A Preliminary Study On Scheffersomyces Stipitis’ Fermentation Capacity Of Monosaccharide And Related Genes

In this thesis, Scheffersomyces stipitis CBS 6054 was research object. Using four different monosaccharides respectively as the sole carbon source of the medium in Scheffersomyces stipitis’ fermentation test. Bacteria’s growth curve, sugar consumption, ethanol production would be measured. Two important genes Ss GSF2 and NG68 which are glycometabolism-related were studied. The results showed that:1. Scheffersomyces stipitis had similar growth curves with high bacteria concentration in xylose and glucose medium, followed by galactose medium and arabinose medium. The fermentation of glucose and xylose are equal, in which sugar reduced to a lower level at 48 h. At 72 h, the sugar utilization ratio of galactose medium and arabinose medium were 90.9%, 21.6%. Ethanol production rate of glucose medium was 0.4774 g/g(ethanol/consumption of sugar) which was 93.6% of the theoretical value. Ethanol production rate of xylose medium was 0.3968 g/g which amounted to 86.3% of the theoretical value. At 72 h, ethanol yield of galactose medium can reach 0.3395 g/g. Arabinose medium produced a small amount of methanol instead of ethanol.2. Relative to glucose medium, Ss GSF2 gene expression of xylose medium had a peak of expression at 24 h and it was similar to glucose medium at other time points. Fusion expression vector and Ss GSF2 expression vector were constructed. They were transformed into Saccharomyces cerevisiae S132. The expression of m RNA were detected and the results showed that Ss GSF2 gene expressed in the engineering strain instead of original strain.3. The bacteria concentration of original strain S132 in descending order were galactose, glucose, xylose, arabinose. Bacteria grew fastest in glucose medium, while bacteria grew slowest in arabinose medium. Growth curve of Ss GSF2 gene transformants in glucose, xylose and galactose medium were consistent. Bacteria grew slower in arabinose medium with the lowest bacteria concentration. In the hexose medium bacteria concentration of S132 strains was higher than Ss GSF2 gene transformants’. In the pentose medium Ss GSF2 gene transformants grew faster than original strain S132. The hexose utilization ratio of Ss GSF2 gene transformants was lower than the original strain. While the pentose utilization ratio of Ss GSF2 gene transformants was higher than the original strain.4. By microscope observation and cultivation experiment found that bacteria morphology of Strain S132 and Ss GSF2 gene transformants were different. Decentralized state of the engineering strain is good and it was not easy settling and coagulation. These characteristics are helpful for better utilization of oxygen and sugar, which will reduce energy consumption during industrial production.5. Bioinformatics analysis showed that NG68 belonged to nuclear genes. ORF length was 4371 bp, encoding 1456 amino acids. The gene was located in the yeast genome chromosome 3, GC content was 44%, containing 144 bp intron and several repeat sequences. NG68 protein contained a signal peptide and five Candida ALS domains. At the end of the protein there was a transmembrane helix structure and the other part of protein were out of the membrane.6. NG68 gene expression were similar in the hexose medium, while in the pentose medium it had a peak of expression at the early stage of fermentation. In addition expression in the xylose and galactose medium were different, indicated that the gene related to metabolism of pentose and had different response degree on different pentose media.