최권영 박현아 2022-08 32247 https://aurora.ajou.ac.kr/handle/2018.oak/21301 학위논문(박사)--아주대학교 일반대학원 :환경공학과,2022. 8 본 연구는 사이토크롬 P450(CYP) 효소의 위치특이적 수산화 활성을 바이오폴리머 합성 분야에 활용한 연구를 다루고 있다. CYP 효소를 사용해 바이오플라스틱 단량체인 α,ω-도데칸다이올과 신규 멜라닌 바이오 폴리머를 생합성하였다. 사이토크롬 P450 수산화 효소는 기질의 위치특이적 수산화 반응을 위한 생촉매로 사용될 수 있으며 기질의 특정 위치에 수산화기를 치환할 수 있다는 점에서 화학적 합성에 비해 이점을 갖는다. 도데칸다이올은 폴리에스터, 폴리우레탄 등의 고분자 단량체로 활용할 수 있고, 산화반응 또는 아미노기 전이반응을 등을 통해 다른 종류의 고분자 단량체로 전환이 가능한 바이오 플라스틱 산업에서 활용도가 높은 물질이다. 대장균 내에 ‘기질 유입 효소-CYP효소-전자전달효소’ 발현 시스템을 모듈화하여 구축하였으며, 1-도데칸올에 대한 omega 위치 특이적 수산화 활성을 갖는 CYP 효소를 전세포반응을 통해 선별하고 도데칸 기질의 omega 수산화 성능 또한 평가하였다. 그 결과, 1-dodecanol과 dodecane에 대한 위치 특이적 수산화가 가능한 효소 CYP153A33과 Nfa22290을 선별하는 데 성공하였으나 전환 수준이 약 23%로 높지 않았고, 과산화(overoxidation) 부산물인 도데칸산이 생성물의 약 10%를 차지하는 한계가 있었다. α,ω-다이올로의 기질전환 비율을 높이기 위한 목적으로 CYP153A33 효소의 돌연변이를 제작하였다. CYP153A33-도데칸올 결합 시뮬레이션을 통해 도출한 모델을 기반으로 하여 구조를 분석하였다. 기존의 CYP153A33 효소 구조변이 관련 선행연구는 기질과 직접 결합하는 수산화 활성부위에 집중되어 이루어졌으나, 본 연구에서는 수산화활성 부위와 더불어 기질이 효소 내부로 유입되는 메커니즘에 집중하였고, 효소 표면구조의 loop가 기질의 유입에 영향을 미칠 수 있음을 증명하였다. Loop를 구성하는 아미노산 서열 중 Proline 135-Proline 136 연속 구조가 loop의 기질유입 핵심부위에 위치함을 확인하였고 이로 인해 유발되는 rigidity를 줄이기 위해 proline 136을 alanine으로 치환함으로써 기질의 유입을 용이케 하였다. 이로써 1-도데칸올의 생전환율을 약 71% 수준으로 3배 이상 높였으며, 그 중 도데칸산이 차지하는 비율은 1% 미만으로 낮추어 CYP153A33 효소의 활용가치를 높였다. Tyr-멜라닌은 아미노산 L-타이로신으로부터 타이로시네이즈 효소 반응으로 전합성이 가능한 바이오폴리머이다. 타이로신을 시작물질로 한 도파크롬 유도체 단량체가 무작위 중합된 형태로, conjugation 구조이며 짙은 갈색을 띄는 특징이 있기 때문에 전자전달이 가능하고 유기반도체 소재, 흑색 유기안료, 자외선 차단 물질 등으로 응용이 가능하다. 무작위 중합으로 고분자의 정확한 구조를 알 수 없는 한계점이 있지만 단량체 구성을 변경하는 방식으로 응용처를 확장해볼 수 있는 가능성이 있는 물질이다. L-트립토판을 시작물질로 하는 indoxyl 유도체를 단량체로 포함시키는 전략으로 기존과 차별된 물성을 갖는 신규 멜라닌을 합성하고자 하였다. 사용한 효소는 CYP102G4로 인돌의 3번 탄소에 위치 특이적으로 수산화를 하는 CYP 효소로, 도파크롬 단량체를 중합하는 타이로시네이즈 효소의 촉매 작용이 가능할 것으로 생각되었다. 새롭게 적용된 CYP102G4 효소와 기존의 Tyr-멜라닌 합성 생촉매인 타이로시네이즈를 동시발현하여 대장균 내에서 신규 멜라닌 합성 경로를 구축하여 생합성하였고, 결론적으로 Tyr-멜라닌 대비 짙은 흑색에 가까운 색의 CYP-멜라닌을 합성하는 데 성공하였다. 멜라닌 생성량 또한 기존의 리터당 수백밀리그램 수준에서 3g 이상으로 크게 증가하였으며, 공극률과 표면적이 높진 않지만 전극물질로써 활용 가능성을 확인하였다. 또한 FT-ICR 분석을 통해 신규 멜라닌이 기존에 비해 높은 비율로 황원소를 포함하는 것을 확인하였다. 결론적으로 본 연구는 CYP 효소를 다이올 형태의 고분자 단량체 및 신규 멜라닌 고분자 생합성에 활용하여 효소의 응용처를 확장하였으며, CYP 효소의 구조 및 기질결합 메커니즘에 기반한 개량을 통해 위치특이적 수산화 활성을 증대함으로써 CYP의 응용과 개량에 관한 학문적 기반을 제공했다는 점에서 의의가 있다. 주요어: P450 효소 (CYP 효소), 위치특이적 수산화, 바이오 단량체, 바이오 폴리머, 도데칸다이올, 멜라닌 Chapter Ⅰ. α,ω-oxyfunctionalization of aliphatic substrates (1-dodecanol and dodecane) via whole-cell biocatalysis of CYP153A33 and Nfa22290 １ A. Abstract ２ B. Introduction ３ C. Materials and Methods ７ 1. Chemicals and reagents. ７ 2. Cloning and expression of CYPs and whole-cell reaction in E. coli ７ 3. UV-vis assays of cytochrome P450 enzymes ８ 4. Whole-cell biotransformation of 1-dodecanol ８ 5. Gas chromatography analysis ９ D. Results and Discussion １１ 1. Construction of the CYP153A33 expression system in E. coli and whole-cell biotransformation of 1-dodecanol by using the recombinant E. coli strain １１ 2. Two transporter systems for FadL and AlkL in 1-dodecanol/ dodecane biotransformation １６ 3. Oxidation product analysis of C12-oxidized intermediates via whole-cell reaction by using the recombinant E. coli ２１ 4. Enhancing the production of α,ω-dodecanaediol via co-factor optimization ２６ 5. Bioinformatic analysis for the selection of 1-dodecanol hydroxylation CYPs from N. farcinica ２８ 6. Spectral feature of Nfa22290, Nfa22930, and Nfa33510: carbon monoxide (CO)-binding assays ３４ 7. Whole-cell production of α,ω-dodecanediol from different concentrations of 1-dodecanol ３６ 8. Whole-cell biotransformation of dodecane to 1-dodecanol and α,ω-dodecanediol ３９ E. Conclusion ４１ Chapter Ⅱ. Structure-guided engineering of CYP153A33 for the control of its over-oxidation activity on aliphatic substrate ４２ A. Abstract ４３ B. Introductiion ４４ C. Materials and Methods ４８ 1. Chemical reagents and media ４８ 2. Site-directed mutagenesis of CYP153A33 and construction of expression system in E. coli ４８ 3. Enzyme expression and whole-cell reaction of primary fatty alcohols by using recombinant E. coli 49 4. Gas chromatography analysis ４９ 5. Structural analysis of enzyme-substrate docking simulation ５０ D. Results and Discussion ５１ 1. Determination of the conversion rate of continuous α- and ω-oxidation of fatty substrates via whole-cell transformation in E. coli ５１ 2. Structure-based analysis of a 1-dodecanol substrate docked model ５４ 3. Geometry of 1-dodecanol substrate-binding pocket and selection of key enzyme residues ６１ 4. Site-directed mutagenesis of CYP153A33 and whole-cell transformation of 1-dodecanol using the CYP153A33 mutant ６６ 5. Evaluation of the substrate specificity of CYP153A33 P136A against medium- and long-chain fatty alkanols ７３ E. Conclusion ８１ Chapter Ⅲ. Hydroxylation of an aromatic substrate (indole) via whole-cell biocatalysis of CYP102G4 monooxygenase and synthesis of novel melanin-based biopolymer ８３ A. Abstract ８４ B. Introduction ８５ C. Materials and Methods ８８ 1. Chemical and reagents ８８ 2. Cloning and expression of melC and cyp102G4 ８８ 3. Whole-cell production of different melanins (Tyr-Melanin, CYP-Melanin, and Sglu-Melanin) ８８ 4. Quantitative and qualitative analysis of CYP-Melanin ８９ 5. Surface characterization of CYP-Melanin ８９ 6. FT-ICR MS analysis and elemental composition assignments ９０ 7. HEMA polymerization for synthetic melanin dyeing ９１ 8. Electrochemical properties of CYP-Melanin ９１ D. Results and Discussion ９３ 1. MelC tyrosinase and CYP102G4 monooxygenase enzymes ９３ 2. Whole-cell bioproduction of the novel CYP-Melanin and dyeing performance of CYP-melanin pigment ９８ 3. Isotherm analysis of CYP-Melanin １０２ 4. Chemical and molecular composition analysis of CYP-Melanin using FT-IR and 15 T FT-ICR mass spectrometry １０６ 5. Electrochemical properties of synthesized CYP-Melanin １１２ E. Conclusion １１６ Chapter Ⅳ. Overall conclusion and discussion １１７ References １２２ eng The Graduate School, Ajou University 아주대학교 논문은 저작권에 의해 보호받습니다. Cytochrome P450 monooxygenase-mediated synthesis of α,ω-diol monomer and aromatic biopolymer CYP 효소를 활용한 α,ω-다이올 단량체 및 방향족 바이오 폴리머의 생합성 Thesis 아주대학교 일반대학원 일반대학원 환경공학과 2022. 8 Doctoral I804:41038-000000032247 https://dcoll.ajou.ac.kr/dcollection/common/orgView/000000032247 biomonomer biopolymer cytochrome P450 (CYP) dodecanediol melanin regiospecific hydroxylation Cytochrome P450 (CYP) monooxygenases can be used as biocatalysts for the hydroxylation of hydrocarbon C-H bonds due to their advantages over chemical catalysts, such as regio and stereoselective activity. My PhD thesis focused on the engineering and utilizing of CYP enzymes for the biosynthesis of building blocks used in high-performance biopolymers. In detail, CYPs were screened and engineered for regiospecific hydroxylation on aliphatic substrates (dodecane and 1-dodecanol) for the synthesis of α,ω-dodecanediol and an aromatic compound (indole) for the synthesis of novel melanin biopolymers. The first target compound I investigated, α,ω-dodecanediol, is a versatile material used in the bioplastics industry. It is widely used as a monomer of polyester and polyurethan, with its production depending on chemical processes. I investigated a new bioprocess, CYP-dependent whole-cell biotransformation, for use in the “membrane protein-CYP-redox protein” expression system. To that end, Escherichia coli cells over-expressing CYP153A33 from Marinobacter aquaeolei VT8 and Nfa22290 from Nocardia farcinica IFM10152 (NFA) in combination with the putida ferredoxin reductase and ferredoxin (CamA/B) redox system from Pseudomonas putida and FadL, a long-chain fatty acid transporter, were examined for dodecane and 1-dodecanol whole-cell biotransformation, respectively. Whole-cell biotransformation of 1-dodecanol and dodecane into α,ω-dodecanediol was successfully performed in these systems, but the conversion yield amounted to only about 23%, and dodecanoic acid accounted for about 10% of the products. I addressed this limitation by performing a site-directed mutagenesis of CYP153A33. The enzyme structure of CYP153A33 was analyzed based on the model derived from the CYP153A33-dodecanol docking simulation. Previous studies related to CYP153A33 enzyme structural mutation have focused on the active site of hydroxylation that binds to a substrate, whereas my work focused on the mechanism underlying the flow of the substrate into the active site of the enzyme. A structural analysis showed that the loop on the enzyme surface can affect the influx of a substrate. Among the peptide bonds constituting the loop, the continuous structure of proline 135-proline 136 was located at the core of the substrate inflow path. The rigidity induced by the structure was reduced by replacing proline 136 with alanine, to facilitate substrate influx. As a result, the bioconversion rate of 1-dodecanol increased more than threefold to about 71%, and the proportion of dodecanoic acid decreased to less than 1%, thereby increasing the utility of CYP153A33. The second target compound I investigated, Tyr-melanin, is a biopolymer that can be fully synthesized from the amino acid L-tyrosine by tyrosinase. Tyr-melanin is a randomly polymerized form of dopachrome derivative monomers converted from tyrosine as a starting material. Because it has a conjugation structure and a dark brown color, it can transfer electrons and thus serve as organic semiconductor material, black organic pigment, and UV-blocking material. Because it is randomly polymerized, the exact structure of the polymer is not known, but its application potential can be expanded by changing its monomer composition. Here, I synthesized a novel melanin compound, with physical properties that differ from those of existing melanin, by including an indoxyl derivative converted from L-tryptophan as a monomer. CYP102G4, a CYP enzyme that regiospecifically hydroxylates a carbon-3 of indole, was used. The indole derivative was expected to be integrated in melanin as a monomer by tyrosinase polymerization. The newly employed CYP102G4 enzyme and the existing Tyr-melanin synthesis biocatalyst, tyrosinase, were co-expressed to construct a new melanin synthesis pathway in E. coli, and novel CYP-melanin was biosynthesized. The CYP-melanin with a dark color close to black was synthesized and comparted to Tyr-melanin. Melanin production increased significantly from several 100 mg/L to more than 3 g/L, and although its porosity was not high and the surface area was not large, CYP-melanin could possibly be used as electrode material. In addition, a FT-ICR analysis showed that CYP-melanin contained more sulfur than Tyr-melanin. Taken together, this study expanded the application of CYPs by utilizing the enzyme for the biosynthesis of diol monomers and novel melanin polymers. In addition, it is significant that it provided an academic basis for the application and improvement of CYPs by increasing site-specific hydroxylation activity through improvement based on the structure and substrate binding mechanism of the CYPs. Keywords: cytochrome P450 (CYP), regiospecific hydroxylation, biomonomer, biopolymer, dodecanediol, melanin