알칸 모노옥시게나아제를 사용한 대장균에서의 12-수산화 라우르산의 생산 연구 및 응용

학위논문(박사) -- 서울대학교대학원 : 공과대학 협동과정 바이오엔지니어링전공, 2021.8. 김병기.최근 석유 고갈 가능성 및 환경오염 문제가 대두되면서 식물 기름유도체로부터 폴리머의 단량체를 생산하는 방법의 중요성이 대두되고 있다. 특히 ω-수산화 지방산은 다양한 바이오폴리머 모노머의 전구체로 사용되는 물질이다. 그렇기 때문에 효소 반응을 사용한 친환경적인 방법으로 ω-수산화 지방산을 생산하는 연구가 전 세계적으로 활발히 연구진행되고 있다. 본 연구는, 대장균에서 12-수산화 라우르산을 메틸 라우르산으로부터 AlkBGT 시스템을 사용해서 생산하는 연구와 생산된 12-수산화 라우르산을 통하여 다양한 바이오폴리머 모노머(ω–아미노 지방산, α,ω-알칸다이올, α,ω-알칸다이아민)를 생산하는 연구를 진행하였다. 우리는 트랜스포터, 프로모터의 개량 및 새로운 알칸 모노옥시게나아제를 대장균에 도입하여 메틸 라우르산으로부터 이상계 반응을 통해 44.8 ± 7.5 mM of 12-수산화 지방산 및 31.8 ± 1.7 mM of 도데칸다이오산을 생산하였다. 또한 다중 효소 연속 반응을 수행하기 위한 3개의 대장균 모듈(수산화 모듈(Cell-Hm), 아미노화 모듈(Cell-Am), 환원 모듈(Cell-Rm))을 구축하였다. 46.3 mM 12-아미노 라우르산, 27 mM 1,12 도데칸다이올, 21.5 mM 1,12-도데칸다이아민이 다중 효소 연속 반응을 통하여 100 mM의 메틸 라우르산으로부터 생산되었다. 퓨도모나스 펠라지아 유래의 알칸 모노옥시게나아제를 (PpAlkB)의 수산화 활성을 증가시키기 위하여 실수 유발 PCR을 통하여 PpAklB의 무작위 돌연변이 라이브러리를 구축하였으며, 활성이 높은 변이주를 고 처리량 분석방법을 통해 선별하였다. 대장균에서 항생제 내성 유전자의 발현을 PpAlkB에 의한 라우르산의 생산양과 결합되어 세포 성장에 의한 고 처리량 분석방법을 통해 생체 내 스크리닝을 진행 할 수 있다. 도데칸과 메틸 라우르산을 기질로 사용하여 2번의 스크리능을 진행한 후 우리는 H292L/F253I/S313P의 돌연변이가 발생한 PpAlkB를 얻을 수 있었다. 12.95 mM (11 mM 12-수산화 라우르산, 1.96 mM 도데칸다이오산)이 100 mM 메틸 라우르산으로부터 PpAlkB 변이주를 포함한 대장균으로부터 생산되었으며, 이는 7.88 mM이 생산된 야생형 PpAlkB를 사용하였을 때보다 64% 증가된 수치이다. 결론적으로 본 연구는 대장균에서 메틸 라우르산을 기질로 사용하여 12-수산화 라우르산을 생산하였으며, 세 종류의 대장균 기반 세포 모듈을 만들어 다중 효소 연속 반응을 수행하여 다양한 바이오폴리머 모노머를 생산할 수 있었다. 또한 생체 내 스크리닝 시스템을 사용하여 메틸 라우르산의 수산화 활성이 높아진 PpAklB의 변이주를 찾을 수 있었다. 본 연구는 AlkB의 효소 공학 및 모듈화된 세포 반응을 통해 대장균에서 지방산 유도체의 생합성 연구 가능하게 했다는 점에서 의의가 있다The production of biochemicals from vegetable oil derivatives has attracted great interest as an alternative mean to develop a sustainable and environmentally-friendly production process known as biorefinery. ω-Hydroxy fatty acids (ω-HFA) are materials frequently used as precursors for various biopolymer monomers. In an effort to make eco-friendly biopolymer monomers, researches are being conducted worldwide to produce such monomers through enzymatic reactions. Here, production of 12-hydroxy dodecanoic acid (12-HDA) from dodecanoic acid methyl ester (DAME) with AlkBGT system in Escherichia coli and biosynthesis of various biopolymer monomers (ω –amino fatty acid, α,ω-alkanediol, α,ω-alkanediamine) with multi-enzymatic cascade reactions were described. In a two-phase reaction system, 44.8 ± 7.5 mM of 12-hydroxy dodecanoic acid (12-HDA) and 31.8 ± 1.7 mM of dodecanedioic acid (DDDA) were produced after transporter engineering, promoter engineering and introduction of novel alkane monooxygenase. For the construction of multi-enzymatic cascade reaction system, three cell-based modules, including a hydroxylation module (cell-Hm), an amination module (cell-Am), and a reduction module (cell-Rm), were designed. 46.3 mM of 12-amino dodecanoic acid, 27 mM of 1,12-dodecanediol, and 21.5 mM of 1,12-diaminododecane were synthesized from 100 mM of DAME and 12-ADA could be isolated using tert-butyloxy carbonyl (Boc)-protection with isolation yield of 66.5%. To improve the hydroxylation activity of PpAlkB, an alkane monooxygenase from Pseudomonas pelgia, a random mutant library was constructed using error-prone PCR and screened for beneficial mutants with a high-throughput assay developed in this study. Expression of the antibiotic resistance gene was coupled with the activity of PpAlkB, of which allowed screening of the mutants in high-throughput manner and in vivo. After two sequential screening with dodecane and DAME as substrates, with a triple mutant, H292L/F253I/S313P, was identified. It produced 12.95 mM (11 mM of 12-HDA and 1.96 mM of DDDA) from 100 mM of DAME, which was 64% increase from 7.88mM by the wild-type. In conclusion, production of 12-HDA with AlkBGT system and biopolymer monomers with multi-enzymatic cascade reaction system were performed using E. coli cell-based modules. In addition, PpAlkB was engineered for enhanced activity toward DAME through the in vivo screening assay. This study presents a new avenue for biosynthesis of fatty acid derivatives in E. coli by utilizing alkB engineering and modularized cell reactions.Chapter 1. Introduction 1 1.1 Biosynthesis of ω-Hydroxy fatty acid for biopolymer monomer 2 1.1.1 Importance of ω-hydroxy fatty acids as a biopolymer monomer 2 1.1.2 Chemical and biological production of ω-hydroxy fatty acids 3 1.2 Non-heme di iron alkane monooxygeanse system (AlkBGT) 7 1.2.1 Structure of AlkB and reaction mechanism of AlkBGT system 9 1.2.2 Biosynthesis of ω-Hydroxy fatty acid with non-heme alkane monooxygenase 15 1.2.3 Previously reported mutant study of AlkB 18 1.3 The scope of thesis 20 Chapter 2. Production of 12-hydroxy dodecanoic acid using a signal peptide sequence-optimized transporter AlkL and a novel monooxygenase 23 2.1 Introduction 24 2.2 Materials and Methods 28 2.2.1 Bacterial strains and materials 28 2.2.2 DNA Manipulation Techniques 29 2.2.3 Identification of novel monooxygenase using bioinformatics tools 29 2.2.4 Cultivation and protein expression 30 2.2.5 Biotransformations with cell extracts 31 2.2.6 Screening of DAME uptake ability of cells through chimeric transporter 32 2.2.7 Whole-Cell Biotransformations 32 2.2.8 Anaylsis by gas chromatography 32 2.3 Results and Discussion 38 2.3.1 Construction of novel chimeric transporters 38 2.3.2 Promoter optimization for efficient expression of the enzymes 48 2.3.3 Identification of new alkane monooxygenase 56 2.3.4 Bioconversion of DAME to 12-HDA using a biphasic reaction system 63 2.4 Conclusion 74 Chapter 3. Multi-enzymatic cascade reactions with Escherichia coli-based modules for synthesizing various bioplastic monomers from fatty acid methyl esters 75 3.1 Introduction 76 3.2 Materials and Methods 83 3.2.1 Bacterial strains and materials. 83 3.2.2 Cultivation and protein expression 83 3.2.3 Concurrent reaction system in shake flask. 84 3.2.4 Carbon source optimization for 1,12-diol biosynthesis in shake flask. 85 3.2.5 Sequential reaction system in shake flask 86 3.2.6 Whole-cell biotransformation in the reactor with constant pH control 86 3.2.7 Analysis by gas chromatography 87 3.2.8 Purification of 12-ADA with Boc protection 89 3.3 Results and Discussion 91 3.3.1 Construction of cell-Hm, cell-Am, and cell-Rm modules for the multilayer enzyme cascade system 91 3.3.2 Biosynthesis of ω-AFA using cell-Hm and cell-Am cascade reaction system 97 3.3.3 Biosynthesis of α,ω-diol in cell-Hm and cell-Rm cascade reaction system 113 3.3.4 Biosynthesis of aliphatic amine using cell-Hm, cell-Rm, and cell-Am in cascade reaction system 123 3.3.5 Efficacy of one-pot reaction system in reactor with constant pH control 139 3.4 Conclusion 159 Chapter 4. Engineering of non-heme alkane monooxygenase with in vivo screening system 160 4.1 Introduction 161 4.2 Materials and Methods 167 4.2.1 Bacterial strains and materials 167 4.2.2 Construction of libraries 167 4.2.3 Growth rate assay in LB media 168 4.2.4 In vivo screening 169 4.2.5 Biotransformations with resting cell reaction 169 4.2.6 Analysis by gas chromatography 170 4.3 Results and Discussion 171 4.3.1 Construction of screening module and assistance module for library screening 171 4.3.2 Growth test of E. coli with screening module in LB media 176 4.3.3 In vivo screening with dodecane 182 4.3.4 Mutant evaluation with DAME as substrate 189 4.3.4 Identification of key residues of PpAlkB with single mutant evaluation 193 4.4 Conclusion 203 Chapter 5. Overall Conclusion and Further Suggestions 204 5.1 Overall conclusion: Biosynthesis of 12-hydroxy lauric acid and biopolymer monomers in Escherichia coli using alkane monooxygenase 205 5.2 Further suggestion: Saturation mutagenesis of PpAlkB 208 6. References 212 Appendix 232 Appendix I. Enzymatic Synthesis of ω-hydroxydodecanoic acid employing a Cytochrome P450 from Limnobacter sp. 105 MED 233 A1.1 Abstract 234 A1.2 Introduction 236 A1.3 Materials and Methods 241 A1.4 Results and Discussion 247 A1.5 Conclusion 272 A1.6 References 273 Abstract in Korean 293박