Title Page
Contents
ABSTRACT 22
Ⅰ. Introduction 25
1.1. Oxylipins 25
1.1.1. Lipid mediators 25
1.1.2. Specialized pro-resolving mediators 27
1.1.3. Plant oxylipins 30
1.2. Lipoxygenases 31
1.2.1. Catalytic mechanisms 31
1.2.2. Regioselectivity 31
1.2.3. Stereoselectivity 32
1.3. Current synthesis method of oxylipins 34
1.4. Purpose of this study 36
Ⅱ. Discovery and engineering of the 15S-lipoxygenase from Archangium violaceum for biosynthesis of lipid mediators from polyunsaturated fatty acids 37
Summary 37
2.1. Introduction 39
2.2. Materials and Methods 43
2.2.1. Materials 43
2.2.2. Preparation of lipid mediators 43
2.2.3. Microbial strain, gene cloning, and site-directed mutagenesis 44
2.2.4. Cell cultivation 44
2.2.5. Enzyme purification and assay 45
2.2.6. Bioconversion of polyunsaturated fatty acids 46
2.2.7. Homology modeling and molecular docking 46
2.2.8. Phylogenetic analysis 46
2.2.9. Analytical methods 47
2.3. Results and Discussion 50
2.3.1. Discovery of the 15S-lipoxygenase 50
2.3.2. Catalytic activity and properties of the 15S-lipoxygenase 58
2.3.3. Identification of lipid mediators produced from polyunsaturated fatty acids by the 15S-lipoxygenase 62
2.3.4. Biosynthesis pathway of lipid mediators formed by the 15S-lipoxygenase 70
2.3.5. Molecular homology model converted from 15S-lipoxygenase to 12S-lipoxygenase by the structure-based protein engineering 73
2.3.6. Identification of lipid mediators produced from polyunsaturated fatty acids by the engineered 12S-lipoxygenase 80
2.3.7. Bioconversion of polyunsaturated fatty acids into lipid mediators by the 15S-lipoxygenase and engineered 12S-lipoxygenase 90
Ⅲ. Discovery and engineering of the 15R-lipoxygenase from Sorangium cellulosum for biosynthesis of lipid mediators from polyunsaturated fatty acids 97
Summary 97
3.1. Introduction 98
3.2. Materials and Methods 101
3.2.1. Materials 101
3.2.2. Preparation of lipid mediators 101
3.2.3. Microbial strain, gene cloning, and cell cultivation 102
3.2.4. Enzyme purification and assay 102
3.2.5. Bioconversion of polyunsaturated fatty acids 103
3.2.6. Homology modeling and molecular docking 103
3.2.7. Analytical methods 104
3.3. Results and Discussion 109
3.3.1. Discovery of the 15R-lipoxygenase 109
3.3.2. Catalytic activity and properties of the 15R-lipoxygenase 115
3.3.3. Molecular homology model converted from double-oxygenating 15R-lipoxygenase to single-oxygenating 15R-lipoxygenase by the structure-based protein engineering 121
3.3.4. Identification of lipid mediators produced from polyunsaturated fatty acids by the double-oxygenating 15R-lipoxygenase and single- oxygenating 15R-lipoxygenase 133
3.3.5. Biosynthesis pathway of lipid mediators formed by the double-oxygenating 15R-lipoxygenase and single-oxygenating 15R-lipoxygenase 145
3.3.6. Bioconversion of polyunsaturated fatty acids into lipid mediators by the double-oxygenating 15R-lipoxygenase 147
3.3.7. Bioconversion of polyunsaturated fatty acids into lipid mediators by the single-oxygenating 15R-lipoxygenase 155
3.3.8. Bioconversion of docosahexaenoic acid-rich fish oil hydrolysate into mono- and dihydroxy fatty acid by the double-oxygenating and single-oxygenating 15R-lipoxygenases 160
Ⅳ. Increased production of resolvin D5 from docosahexaenoic acid-enriched oil using a bioreactor by the 15S-lipoxygenase from Archangium violaceum 163
Summary 163
4.1. Introduction 165
4.2. Materials and Methods 167
4.2.1. Materials 167
4.2.2. Microbial strain and cell cultivation 167
4.2.3. Hydrolysis of docosahexaenoic acid-enriched oil and purification 168
4.2.4. Optimization of polymers and solvents 168
4.2.5. Optimization of cell and substrate concentrations 168
4.2.6. Bioconversion of docosahexaenoic acid into resolvin D5 169
4.2.7. Purification of resolvin D5 169
4.2.8. Analytical methods 170
4.3. Results and Discussion 171
4.3.1. Hydrolysis of docosahexaenoic acid-enriched oil and docosahexaenoic acid purification 171
4.3.2. Optimization of reaction conditions for the improved production of resolvin D5 174
4.3.3. Bioconversion of reagent-grade and docosahexaenoic acid in docosahexaenoic acid-enriched oil hydrolysate into resolvin D5 in a baffled flask, respectively 179
4.3.4. Bioconversion of reagent-grade and docosahexaenoic acid in docosahexaenoic acid-enriched oil hydrolysate into resolvin D5 in a bioreactor, respectively 181
4.3.5. Purification of resolvin D5 from bio-transformed solution by filtration and resin extraction 185
Ⅴ. Increased production of C18 trihydroxy fatty acids from safflower oil using a bioreactor by the linoleate 13S-lipoxygenase from Archangium violaceum 188
Summary 188
5.1. Introduction 190
5.2. Materials and Methods 193
5.2.1. Materials 193
5.2.2. Microbial strain and cell cultivation 193
5.2.3. Enzyme purification and assay 194
5.2.4. Hydrolysis of safflower oil into linoleic acid 195
5.2.5. Bioconversion of C18 polyunsaturated fatty acids 195
5.2.6. Analytical methods 196
5.3. Results and Discussion 199
5.3.1. Preparation of linoleic acid from safflower oil by lipase treatment with resin 199
5.3.2. Catalytic activity and properties for C18 polyunsaturated fatty acids 204
5.3.3. Identification of THFAs as oxylipins produced from C18 polyunsaturated fatty acids by the linoleate 13S-lipoxygenase 207
5.3.4. Bioconversion of C18 polyunsaturated fatty acids into trihydroxy fatty acids in a baffled flask. 214
5.3.5. Bioconversion of linoleic acid in safflower oil hydrolysate into 11R,12R,13S-trihydroxyoctadecenoic acid in a bioreactor 218
Ⅵ. Production of resolvin E-series from eicosapentaenoic acid by arachidonate 15-lipoxygenases 221
Summary 221
6.1. Introduction 222
6.2. Materials and Methods 224
6.2.1. Materials 224
6.2.2. Microbial strain, site-directed mutagenesis, and cell cultivation 224
6.2.3. Optimization of solvents and polymers 224
6.2.4. Optimization of cell and substrate concentrations 225
6.2.5. Bioconversion of eicosapentaenoic acid into resolvin E4 and its enantiomer by wild-type 15-lipoxygenases 226
6.2.6. Homology modeling and molecular docking 226
6.2.7. Enzyme purification and assay 227
6.2.8. Bioconversion of eicosapentaenoic acid and 18-eicosapentaenoic acid 227
6.2.9. Analytical methods 228
6.3. Results and Discussion 233
6.3.1. Optimization of reaction conditions for the improved production of resolvin E4 and its enantiomer 233
6.3.2. Bioconversion of eicosapentaenoic acid into resolvin E4 or enantiomer of resolvin E4 by cells expressing Archangium violaceum 15S- lipoxygenase or Sorangium cellulosum 15R-lipoxygenase 237
6.3.3. Homology models for the change of 15-lipoxygenase to 18-lipoxygenase by structure-guided protein engineering 244
6.3.4. Bioconversion of eicosapentaenoic acid into 18-eicosapentaenoic acids by the engineered 18-lipoxygenases 250
6.3.5. Bioconversion of the prepared 18-eicosapentaenoic acids into resolvin E2 and 18S-resolvin E2 by Danio rerio 5S-lipoxygenase 257
Ⅶ. Conclusions 262
References 265
Abstract (in Korean) 277
Table 2.1. Abbreviations used in Chapter 2. 41
Table 2.2. Regression equations for calibration curves of DiHFAs obtained from LOXs from A. violaceum. 48
Table 2.3. Primer design for amplification of specific DNA sequences and site-directed mutagenesis of specific DNA sequences. 49
Table 2.4. Specific activities and kinetic parameters of the wild-type 15-LOX and engineered 12-LOX from A. violaceum. 60
Table 2.5. Specific activities of LOXs from soybean, mouse, B. thailandensis, and A. violaceum. 61
Table 2.6. 1D NMR data of 7S,17S-DiHDPA (Resolvin D5n₋₃ DPA, RvD5n₋₃ DPA) in CDCl₃ (850 MHz NMR).[이미지참조] 68
Table 2.7. 1D NMR data of 7S,17S-DiHDHA (Resolvin D5, RvD5) in CDCl₃ (850 MHz NMR). 69
Table 2.8. Specific activities and regio-isomeric ratios to products of the wild-type and variant LOXs from A. violaceum for the production of LMs. 77
Table 2.9. 1D NMR data of 7S,14S-DiHDPA (7S-MaR1n₋₃ DPA) in CDCl₃ (850 MHz NMR).[이미지참조] 87
Table 2.10. 1D NMR data of 7S,14S-DiHDHA (7S-MaR1) in CDCl₃ (850 MHz NMR). 88
Table 2.11. Specific productivity, volumetric productivity, and yield of recombinant cells for the production of DiHFAs. 96
Table 3.1. Abbreviations used in Chapter 3. 100
Table 3.2. Regression equations for calibration curves of PUFAs, MonoHFAs, and DiHFAs. 106
Table 3.3. Primer design for amplification of specific DNA sequences. 107
Table 3.4. Docking energy and distance of ARA from 10 LOXs existed in S. cellulosum. 110
Table 3.5. Specific activities and kinetic parameters of LOX. 117
Table 3.6. Specific activities of double-oxygenating LOXs from mouse, E. numazuensis, A. violaceum, and S. cellulosum. 120
Table 3.7. Specific activities of the Leu606 variant LOXs from S. cellulosum for ARA and 15R-HETE and ratio of 5R,15R-DiHETE as final products. 132
Table 3.8. 1D NMR data of 5R,15R-DiHETE in MeOD (850 MHz NMR). 139
Table 3.9. 1D NMR data of 7R,17R-DiHDHA in MeOD (850 MHz NMR). 140
Table 3.10. 1D NMR data of 5R,15R-DiHEPA in MeOD (850 MHz NMR). 141
Table 3.11. Specific productivity, volumetric productivity, and yield of recombinant cells for the bioconversion of DiHFAs. 154
Table 4.1. Composition of oil hydrolysate obtained from lipase without and with resin and solvent extraction. 172
Table 4.2. Reaction performance of the recombinant cells for the production of DiHFAs from DHA in a baffled flask or bioreactor. 180
Table 4.3. Serial purification of RvD5 from cell-removed supernatants by filtration and treatments of resins. 187
Table 5.1. Abbreviations used in Chapter 5. 192
Table 5.2. Regression equations for the calibration curves of the peak areas in HPLC chromatograms to the amount of PUFAs, MonoHFAs, MonoHpFAs, EHFAs, and THFAs. 198
Table 5.3. Composition of safflower oil hydrolysate obtained from hydrolysis of safflower oil by treating lipase in the absence and presence of the adsorbent resin SP207. 201
Table 5.4. Specific activities and kinetic parameters of LOXs for isomerization of PUFAs into MonoHpFAs and EHFAs. 206
Table 5.5. 1D NMR data of 12S,13S-EHODE(γ) in CDCl₃(850 MHz NMR). 212
Table 5.6. 1D NMR data of 11R,12R,13S-TriHODE(γ) in CDCl₃(850 MHz NMR). 213
Table 5.7. Performance of microbial biosynthesis systems to produce THFAs. 217
Table 6.1. Primer design for site-directed mutagenesis of specific DNA sequences. 230
Table 6.2. Regression equations for calibration curves of MonoHFAs and DiHFAs 232
Table 6.3. Specific productivity, volumetric productivity, and yield of recombinant cells for the bioconversion of EPA into RvEs. 243
Table 6.4. Specific activities and regio-isomeric ratios to products of wild-type and variant LOXs from S. cellulosum and A. violaceum, respectively. 247
Table 6.5. Specific activities and kinetic parameters of wild-type 15-LOXs and engineered 18-LOXs from S. cellulosum and A. violaceum. 248
Figure 1.1. Outline of the essential LMs, including SPMs, switch involved in the return to homeostasis. The figure was referenced from title [The protectin family... 26
Figure 1.2. Pathways and chemical structures of human SPMs. The figure was referenced from title [Molecular insights into lipoxygenases for biocatalytic... 29
Figure 1.3. Overall mechanisms of oxygenation and isomerization reactions by LOX for the biosynthesis of LMs. 33
Figure 2.1. HPLC of reaction products obtained from the conversion of DHA by whole A. violacum cells. HPLC chromatograms of standards and reaction... 51
Figure 2.2. LC-MS chromatograms of unknown products obtained from the conversion of DHA by whole cell conversion of A. violaceum. (A) LC-MS and LC-... 53
Figure 2.3. Chiral phase-HPLC chromatograms of the first- and second-oxygenated product obtained from conversion of DHA by ARA 15S-LOX from A.... 54
Figure 2.4. Alignment of amino acid sequence of human ARA 5-LOX with LOXs existed in A. violaceum. GenBank accession numbers of human arachidonate... 55
Figure 2.5. HPLC chromatograms of reaction products from the conversion of DHA by three candidate LOXs from A. violaceum that may produce SPMs. HPLC... 56
Figure 2.6. Phylogenetic tree based on amino acid sequences of LOXs. The numbers (1), (2) and (3) represent LOX1, which is identified as ARA 15-LOX,... 57
Figure 2.7. SDS-PAGE analysis and gel filtration chromatography of A. violaceum LOX expressed in E. coli C2566. (A) SDS-PAGE of A. violaceum... 59
Figure 2.8. LC-MS/MS profiles of LMs obtained from the conversion of PUFAs by 15S-LOX from A. violaceum. (A) Products from arachidonic acid. (B) Products... 64
Figure 2.9. Chiral phase-HPLC chromatograms of the first- and second-oxygenated product obtained from the conversion of ARA by 15S-LOX from A.... 65
Figure 2.10. 1D and 2D NMR data of 7S,17S-DiHDPA (resolvin D5n₋₃ DPA, RvD5 n-₃ DPA) in CDCl₃ (850 MHz NMR). (A) ¹H NMR. (B) ¹³C NMR. (C) Correlation...[이미지참조] 66
Figure 2.11. 1D and 2D NMR data of 7S,17S-DiHDHA (resolvin D5) in CDCl₃ (850 MHz NMR). (A) ¹H NMR. (B) ¹³C NMR. (C) Correlation spectroscopy... 67
Figure 2.12. Biosynthetic pathways of DiHFAs from PUFAs by the wild-type 15S-LOX from A. violaceum. Navy blue arrows indicate the conversion of PUFAs... 71
Figure 2.13. HPLC chromatograms of products obtained from the conversion of ARA by 15S-LOX from A. violaceum. (A) HPLC chromatograms for the... 72
Figure 2.14. Alignment of the amino acid sequence of 15S-LOX from A. violaceum with the previously reported 12S-LOXs and 15S-LOXs. Comparison... 75
Figure 2.15. Selection of five non-catalytic residues interacted with the substrate DHA in active-site pocket by molecular modeling. (A) Interaction of... 76
Figure 2.16. Docking models of DHA as a substrate in the substrate-binding pockets of the wild-type and engineered LOXs. (A) Docking model of DHA as a... 79
Figure 2.17. HPLC chromatograms of products obtained from the conversion of ARA by engineered ARA 12-LOX from A. violaceum. (A) HPLC chromatograms... 81
Figure 2.18. LC-MS/MS profiles of LMs obtained from the conversion of PUFAs by engineered 12S-LOX from A. violaceum. (A) Products from arachidonic acid.... 83
Figure 2.19. Chiral phase-HPLC chromatograms of the first- and second-oxygenated reaction products obtained from the conversion of ARA by... 84
Figure 2.20. 1D and 2D NMR data of 7S,14S-DiHDPA (7S-MaR1n₋₃ DPA) in CDCl₃ (850 MHz NMR). (A) ¹H NMR. (B) ¹³C NMR. (C) Correlation spectroscopy...[이미지참조] 85
Figure 2.21. 1D and 2D NMR data of 7S,14S-DiHDHA (7S-MaR1) in CDCl₃ (850 MHz NMR). (A) ¹H NMR. (B) ¹³C NMR. (C) Correlation spectroscopy (COSY).... 86
Figure 2.22. Biosynthetic pathways of DiHFAs from PUFAs by the engineered 12S-LOX from A. violaceum. Navy blue arrows indicate the conversion of PUFAs... 89
Figure 2.23. Effects of pH, temperature, and cysteine concentration on 5S,15S-DiHETE production by recombinant E. coli cells expressing ARA 15S-LOX from... 92
Figure 2.24. Biotransformation of ARA to 5S,15S-DiHETE by recombinant E. coli cells expressing ARA 15S-LOX from A. violaceum without or with cysteine.... 93
Figure 2.25. Biotransformation of PUFAs to DiHFAs by recombinant E. coli cells expressing wild-type 15S-LOX from A. violaceum. (A) Biotransformation of ARA.... 94
Figure 2.26. Biotransformation of PUFAs to DiHFAs by recombinant E. coli cells expressing the engineered 12S-LOX from A. violaceum. (A) Biotransformation... 95
Figure 3.1. Reverse-phase HPLC profiles of MonoHFAs obtained from the conversion of ARA by the selected 6 LOXs derived from S. cellulosum with ARA... 111
Figure 3.2. Chiral-phase HPLC profiles of MonoHFAs obtained from the conversion of ARA by the selected 3 putative LOXs derived from S. cellulosum... 112
Figure 3.3. 15R-LOX sequence derived from S. cellulosum so ce1871. The sequence of this gene was not the same as that of the gene encoding S4XZS0,... 113
Figure 3.4. Reverse- and chiral-phase HPLC profiles of the reaction products obtained from the conversion of ARA by the putative LOX cloned with the primer... 114
Figure 3.5. SDS-PAGE analysis and gel filtration chromatography of S. cellulosum 15R-LOX expressed in E. coli C2566. (A) SDS-PAGE of S.... 116
Figure 3.6. Alignment of amino acid sequence of S. cellulosum 15R-LOX with those of H. sapiens 5S-LOX, M. musculus 8S-LOX, M. xanthus 11S-LOX, P.... 119
Figure 3.7. Reliability of the models of 15R-LOX from S. cellulosum built by Alphafold and DS. (A) Alphafold model. Blue color represents high reliability,... 124
Figure 3.8. Confirmation of the active site in 15R-LOX from S. cellulosum using DS model by Alphafold model. The catalytic residues... 125
Figure 3.9. Selection of five favorable residues (Gln354, Leu413, Val602, Met603, and Leu606) interacted with the substrate 15R-HETE in substrate-... 126
Figure 3.10. Reverse-phase HPLC profiles of the products obtained from the conversion of ARA by the wild-type and variant LOXs at position 354 from S.... 127
Figure 3.11. HPLC profiles for reaction products obtained from the conversion of ARA by the L413F, V602F, M603F, and L606F variant and wild-type 15R-LOXs... 128
Figure 3.12. Proposed schematic drawing of the second oxygenation of 15R-HETE by wild-type 15R-LOX, L606F, and L606W variant LOXs. In panels A and B, the substrate-binding pocket is shown as brown-blue clouds, and the blue sphere represents... 129
Figure 3.13. HPLC profiles for the products obtained from the conversion of ARA and 15R-HETE by the variants at position 606. HPLC profiles for the products... 131
Figure 3.14. LC-MS/MS profiles of MonoHFAs and DiHFAs obtained from the conversion of PUFAs by 15R-LOX from S. cellulosum. (A) Products from... 136
Figure 3.15. Normal-phase HPLC profiles of DiHFA obtained from the bioconversion of ARA by S. cellulosum 15R-LOX with DiHFA standards. DiHFAs... 137
Figure 3.16. 1D and 2D NMR data of 5R,15R-DiHETE (isomer of leukotriene B4) in MeOD (850 MHz NMR). (A) ¹H NMR. (B) ¹³C NMR. (C) Correlation... 138
Figure 3.17. 1D and 2D NMR data of 7R,17R-DiHDHA (enantiomer of RvD5) in MeOD (850 MHz NMR). (A) ¹H NMR. (B) ¹³C NMR. (C) Correlation spectroscopy... 142
Figure 3.18. 1D and 2D NMR data of 5R,15R-DiHEPA (enantiomer of RvE4) in MeOD (850 MHz NMR). (A) ¹H NMR. (B) ¹³C NMR. (C) Correlation spectroscopy... 143
Figure 3.19. Comparison analysis of 1D NMR (850 MHz NMR) data of 5R,15R-DiHETE (blue-line) obtained from the conversion of ARA by S. cellulosum 15R-... 144
Figure 3.20. Pathways of PUFAs into DiHFAs by wild-type and engineered ARA 15R-LOXs from S. cellulosum. (A) Metabolism of ARA by 15R-LOX. (B)... 146
Figure 3.21. Optimization of pH, temperature, substrate, and cell concentration on the production of 5R,15R-DiHETE by E. coli cells expressing double-... 149
Figure 3.22. Binding of absorbent resins to ARA as substrate. (A) Selection of absorbent resins for ARA as substrate. Adsorbent resins were used to test... 150
Figure 3.23. Effect of the absorbent resin SP825 concentration on the production of 5R,15R-DiHETE from ARA by 15R-LOX from S. cellulosum with... 151
Figure 3.24. Bioconversion of PUFAs into DiHFAs by E. coli cells expressing wild-type double-oxygenating 15R-LOX from S. cellulosum with the addition of... 153
Figure 3.25. Optimization of pH, temperature, substrate, and cell concentration on the production of 15R-HETE by E. coli cells expressing engineered 15R-LOX... 156
Figure 3.26. Optimization of cell and substrate concentration on the bioconversion of ARA into 15R-HETE by E. coli cells expressing engineered... 157
Figure 3.27. Bioconversion of PUFAs into MonoHFAs by E. coli cells expressing engineered single-oxygenating 15R-LOX (L606F variant) from S. cellulosum... 159
Figure 3.28. Biotransformation of DHA-rich fish oil hydrolysate into 7R,17R-DiHDHA and 17R-HDHA by E. coli cells expressing wild-type double-... 162
Figure 4.1. HPLC profiles of glycerol in oil hydrolysate with glycerol standard. (A) HPLC profile of glycerol standard. (B) HPLC profile of glycerol in oil... 173
Figure 4.2. Effects of polymer and solvent on the production of RvD5 from DHA in oil hydrolysate as substrate by E. coli cells expressing A. violaceum 15S-LOX.... 175
Figure 4.3. Optimization of the concentrations of PVP and DMSO on the production of RvD5 from DHA in oil hydrolysate as substrate by E. coli cells... 176
Figure 4.4. Optimization of cells and the substrate concentrations for the production of RvD5 from reagent-grade DHA or DHA in oil hydrolysate as... 178
Figure 4.5. Optimization of the concentrations of reagent-grade DHA or DHA in oil hydrolysate as substrate and cells at the optimal ratio of cells to substrate on... 183
Figure 4.6. Bioconversion of reagent-grade DHA and DHA in oil hydrolysate as substrate into RvD5 by E. coli cells expressing A. violaceum 15S-LOX in a... 184
Figure 4.7. HPLC profiles at purification steps of RvD5 obtained from the bioconversion of DHA in the oil hydrolysate in a bioreactor. (A) HPLC profile of... 186
Figure 5.1. Optimization of safflower oil concentration on the production of LA in safflower oil hydrolysate by lipase. The hydrolysis reactions were performed... 200
Figure 5.2. Optimization of the ratio of the safflower oil and adsorbent resin SP207 with lipase on the concentration of LA in recovered safflower oil... 202
Figure 5.3. GC profiles for determining the effect of adsorbent resin SP207 on the yield of safflower oil hydrolysis. The adsorbent resin bound linoleic acid (LA) and oleic acid (OA) but not glycerol or palmitic acid, and the bound LA and OA were... 203
Figure 5.4. Chiral phase-HPLC profiles of the product obtained from the bioconversion of LA by purified A. violaceum LA 13S-LOX in the presence of... 205
Figure 5.5. LC-MS/MS analysis of the products obtained from the bioconversion of PUFAs, including (A) LA, (B) ALA, and (C) GLA by E. coli cells co-expressing... 208
Figure 5.6. Designed bioconversion pathways by the recombinant E. coli cells co-expressing LA 13S-LOX from A. violaceum and EH from M. xanthus. 209
Figure 5.7. NMR profiles of 12S,13S-EHODE(γ) by NMR in CDCl₃. (A) ¹H NMR peak. (B) ¹³C NMR peak. (C) 2D NMR profiles (850 MHz NMR). 210
Figure 5.8. NMR profiles of 11R,12R,13S-TriHODE(γ) by NMR in CDCl₃. (A) ¹H NMR peak. (B) ¹³C NMR peak. (C) 2D NMR profiles (850 MHz NMR). 211
Figure 5.9. Optimization of temperature, pH, cell, and substrate concentration on the production of 11R,12R,13S-TriHODE(γ) by E. coli cells co-expressing A.... 216
Figure 5.10. Optimization of substrate concentration on the bioconversion from LA in safflower oil hydrolysate obtained from lipase and resin treatment into... 219
Figure 5.11. Bioconversion of linoleic acid in safflower oil hydrolysate obtained from lipase and resin treatment into 11R,12R,13S-TriHODE(γ) by E. coli cells co-... 220
Figure 6.1. Effects of solvents and polymers on the production of RvE4 and its enantiomer from EPA as substrate by E. coli cells expressing A. violaceum 15S-... 234
Figure 6.2. Optimization of the concentrations of PVA and DMSO; or PVP and EtOH on the production of RvE4 and its enantiomer from EPA as substrate by E.... 235
Figure 6.3. Optimization of cells and the substrate concentrations for the production of RvE4 and its enantiomer from EPA by E. coli cells expressing A... 236
Figure 6.4. Bioconversion of EPA as substrate into RvE4 and its enantiomer by E. coli cells expressing A. violaceum 15S-LOX and S. cellulosum 15R-LOX,... 239
Figure 6.5. Bioconversion of EPA as substrate into RvE4 and its enantiomer by E. coli cells expressing A. violaceum 15S-LOX and S. cellulosum 15R-LOX,... 240
Figure 6.6. Optimization of the concentrations of EPA as substrate and cells at the optimal ratio on the production of RvE4 and its enantiomer by E. coli cells... 241
Figure 6.7. Bioconversion of EPA as substrate into RvE4 and its enantiomer by E. coli cells expressing A. violaceum 15S-LOX and S. cellulosum 15R-LOX,... 242
Figure 6.8. Selection of four favorable residues (Leu423, Leu424, Leu568, and Met603) interacted with the substrate EPA in substrate-binding pocket of S.... 246
Figure 6.9. Distances of EPA as a substrate to the active sites of wild-type and engineered LOXs from S. cellulosum and A. violaceum in the docking models.... 249
Figure 6.10. Chiral phase-HPLC profiles of the product obtained from the bioconversion of EPA by the engineered 18-LOXs from S. cellulosum and A.... 251
Figure 6.11. LC-MS/MS profiles of 18-HEPA obtained from the conversion of EPA by engineered 18-LOXs from S. cellulosum and A. violaceum. (A) Products... 252
Figure 6.12. Effects of pH and temperature on the bioconversion of EPA into 18R- and 18S-HEPAs by E. coli cells expressing engineered 18-LOXs from S.... 254
Figure 6.13. Optimization of substrate and cell concentrations for the bioconversion of EPA into 18R- and 18S-HEPAs by E. coli cells expressing... 255
Figure 6.14. Bioconversion of EPA into 18R- or 18S-HEPAs by E. coli cells expressing engineered 18R-LOX from S. cellulosum and engineered 18S-LOX... 256
Figure 6.15. Optimization of substrate concentrations for the bioconversion of 18R- and 18S-HEPAs into 5S,18R-DiHEPA and 5S,18S-DiHEPA by E. coli cells... 258
Figure 6.16. Bioconversions of 18R- and 18S-HEPAs into 5S,18R-DiHEPA and 5S,18S-DiHEPA by E. coli cells expressing 5S-LOX from D. rerio, respectively.... 259
Figure 6.17. Chiral phase-HPLC profiles of the product obtained from the bioconversion of 18R- and 18S-HEPA by 5S-LOX from D. rerio in the presence... 260
Figure 6.18. LC-MS/MS profiles of 5,18-DiHEPA obtained from the bioconversions of 18R- and 18S-HEPAs by 5S-LOX from D. rerio. (A) LC-MS/MS... 261