Redesigning gut bacterial oxidoreductases for biosynthesis of functional soy phytoestrogens in the whole-cell biotransformation system : 장내 미생물 유래 산화환원효소의 재설계 및 전세포 생산 시스템 구축을 통한 기능성 대두 파이토에스트로겐의 생산 연구

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dc.description학위논문 (박사)-- 서울대학교 대학원 : 공과대학 화학생물공학부, 2018. 8. 김병기.-
dc.description.abstractSoy isoflavones are naturally occurring phytochemicals, which are biotransformed into functional derivatives through oxidative and reductive metabolic pathways of diverse microorganisms. Such representative derivatives, equols and ortho-dihydroxy isoflavones (ODIs), have attracted great attention for their versatile health benefits since they were found from soybean fermented foods and human intestinal fluids. Recently, scientists in food technology, nutrition, and microbiology began to understand their correct biosynthetic pathways and nutraceutical values, and have attempted to produce the valuable bioactive compounds using microbial fermentation and enzyme/whole-cell-based biotransformation. Furthermore, artificial design of microbial catalysts and/or protein engineering of oxidoreductases were also conducted to enhance production efficiency and regioselectivity of products.

In this thesis, reductive metabolite equols and oxidative derivative ODIs were selected as production targets and efficient biosynthetic strategies were introduced. Primarily, recombinant E. coli strain retaining equol production capability was constructed and engineered for synthesis of equol and its derivatives. Because the reductive pathway is highly dependent on intracellular reductive potential comprised of NAD(P)H, whole-cell biotransformation was recognized as efficient and low-cost bioprocess. On the other hand, regioselective ODI production was investigated with engineered tyrosinase. Since the biocatalysis depends on monooxygenase activity of tyrosinase, inhibition of second oxidation step (or pigmentation) of tyrosinase should be immediately supplemented in the reaction buffer.

For microbial productions of equols using the recombinant E. coli strain, four key enzymes in equol production pathway, daidzein reductase (DZNR), dihydrodaidzein racemase (DDRC), dihydrodaidzein reductase (DHDR) and tetrahydrodaidzein reductase (THDR) were manipulated. Then, rate-determining enzymes have been identified for the biotransformation with low and high initial substrate loads, respectively. Hydrophilic polymer supplementation, reaction compartmentalization and computationally designed enzyme engineering were also introduced to achieve g/L level production of equol derivatives. As a result, equol derivatives could be produced with fine yields, 1.9 (for equol) and 1.3 g/L (for 5-hydroxy-equol), showing remarkable productivities. The biotransformation system also takes advantage of aerobic manipulation, a favorable fermentation process for industrial-scale production.

While, to achieve the massive production of ODIs, mono-oxygenase activity of a bacterial tyrosinase has been exploited. Because the wild-type tyrosinase has poor regioselectivity and catalytic activity, circular permutation (CP) and site-directed mutagenesis were performed. In results, a CP variant with enhanced polyphenol hydroxylation activity was demonstrated for 1.5 g/L of 3-hydroxygenistein production, and several mutants with some amino acid substitutions were verified to produce 6 or 8-hydroxyformononetin with increased regioselectivity.

In short, this study explored efficient biosynthetic methods of functional isoflavone derivatives, equols and ODIs using artificially reconstructed enzymes or microbial catalysts. It would provide useful catalytic platforms to produce various bioactive isoflavone derivatives.

Abstract v

List of Tables xii

List of Figures xiii

Chapter 1. Introduction 1

1.1 Microbially modified isoflavone derivatives 2

1.1.1 Equols 2

1.1.2 Ortho-dihydroxyisoflavones (ODIs) 4

1.2 Microbial Synthesis of isoflavone derivatives 7

1.2.1 Equol synthesis 7

1.2.2 Regioselective bioconversion of ODIs 11

1.3 The scope of thesis 19

Chapter 2. Construction of equol-producing recombinant Escherichia coli and production of (S)-equol with enhanced dihydrodaidzein reductase mutant P212A 22

2.1 Introduction 23

2.2 Materials and Methods 27

2.2.1 Chemicals 27

2.2.2 Cloning and construction of recombinant strain 28

2.2.3 Site-directed mutagenesis of DHDR 30

2.2.4 Whole-cell reaction with (S)-equol producing recombinant E. coli…. 32

2.2.5 HPLC analysis 33

2.2.6 Recombinant enzyme expression and purification 35

2.2.7 Enzyme assays 36

2.2.8 Enzymatic synthesis of trans- and cis-THD 38

2.3 Results 40

2.3.1 Daidzein to (S)-equol biosynthesis using recombinant E. coli BL21 (DE3). 40

2.3.2 pH effect and kinetics of DHDR 46

2.3.3 Construction and characterization of DHDR P212A mutant 51

2.3.4 Enantioselectivity of DHDR and production of (3S,4R)-trans-THD.. 55

2.3.5 Efficient (S)-equol production using the DHDR P212A mutant 60

2.4 Discussion 63

2.5 Conclusion 66

Chapter 3. Biosynthesis of (-)-5-hydroxy-equol and 5-hydroxy-dehydroequol from genistein using equol-producing recombinant E. coli 68

3.1 Introduction 69

3.2 Materials and Methods 73

3.2.1 Chemicals 73

3.2.2 Biotransformation and preparation of 5-hydroxy-equol and 5-hydroxy-dehydroequol 73

3.2.3 HPLC and GC/MS analysis 75

3.2.4 Chirality studies of biosynthesized 5-hydroxy-equol 75

3.2.5 Estrogen receptor binding assay 76

3.3 Results and Discussion 77

3.3.1 Conversion of genistein into 5-hydroxy-equol by tDDDT 77

3.3.2 Determination of absolute configuration of biosynthesized 5-hydroxy-equol 82

3.3.3 Compartmentalization strategy to produce 5-hydroxy-equol with high selectivity over 5-hydroxy-dehydroequol 86

3.3.4 Selective production of 5-hydroxy-dehydroequol in the absence of THDR 91

3.3.5 Phytoestrogenic property of (-)-5-hydroxy-equol 94

3.4 Conclusion 98

Chapter 4. Protein engineering and isoflavone solubilization strategy for g/L scale production of equol and 5-hydroxy-equol 99

4.1 Introduction 100

4.2 Materials and Methods 105

4.2.1 Chemicals 105

4.2.2 Solubility test 105

4.2.3 Whole-cell biotransformation study 106

4.2.4 Homology modeling and docking simulation 107

4.2.5 Site-directed mutagenesis of THDR 107

4.3 Results 109

4.3.1 Isoflavone solubility in HP supplemented solution 109

4.3.2 HP addition effect on (S)-equol bioconversion system 113

4.3.3 Application of HP into other polyphenols solubilization 117

4.3.4 Homology modeling of THDR and docking simulation 117

4.3.5 Site-directed mutagenesis of THDR and biotransformation using the mutants 123

4.4 Discussion 128

4.5 Conclusion 133

Chapter 5. Regioselective ortho-hydroxylation of isoflavones and equols using engineered tyrosinase 134

5.1 Introduction 135

5.2 Materials and Methods 140

5.2.1 Chemicals 140

5.2.2 Construction of CP library 140

5.2.3 Tyrosinase activity assay 142

5.2.4 Enzyme expression and purification 142

5.2.5 Ortho-hydroxylation using tyrosinase variants 144

5.2.6 Computational analysis and site-directed mutagenesis 145

5.2.7 Whole-cell biotransformation 146

5.3 Results and Discussion 147

5.3.1 Construction of smart CP library of BmTy 147

5.3.2 Chaperone-assisted expression of CP variants 152

5.3.3 Determination of kinetic characters and substrate specificity 156

5.3.4 Quantitative preparation of 3-hydroxygenistein (orobol) using cp48 163

5.3.5 Regioselective hydroxylation of formononetin using tyrosinase mutants 165

5.3.6 Oxidoreductive biotransformation for regioselective prepration of ortho-hydroxyequols 175

5.4 Conclusion 178

Chapter 6. Overall Conclusion and Further Suggestions 179

6.1 Production of equol derivatives using recombinant E. coli 180

6.2 Regioselective preparation of ODIs and OHEs using tyrosinase 183

6.3 Further suggestion 1: Catalytic mechanism of tetrahydrodaidzein reductase 184

6.4 Further suggestion 2: Equol-producing probiotics 187

References 188

Abstract in Korean 217
dc.publisher서울대학교 대학원-
dc.titleRedesigning gut bacterial oxidoreductases for biosynthesis of functional soy phytoestrogens in the whole-cell biotransformation system-
dc.title.alternative장내 미생물 유래 산화환원효소의 재설계 및 전세포 생산 시스템 구축을 통한 기능성 대두 파이토에스트로겐의 생산 연구-
dc.contributor.AlternativeAuthorPyung-Gang Lee-
dc.contributor.affiliation공과대학 화학생물공학부-
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College of Engineering/Engineering Practice School (공과대학/대학원)Dept. of Chemical and Biological Engineering (화학생물공학부)Theses (Ph.D. / Sc.D._화학생물공학부)
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