1. Improved Galactose Fermentation of Saccharomyces cerevisiae Through Inverse Metabolic Engineering 1 조 조장 : 우대균 조원 : 김상민 박동주 박지연 이혁 길영욱

2. Contents Introduction Materials & Methods Results Discussion

3. Introduction Biofuel Biomass 로부터 연소나 발효 과정에 의해 생성 되는 연료

4. Terrestrial biomass (soybean, corn) Marine plant biomass (seaweed) 1.Production yields per unit area are very high 2.Marine biomass can be depolymerized easily 3.The rate of carbon dioxide fixation by marine biomass is very high Introduction

5. Marine plant biomass (seaweed) Main sugar is galactose galactose Ethanol yield and productivity from galactose glucose are lower than those from glucose in Saccharomyces cerevisiae Introduction

6. Leloir pathway glucose-6-phosphate glycolytic pathway pentose phosphate pathway Introduction

7. upstream activation sites (UAS) upstream repression sites (URS) GAL4 / GAL80 : transcriptional regulators Protein complex When glucose is present Introduction

8. First, engineering of the regulatory network controlling the expression of the GAL genes Result 19% increase in specific galactose uptake rate (3.57 vs. 3.01mmol gal/g cell h) 154% increase in specific ethanol production rate (4.19 vs. 2.71 mmol ethanol/g cell h) increase metabolic flux Introduction

9. Second, to overexpress PGM2, a metabolic gene coding for phosphoglucomutase Result Overexpression of PGM2 led to a 70% increase in galactose uptake rate perturbation of a single metabolic enzyme Introduction

10. Based on the inverse metabolic engineering approach, we have identified three gene targets whose overexpression elicited improved galactose utilization 1.SEC53 coding for phosphomannomutase (PGM2) which exhibits also phosphoglucomutase activity 2.SNR84 coding for a small nuclear RNA 3.truncated form of TUP1 coding for a global repressor of transcription Introduction

11. Materials & Methods 1) Strains and plasmids -Saccharomyces cerevisiae CEN.PK2- 1D - S. cerevisiae L2612 - S. cerevisiae SC288C -E.coli DH5α Galactose fermentation Genomic Libraries Gene cloning, manipulation

12. YP medium 30 ℃ + glucose Select transformants TRP1 selectable marker, YSC medium Fermentation YSC medium 2) Media and Culture Condition Materials & Methods

13. 3) Construction of S. cerevisiae Genomic Library pRS42 4 Materials & Methods

14. 4) Construction of PGM2 Overexpression strain TUP1 knockout strain pRS424TEF BamHI XhoI ORF of PGM2 Materials & Methods

15. 5) mRNA Abundance Measurement by RT-PCR RNAcDNART-PCR Materials & Methods 6) Analytical Methods Glucose and Galactose High-performance liquid chromatography EthanolGas- chromatography Cell GrowthA 600

16. Results 1) Screening of Transformants Exhibiting Improved Galactose Fermentaion Capabillity,

17. 1) Screening of Transformants Exhibiting Improved Galactose Fermentation Capabillity Results

18. 1) Screening of Transformants Exhibiting Improved Galactose Fermentaion Capabillity Results

19. 1) Screening of Transformants Exhibiting Improved Galactose Fermentaion Capabillity Results

20. 2) Improved Growth and Fermentation on Galactose by the Amplification of SNR84 Control Amplification of SNR84 Results

21. 2) Improved Growth and Fermentation on Galactose by the Amplification of SNR84 Amplification of SNR84 Control Results

22. 3) Improved Growth and Fermentation on Galactose by Overexpressing a truncated form of TUP1 Truncated TUP1 Results

23. 3-1) What is truncated TUP1 ?. Results

24. 3-2) Improved Growth and Fermentation on Galactose by Overexpressing a truncated form of TUP1 ControlOverexpressed tTUP1 Results

25. 3-2) Improved Growth and Fermentation on Galactose by Overexpressing a truncated form of TUP1 Control Overexpressed tTUP1 Results

26. 3-3) Comparison of cell growth and ethanol production of the TUP1 knockout mutant TUP1 Cell Growth Ethanol Production Results

27. ControlOverexpressed tTUP1 3-4) Measured the expression levels of the GAL genes by RT-PCR Results

28. 3-5) Comparison of cell growth and ethanol production of the transformant overexpressing tTUP1 using various carbon sources. Control Overexpressed tTUP1 Results

29. 3-5) Comparison of cell growth and ethanol production of the transformant overexpressing tTUP1 using various carbon sources. Control Overexpressed tTUP1 Results

30. 3-5) Comparison of cell growth and ethanol production of the transformant overexpressing tTUP1 using various carbon sources. Control Overexpressed tTUP1 Results

31. 3-6) Investigated synergistic effects. ControlPGM2 overexpression PGM2 + SNR84 overexpression PGM2 + tTUP1 overexpression Results

32. Two types of genetic perturbation 1.Improves ethanol production 2. Improves ethanol production & cell growth Deletion of GAL80, MIG1, GAL6 Discussion Overexpression of PGM2, SEC53 SNR84 (Small Nuclear RNA) tTUP1 (truncated TUP1)

33. References Ki-Sung Lee. et al., “Improved Galactose Fermentation of Saccharomyces cereisiae Through inverse Metabolic Engineering,” Biotechnology and Bioengineering, Vol. 108, No.3, pp621-631, 2011 http://en.wikipedia.org/wiki/Leloir_pathway