Effects of overexpression and disruption of transketolase genes in Saccharomyces cerevisiae

• Main Authors: Hsieh, Wen-Hao, 謝文豪
• Other Authors: Jiann-Hwa Chen
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/61849037048623788847

碩士 === 國立中興大學 === 分子生物研究所 === 84 === Transketolase, together with transaldolase, catalyzes the key step in the non-oxidative pentose phosphate pathway in living organisms. In the baker's yeast Saccharomyces cerivisiae two genes, TKT1 and TKT2, encode for the major and minor transketolase activity separately and both genes share 71% identity at the amino acid sequence level. In our laboratory, the TKT1 gene was PCR-cloned and a tkt1- mutant was generated previously. In this study, the TKT2 gene was also PCR-cloned and a tkt2- mutant as well as a tkt1- tkt2- double mutant were generated. The tkt2- mutant showed about 75% of the transketolase activity of the wild-type cells, whereas both the tkt1- mutant and the tkt1-tkt2- mutant showed undetectable level of the transketolase activity in their cell extracts. Cellular growth of the tkt1-, tkt2- and tkt1- tkt2- mutants as wll as the wild-type cells in different medium was measured. In rich medium, they did not show any difference in growth. In minimum medium, however, thetkt1-tkt2-double mutant showed slower growth while the tkt1-, tkt2- and the wild-type cells still showed similar growth. The result indicated that small amout of transketolase activity is required for normal growth in minimum medium, but not required in rich medium. For each mutant, transaldolase activity of the cell extract was also measured. Again, I found that there was no significant difference in the transaldolase activity in the cell extracts between the tkt1-, tkt2- and the wild-type cells. But the tkt1- tkt2- double mutant showed about one sixth of the transaldolase activity of the wild-type cells in the cell extract. The result indicated that small amount of transketolase activity is also required for normal transaldolase activity in vivo. In order to generate the TKT1 or TKT2 over-production mutant, two strategies were used. In one way, either the TKT1 or TKT2 gene was cloned into a high-copy plasmid JDB207 and transformed into the wild-type cells seperately. In the other way, promoter of the TKT1 gene or TKT2 gene in an YEP13-based vector was replaced with an inducible Gal1 promoter, transformed into the wild-type cells and the transformant was grown in galactose induction medium. In either way, I found that the TKT1 overproduction mutant did not show significant increase in the transketolase activity, despite thau the TKT1 protein was largely over- produced in both cases as demonstrated by Western analysis. There was no significant increase in the transaldolase activity in the cell extract of the TKT1 overproduction mutant. On the contrary, the JDB207-based TKT2 overproduction mutant showed 79% increase in the trans-ketolase activity and the galactose- inducible TKT2 overproduction mutant showed ten-fold increase in the transketolase activity upon galactose induction. The former showed 48% increase of the transaldolase activity and the latter showed 50% increase of the transaldolase activity in their cell extracts. The result showed that increase in transketolase activity from 79% to ten-fold could result in about 50% increase in the transaldolaseactivity in the cell extracts. The reason why the TKT1 overproduction mutant did not show increase in tansketolase activity was probably because that the PCR-cloned TKT1 gene was not a functional gene. The TKT2 gene, although also PCR-cloned, had been carefully sequenced and the errors been corrected according to the published TKT2 sequences and therefore was a functional gene in the TKT2 overproduction mutant.