Title Page
Contents
ABSTRACT 11
Ⅰ. Introduction 13
Ⅱ. Materials and Methods 17
2.1. Chemicals and enzymes, genes 17
2.2. E. coli lysate preparation for cell-free protein synthesis 17
2.3. Enzyme expression by E. coli lysate-based cell-free protein synthesis 18
2.4. Cell-free expressed enzyme quantification 19
2.5. Liposome preparation 19
2.6. In vitro lycopene biosynthesis starting from GPP 20
2.7. Lycopene extraction and analysis 20
2.8. In vitro lycopene biosynthesis starting from glucose 21
Ⅲ. Results 22
3.1. Establishment of CFPS system using GFP and TA as a model protein 22
3.2. Establishment of cell-free expressed enzymes quantification by split GFP assay and SDS-PAGE gel-based software analysis 28
3.3. Development of an in vitro synthesis of lycopene from GPP 34
3.4. Development of UV-spectrophotometric analysis of lycopene for high-throughput screening 40
3.5. Optimization of the in vitro lycopene synthesis reaction 45
3.6. Rapid homolog enzymes screening in the lycopene synthetic pathway 49
3.7. Applying the in vitro lycopene synthetic enzymes as a GPP sensor 55
3.8. Expanding and additional homolog screening of the lycopene synthetic pathway starting from glucose 57
3.9. Comparison of rapid screening of in vitro lycopene synthetic enzymes with different strategies 64
Ⅳ. Discussion 69
Ⅴ. Conclusion 71
References 72
Abstract (in Korean) 77
Table 1. Methods approaches for in vitro lycopene synthesis. 37
Table 2. Homolog enzymes used for lycopene synthetic pathway screening. 50
Table 3. Homolog enzymes used for MVA pathway screening. 60
Figure 1. SDS-PAGE gel of the expression of eGFP by CFPS system. Percentage indicates the concentration of the target DNA as PCR product form. 100 %=19.3 ng/µL,... 24
Figure 2. Fluorescence of the expression of eGFP by CFPS system. Percentage indicates the concentration of the target DNA as PCR product form. 100 %=19.3 ng/µL, 80 %=... 25
Figure 3. The result of red color assay with CVTA expressed by CFPS system. Reaction condition : Kpi buffer pH 7.5, Benzaldehyde; 5 mM, PLP; 0.5 mM, 1 ; 12.5 mM, 30℃,... 27
Figure 4. (A) Relationship between the concentration of purified GFP₁₁ tagged CrtI and fluorescence. (B) Calculation of cell-free expressed CrtI based on the standard... 31
Figure 5. SDS-PAGE gel of cell-free expressed CrtE, CrtB, CrtI with various tags. 32
Figure 6. (A) Standard curve of the SDS-PAGE gel band Intensity between BSA concentration. (B) Average concentration of cell-free expressed sfGFP. 33
Figure 7. Color of the organic solvent to extract lycopene from in vitro lycopene synthesis using lycopene producible engineered E. coli lysate. 36
Figure 8. Result of in vitro lycopene synthesis method approach 12. 38
Figure 9. In vitro lycopene synthesis with the employment of liposome at a gradient concentration. (A) Lycopene synthesis by engineered E. coli lysate. (B) Lycopene... 39
Figure 10. Lycopene extracted by dodecane and loaded in 96-well plate. 42
Figure 11. HPLC chromatogram of authentic lycopene, lycopene extracted from engineered E. coli, and lycopene extracted from in vitro reactions. (A) Chromatogram... 43
Figure 12. Comparison of the absorbance at 472 nm and the area of the HPLC peak at 25.5 minute of the extract of in vitro lycopene synthesis reaction. 44
Figure 13. Relationship between liposome concentration and lycopene productivity detected at 472 nm. 46
Figure 14. Time profiling of in vitro lycopene synthesis reaction. 47
Figure 15. Relationship between lycopene productivity and enzyme concentration. (A) Half concentration of each enzyme. (B) Double concentration of each enzyme.... 48
Figure 16. Average concentration of each soluble enzymes expressed by CFPS system. 52
Figure 17. Comparison of lycopene production between each standard and homolog enzymes. Reaction condition : 100 mM Tris-HCl (pH 8.0), 4 mM MgCl₂, 4 mM... 53
Figure 18. Comparison of the enzyme combination of lycopene synthetic pathway. (A) relative absorbance between the standard and the optimized group at 4 hour reaction.... 54
Figure 19. Standard curve of lycopene production by optimized in vitro lycopene synthesis depending on the concentration of GPP. 56
Figure 20. Comparison of lycopene productivity between standard and optimized combination of in vitro lycopene synthetic enzymes with the addition of cell-free... 59
Figure 21. Comparison of lycopene production between each standard and homolog enzymes of ACAT, HMGS, HMGR, MK. Reaction condition : 100 mM Tris-HCl pH... 62
Figure 22. Comparison of lycopene production between each standard and homolog enzymes of PMK, PMD, IDI, GPPS. Reaction condition : 100 mM Tris-HCl pH 8.0,... 63
Figure 23. Comparison of lycopene production between each standard and homolog enzymes using the 'down to up' screening strategy. Reaction condition : 100 mM Tris-... 66
Figure 24. Comparison of lycopene production between each standard and homolog enzymes using the 'up to down' screening strategy. Reaction condition : 100 mM Tris-... 67
Figure 25. Comparison of lycopene productivity between best enzyme combination of in vitro lycopene synthetic pathway with different strategies. (A) Relative absorbance... 68
Scheme 1. Reaction pathway of lycopene starting from GPP. 16
Scheme 2. Reaction pathway of the red color assay for TA. 26
Scheme 3. Reaction pathway of lycopene starting from Glucose. 58