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title page
Abstract
Contents
I. Introduction 11
1. Xylitol and Xylitol production 12
2. Heterologous expression in yeasts 17
3. Cofactor regeneration system 19
3.1. Cofactor regeneration system by expression of PTDH 20
3.2. Requirement of the NADPH regeneration as reducing power for efficient xylitol production 23
4. Objectives 25
4.1. Construction of xylitol dehydrogenase expression system 25
4.2. Expression of phosphate dehydrogenase in Cadida tropicals 25
II. Construction of xylitol dehydrogenase expression system in Candida tropicalis 27
1. Material and method 28
1.1. Strains and culture conditions for genetic manipulation 28
1.2. Selection of uracil auxotroph 30
1.3. Construction of expression cassete for XDH expression 32
1.4. Transformation of yeast 35
1.5. Culture conditions for XDH activity assay 37
1.6. Assay of xylitol dehydrogenase (XDH) 37
2. Results and discussion 38
2.1. Selection of uracil auxotroph using FOA plate in XDH-depleted mutant 38
2.2. Xylitol dehydrogenase (XDH) expression and activity assay 40
3. Conclusions 45
II. Expression of phosphite dehydrogenase in Candida tropicals 47
1. Material and method 48
1.1. Strains and culture conditions for genetic manipulation 48
1 2. Consturction of the cassettes for PTDH expression 48
1.3. Transformation of yeast 51
1.4. Culture conditions for PTDH and codon-optimized PTDH activity assay 51
1.5. Assay of PTDH and codon-optimized PTDH 51
2. Results and discussion 53
2.1 Phosphite dehydrogenase expression and activity assay 53
3. Conclusions 60
국문요약(SUMMARY) 61
References 63
감사의 글 68
Table 1. The C. tropicals strains used in this study. 29
Table 2. The primers used in this XDH expression experiments. 34
Table 3. The primers used in this PTDH expression experiments. 50
Table 4. Codon utilization in PTDH from P. stutzeri. 57
Figure 1. Molecular structure of D-xylose, xylitol, and D-xylulose 15
Figure 2. Schematic diagram of D-xylose metabolism 16
Figure 3. Schematic representation of the use of phosphite dehydrogenase (PTDH) as an NADPH-regenerating enzyme in general system. 22
Figure 4. Physical maps of formation of the uracil auxotroph by homologous recombination. Arrows indicate the gene specific primers. 31
Figure 5. Physical maps of the expression cassette for specific integration of hisG gene in C. tropicals XYL2-disrupted site. 36
Figure 6. Confirmation of formation of the uracil auxotroph through homologous recombination by PCR. 39
Figure 7. Cell growth of following cultures in xylose minimal media. 41
Figure 8. Enzymatic confirmation of XDH expression. 44
Figure 9. Confirmation of transcription of PTDH gene from P. stutezeri by RT-PCR. 54
Figure 10. Schematic representation of codon optimization of PTDH 56
Figure 11. Enzymatic confirmation of PTDH expression. Specific activities of PTDH in YM media were measured. 59
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