current understanding about yeast SPB structure and function, but also aids investigation of its homologues, and hence the MTOC in higher eukaryotes.In this work, the role of Sfi1p, as a component of SPB, during meiosis and karyogamy was investigated. We investigated the sporulation efficiency and the distribution of genetic materials into meiotically-produced haploid spores of the diploid mutant strains homogenous for specific SFI1 ts alleles at 28℃and 23℃, respectively. Some C terminal ts mutants showed severe defects in sporulation efficiency even at permissive temperature and a significant portion of the sporulated mutant cells gave rise to dyads, the asci containing only two spores. Fluorescence microscopy analysis of microtubes,nuclei and forespore membranes of SFI1 ts mutants whose sporulation efficiency was significantly impaired showed that the C terminal ts mutantion of Sfi1p resulted in an abnormal function during meiosis II in that the SPBs could not separate and only two forespore membranes are intact and, as a result, dyads were formed. This phenomenon demonstrated that, in addition to its characterized function in SPB duplication, Sfi1p also plays an important role in yeast sporulation and loss of this function renders yeast cells defects in meiosis.At 28℃and 23℃, we also observed that some Sfi1p ts mutants displayed a significant defect in zygotes formation during mating. Sfi1p localized at Half-Bridge which is linked to microtubes during mating and disfunction of Sfi1p could affect proper organization of microtubes. Fluorescence microscopy study of nuclei and microtubes showed that nuclear congression slowed and the efficiency of nuclear fussion declined in the mutants and hence the number of zygotes decreased. These observations suggested that Sfi1p plays an important role in karyogamy.