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dc.contributor.advisor박진병-
dc.contributor.author전은영-
dc.creator전은영-
dc.date.accessioned2017-09-10T00:05:25Z-
dc.date.available2017-09-10T00:05:25Z-
dc.date.issued2016-
dc.identifier.otherOAK-000000126840-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000126840en_US
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/236803-
dc.description.abstract중쇄 오메가 하이드록시카르복실산, 알파, 오메가 디카르복실산 등의 중쇄 카르복실산은 엔지니어링 플라스틱이나 유화제, 동결방지제, 부식방지제 생산에 사용이 가능하며 또한, 식품이나 화장품에서 항균 소재로 이용되고 있다. 재생 가능한 지방산으로부터 중쇄 카르복실산을 생산하는 생합성 경로는 S. maltophilia 유래 hydratase, Micrococcus luteus NCTC2665 유래 ADH, Pseudomonas putida KT2440 유래 BVMO, P. fluorescens SIK WI 유래 esterase 로 이루어진다. 본 연구에서는 장쇄 지방산으로부터 중쇄 카르복실산 생산하는 생합성 경로를 발현하는 재조합 Escherichia coli 기반 생촉매의 개량에 관한 연구를 진행하였다. 연구 2에서는 생합성 경로의 핵심 효소인 BVMO 의 안정성과 반응활성을 위해 고세균 유래의 샤페론을 도입하였다. 심층 해수 고세균 유래의 샤페론인 γ-prefoldin 과 thermosome 을 생합성 경로의 ADH와 BVMO를 발현시킨 E. coli 생촉매에 동시 발현시킨 결과 ricinoleic acid 를 기질로 이용하는 생물전환 속도가 증가하였다. 또한 고세균 샤페론의 발현수준에 따라 생물전환 속도가 영향을 받으며, 샤페론 최대 발현수준의 46% 발현될 때, 2배 증가한 12 μmol g dry cells-1 min-1 전세포 생물전환 활성을 보였다. 따라서, 적절한 수준의 샤페론 동시발현을 통해 전세포 생물전환 활성과 안정성을 개선하였다. 연구 3에서는 생합성 경로에 관여하는 ADH 와 BVMO 를 결합하여 융합 효소를 구축하였다. M. luteus 유래 ADH 는 상당히 수용성 발현이 용이한데 반해, P. putida 유래의 BVMO 는 불용성 발현이 된다. 이 두 효소를 융합시킨 결과, BVMO 의 수용성 발현이 상당히 증가하였고, 이로 인해 다양한 장쇄 이차 알코올에 대한 생물전환 속도가 향상되었다. 특히, 유동성을 갖는 링커로 융합 효소를 구축한 결과 Ricinoleic acid 전세포 생물전환 속도는 단독발현 보다 40 % 증가한 22 μmol g dry cells-1 min-1 를 보였다. 이러한 증가는 효소의 수용성 발현 증가와 융합 효소의 활성 부위가 가까워짐에 따라 중간체의 이동의 용이함에 따라 나타난 것으로 판단된다. 연구 4에서는 E. coli 의 외막에 존재하는 장쇄 지방산 운반단백질인 FadL 을 추가로 발현시켜 기질인 불용성 장쇄 지방산을 전세포 생촉매내로의 유입속도를 증가시키고자 하였다. FadL 추가 발현 단일 효소 생촉매의 생물전환 결과 oleic acid, 12-hydroxystearic acid, 10-hydroxystearic acid 의 생물전환 속도가 상당히 증가하였다. 중쇄 카르복실산 생산을 위한 E. coli 생촉매에 FadL 을 추가 발현 시킨 결과 oleic acid 의 생물전환 속도가 5배 이상 증가하였다. 따라서 추가적인 FadL 의 발현이 지방산을 이용한 생물전환 속도를 증가로 이어졌다. 이러한 연구들은 지방산을 이용하여 다중 효소 합성경로를 거쳐 중쇄 카르복실산을 생산하는 친환경적 전세포 생촉매를 개발하는데 기여할 것으로 기대된다. ;A variety of carboxylic acids including C9 to C13 ω-hydroxycarboxylic acids, ω-aminocarboxylic acids, and α,ω-dicarboxylic acids can be produced from renewable long chain fatty acids via Escherichia coli-based biocatalysis, expressing a fatty acid double bond hydratase, a long chain secondary alcohol dehydrogenase (ADH), a Baeyer-Villiger monooxygenase (BVMO), and an esterase. However, the productivities are limited by low stability and activity of the catalytic enzymes in particular the BVMOs, which catalyze oxidative C-C bond cleavage reaction. In addition, the productivities are affected by inefficient transport of fatty acid substrates into the enzymes inside whole-cells. Thereby, this study has focused on engineering of the BVMOs and E. coli cells to enhance fatty acid biotransformation performance. The first approach was to introduce the archaeal chaperones (i.e., γ-prefoldin and thermosome) into the recombinant E. coli expressing an ADH of Micrococcus luteus and a BVMO of Pseudomonas putida KT2440. Ricinoleic acid biotransformation activity of the recombinant E. coli was improved significantly with co-expression of γ-prefoldin or recombinant thermosome (r-thermosome) originating from the deep-sea hyperthermophile archaea Methanocaldococcus jannaschii. Furthermore, the degree of enhanced activity was dependent on the expression levels of the chaperones. For example, whole-cell biotransformation activity was highest at 12 μmol g dry cells-1 min-1 when γ-prefoldin expression level was approximately 46% of the theoretical maximum. This value was approximately two-fold greater than that in E. coli, where the γ-prefoldin expression level was zero or set to the theoretical maximum. Therefore, it was assumed that co-expression of the chaperones to an optimal level may improve the biotransformation activity of whole-cell biocatalysts. The second approach was to construct fusion enzymes between soluble ADH enzyme of M. luteus and less soluble BVMO of P. putida KT2440. The recombinant E. coli, expressing the fusion enzymes, showed significantly greater bioconversion activity with long chain sec-alcohols (e.g., 12-hydroxyoctadec-9-enoic acid (1a), 13-hydroxyoctadec-9-enoic acid (2a), 14-hydroxyicos-11-enoic acid (4a) described in Scheme 2) when compared to the recombinant E. coli expressing the ADH and BVMOs independently. For instance, activity of the recombinant E. coli expressing the ADH-Gly-BVMO, in which glycine-rich peptide was used as the linker, with 1a was increased up to 22 μmol g dry cells-1 min-1. This value is over 40% greater than the recombinant E. coli expressing the ADH and BVMO independently. The substantial improvement appeared to be driven by an increase in the functional expression of the BVMOs and/or an increase in mass transport efficiency by localizing two active sites in close proximity. Another approach was to improve transport of fatty acid substrates into the recombinant E. coli cells by increased expression of a long chain fatty acid transporter FadL in the recombinant. Increased expression of the FadL in recombinant E. coli expressing a hydratase of Stenotrophomonas maltophilia or the ADH of M. luteus allowed to reach higher biotransformation activity with oleic acid, 10-hydroxystearic acid, and 12-hydroxystearic acid. This approach also led to a significant enhancement of the biotransformation rate of ricinoleic acid (1a) into the ester (1c) as well as of oleic acid (6) into the ester (6c). For instance, increased expression of the FadL in the recombinant E. coli expressing the hydratase of S. maltophilia or the ADH of M. luteus, and the BVMO of P. putida KT2440 resulted in 5.3-fold higher biotransformation rate with oleic acid. These results indicated that additional expression of the FadL might lead to a significant increase of whole-cell biotransformation rate of fatty acids. This study will contribute to development of an environmentally friendly whole cell biocatalytic process for the production of valuable carboxyl synthons, such as medium chain ω-hydroxycarboxylic acids and α, ω-dicarboxylic acids from renewable fatty acids.-
dc.description.tableofcontentsAbstract 1 Chapter Ⅰ 4 Introduction 4 A. Enzyme engineering and soluble expression 8 1. Coexpression of chaperones 8 2. Construction of fusion enzymes 9 B. Increase of permeability of E. coli whole cell biocatalysts 13 1. Physical method 14 2. Chemical method 14 3. Molecular engineering 17 C. Research objectives 20 References 22 Chapter Ⅱ 35 Expression levels of chaperones influence biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and Pseudomonas putida Baeyer Villiger monooxygenase 35 Abstract 36 A. Introduction 37 B. Materials and Methods 39 1. Microbial strains and culture media 39 2. Plasmid construction 41 3. Ricinoleic acid biotransformation 41 4. Product analysis by gas chromatography/mass spectroscopy 41 5. 5-UTR modification 42 6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses 48 C. Results 49 1. Biotransformation of ricinoleic acid (1a) into ester (1c) 49 2. Effect of chaperones on E. coli biotransformation activity 53 3. Effect of target protein structure on chaperone activity 56 4. Effect of chaperone expression level on biocatalytic activity 59 D. Discussion 64 E. Conclusion 70 Acknowledgement 71 References 72 Chapter Ⅲ 76 Enzyme fusion for whole-cell biotransformation of long-chain sec-alcohols into esters 76 Abstract 77 A. Introduction 78 B. Materials and Methods 84 1. Microbial strains and culture media 84 2. Reagents 87 3. Gene cloning 87 4. Purification of ADH 92 5. Activity assay of ADH 92 6. Protein electrophoresis 93 7. Whole cell biotransformation 93 8. Product analysis by GC/MS 93 C. Results 95 1. Design and construction of the fusion enzymes 95 2. Catalytic activity of the fusion enzymes 98 3. Effect of induction temperature on biocatalytic activity 103 4. Biotransformation activity of the fusion enzymes 106 5. Fusion of R. jostii BVMO and M. luteus ADH 109 D. Discussion 114 References 116 Chapter Ⅳ 121 Improving fatty acid biotransformation activity of recombinant Escherichia coli-based biocatalysts by enhanced expression of a long chain fatty acid transporter FadL 121 Abstract 122 A. Introduction 123 B. Materials and Methods 126 1. Microbial strains and culture medium 126 2. Gene cloning 126 3. Determination of cell concentration 134 4. ADH purification 135 5. NAD assay of isolated alcohol dehydrogenase (ADH) 135 6. Product analysis by gas chromatography/mass spectrometry (GC-MS) 136 7. Protein electrophoresis 136 C. Results 138 1. Effect of FadL overexpression on whole-cell biotransformation of fatty acids 138 2. FadL application on multi-step whole-cell biocatalysis 144 3. Biotransformation at high cell density culture 152 D. Discussion 155 E. Conclusion 162 References 163 CHAPTER V 170 CONCLUSION 170 Abstract in Korean (국문초록) 172-
dc.formatapplication/pdf-
dc.format.extent1920823 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.subject.ddc600-
dc.titleEngineering of Whole Cell Biocatalysts for the Production of ω-Hydroxycarboxylic Acids from Renewable Fatty Acids-
dc.typeDoctoral Thesis-
dc.format.pagexiii, 174 p.-
dc.description.localremark박188-
dc.identifier.thesisdegreeDoctor-
dc.identifier.major대학원 식품공학과-
dc.date.awarded2016. 8-
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