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研究生： 李俐慧
Ramli, Theresia Cecylia
論文名稱： E.coli 轉化合成羥基十二烷酸甲酯
SYNTHESIS OF 12-HYDROXY DODECANOIC ACID METHYL ESTER THROUGH WHOLE-CELL BIOTRANSFORMATION IN ESCHERICHIA COLI
指導教授： 胡育誠
HU, YU-CHEN
口試委員: 沈若樸
SHEN, CLAIRE-ROAPU
宋立瑜
SUNG, LI-YU
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2020
畢業學年度： 108
語文別： 英文
論文頁數： 43
中文關鍵詞： CRISPR Cas9 、羟基十二烷酸 、大肠杆菌
外文關鍵詞： CRISPR Cas9, DAME, HDAME
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    • 末端氧化的12-羟基脂肪酸（w-OHFAs），例如12-羟基十二酸（w-OHC12），是尼龙12（聚酰胺）的重要组成部分，它是通用的，具有高耐久性和优异的耐磨性。当前，生产w-OHC12的常规方法不仅需要数步艰巨的步骤，而且价格昂贵，并且效率低且选择性差。全细胞生物转化可以替代传统方法，因为该方法可以直接氧化十二烷酸（C12脂肪酸）以高选择性生产w-OHC12。由于十二烷酸已知对微生物有毒，因此将其酯形式即十二烷酸甲酯用作底物。因此，产物w-OHC12也呈酯形式，即12-羟基十二烷酸甲酯（HDAME）。用大肠杆菌中表达的水生Marinobacter quaeolei VT8的细菌酶CYP153A进行生物转化。我们还尝试首先提高转化率，首先使该酶（A231G）发生突变，使其更适合C12转化，其次将其与来自巨大芽孢杆菌的P450 BM3的还原酶结构域融合。这种结合产生了0.48g / L的HDAME。为了在没有选择标记的情况下在大肠杆菌中进一步表达CYP153A，我们使用CRISPR Cas9系统将CYP153A基因敲入大肠杆菌基因组。

    Terminally oxidized 12-hydroxy fatty acids (w-OHFAs), such as 12-hydroxydodecanoic acid (w-OHC12), are important building block of nylon 12 (polyamide) which is versatile, has a high durability, and excellent abrasion resistance. Currently , conventional way to produce w-OHC12 not only require several arduous steps, but also expensive and suffer from low efficiencies and poor selectivity. Whole cell biotransformation serves as a promising alternative to the conventional method as this method can directly oxidize dodecanoic acid (C12 fatty acid) to produce w-OHC12 with high selectivity. As dodecanoic acid is known to be toxic to microbes, its ester form which is, dodecanoic acid methyl ester is used as the substrate. Therefore the product, w-OHC12 is also in ester form , 12-hydroxydodecanoic acid methyl ester (HDAME). The biotransformation is performed with bacterial enzyme CYP153A from Marinobacter aquaeolei VT8 expressed in Escherichia coli. We also attempted to increase the conversion b y first, mutating this enzyme (A231G) to make it more suitable for C12 conversion and second, fusing it with the reductase domain of P450 BM3 from Bacillus megaterium. This combination resulted in the production of 0.48 g/L HDAME. To further express CYP153A in E.coli without selection marker, we used CRISPR Cas9 system to knock in CYP153A gene to E.coli genome.

    摘要 i ABSTRACT ii TABLE OF CONTENT iii LIST OF FIGURES v LIST OF TABLES vi CHAPTER 1: INTRODUCTION 1 CHAPTER 2: LITERATURE REVIEW 3 2.1. Substrate 3 2.2. Whole-Cell Biotransformation 4 2.2.1. Microorganism 4 2.2.2. Enzymes 5 2.2.3. Regulator protein 6 2.2.4. Transport protein 7 2.3. CRISPR/Cas system 7 2.3.1. Class 2 Type II CRISPR 8 CHAPTER 3: EXPERIMENTAL DESIGN 15 3.1. Chemical and materials 15 3.2. E.coli growth test in dodecanoic acid (DA) and dodecanoic acid methyl ester (DAME) 15 3.3. Construction of plasmid expressing CYP153A and its variation 15 3.4. Determination of CYP153A expression with SDS-PAGE 17 3.5. Determination of CYP153A expression using CO spectral assay 17 3.6. Shake flask biotransformation 17 3.7. Analysis 18 3.8. Construction of CRISPR system to induce DSB in E.coli 19 3.9. Integration of selected enzymes to genomic DNA with CRISPR 20 3.10. Curing Tc resistant gene from E.coli genome 20 3.11. Determination of alkL expression using real time polymerase chain reaction (RT-qPCR) 20 3.12. Two liquid phase biotransformation 21 CHAPTER 4: EXPERIMENTAL RESULT 22 4.1. E.coli W3110 growth rate in the presence of dodecanoic acid and dodecanoic acid methyl ester 22 4.2. Expression of CYP153A gene in E.coli W3110 22 4.3. Biotransformation with E.coli W3110 23 4.4. Integration of CYP153A cassette into E.coli W3110 genome with CRISPR Cas9 23 4.5. Using E.coli MG1655 as host to replace E.coli W3110 24 4.6. Integration of CYP153A cassette into E.coli MG1655 24 4.7. Expression of alkL and CYP153A in E.coli MG1655#1 24 CHAPTER 5: DISCUSSION 32 CHAPTER 6: FUTURE WORK 34 6.1. Expression of CYP153A in E.coli 34 6.2. Beta-oxidation cycle 34 6.3. Two Liquid Phase biotransformation 35 REFERENCES 39

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