“Patent pending, temporarily not
authorized for public access”
摘　　要
丁酸和正丁醇具有許多重要的應用。前者用於製藥，化工，食品等行業，而後者則是在油墨，塑料和塗料行業的應用。特別是，丁酸可以作為前體的正丁醇，一個潛在的生物燃料的生產。相比，乙醇，正丁醇具有更高的能量密度，更低的蒸汽壓，和在水中的溶解度較低。可再生的生物燃料來源，最近由於化石燃料價格暴漲吸引了顯著關注人類社會，石油儲量的不斷枯竭，溫室氣體的高排放和石油供應的不穩定性。在生物技術領域，丁酸和正丁醇已被常規地由梭狀芽胞桿菌物種產生。然而，在生產中的丁酸和正丁醇的預先因為有限的遺傳工具，無法提供生理信息，以及複雜的發酵模式受阻梭菌。
大腸桿菌是一個行業友好的細菌，並有許多吸引人的特性，如在一個簡單的培養基中生長速度快，發達的遺傳工具，和已知的生理學。在這項研究中，丁酸和正丁醇生產平台在大腸桿菌中通過代謝工程開發的。它是已知的質粒是有問題的，因為它具有不穩定性，細胞 - 細胞變異和代謝負擔的問題。這個問題是由遺傳工具箱基因組工程的發展解決。該工具箱基本上包括attP位基於站點的整合載體和attB位點為基礎的模板載體，兩者都配有一個選擇標記由兩個突變型loxP（即LE*和RE*）兩側。與此工具箱，它包括正丁醇的合成途徑的異源基因導入大腸桿菌基因組中。因此，正丁醇的生產滴度都達到了3.7 g/L。這表明，我們的基因工程技術是可行和有效的大腸桿菌的代謝工程。
在此研究的第二部分，丁酸的合成途徑由基因工程技術重建於大腸桿菌。此外，除去依賴NADH的代謝途徑，以增加氧化還原當量和導致浪費的副產物不期望的信號通路被消除，以節約碳的可用性。其結果是，該重組大腸桿菌能產生丁酸鹽具有高滴度，生產率和選擇性。丁酸的生產滴度為10 g/L的同一個轉化率達到理論產率的85.5％。丁酸的選擇性，其定義為丁酸酯為乙酸鹽的重量比，最後達到143。
在這項研究中的第三部分，我們提出正丁醇的新的生產平台。此工作由丁酸的生物轉化，以正丁醇。內源性atoDA和clotridial adhEII被overexprssed在大腸桿菌。除去不希望的途徑之後，工程化的大腸桿菌菌株（該丁酸轉化菌株）能夠從補充丁酸生產正丁醇。正丁醇的生產滴度為6.9 g/L與理論產率的92％。最後，2大腸桿菌菌株，從第二部分的丁酸產生菌和丁酸轉化菌株，共培養在葡萄糖的存在沒有丁酸補充。這導致生產正丁醇5.5 g/L，佔理論產率的69％。
在這項研究中的最後一部分，提出了從葡萄糖在大腸桿菌中提高正丁醇的新戰略，實現了氧化還原平衡。從本研究的第一部分中的正丁醇生產菌株被用來達到這個目的。其結果是，正丁醇滴度達到6.2 g/L與理論產量為與其中心代謝被操縱的應變為76％。
總之，在本研究中我們提出的策略是有希望的。它可能在生物技術打開了新的途徑進行有效的生產丁酸和正丁醇的大腸桿菌。

键词: 大肠埃希氏菌，丁酸，正丁醇。

“Patent pending, temporarily not
authorized for public access”
Abstract
Butyric acid and n-butanol have many important applications. The former is used in the pharmaceutical, chemical and food industries while the latter is applied in the ink, plastic, and paint industries. In particular, butyric acid can serve as a precursor for the manufacture of n-butanol, a potential biofuel. As compared to ethanol, n-butanol has higher energy density, lower vapor pressure, and lower solubility in water. Renewable biofuel sources have recently attracted significant attention in human society due to the skyrocketing price of fossil fuels, continued depletion of oil reserves, high emission of greenhouse gas, and instability of oil supply. In the biotechnology field, butyric acid and n-butanol have been conventionally produced by Clostridium species. However, the advance in production of butyric acid and n-butanol is hampered in Clostridium because of limited genetic tools, unavailable physiological information, and the complicated fermentation pattern.
Escherichia coli is an industry-friendly bacterium and has many attractive traits such as fast growth in a simple medium, well-developed genetic tools, and known physiology. In this study, a production platform of butyric acid and n-butanol was developed in E. coli by metabolic engineering. It is known that plasmid is problematic because it has a problem of instability, cell-cell variation, and metabolic burden. This issue was addressed by development of a genetic toolbox for genomic engineering. The toolbox essentially includes the attP site-based integration vectors and the attB site-based template vectors, both equipped with a selective marker flanked by two mutant loxP sites (i.e., LE* and RE*). With this toolbox, heterologous genes comprising the synthetic pathway of n-butanol were introduced into E. coli genome. Consequently, the production titer of n-butanol reached 3.7 g/L. This indicates that our genomic engineering technique is feasible and effective for metabolic engineering of E. coli.
In the second part of this study, a synthetic pathway of butyric acid was reconstructed in E. coli by the genomic engineering technique. In addition, the NADH-dependent metabolic pathways were removed to increase availability of the redox equivalent and undesired pathways leading to waste byproducts were eliminated to conserve carbon. As a result, the recombinant E. coli was able to produce butyrate with high titer, productivity, and selectivity. The production titer of butyrate was 10 g/L with a conversion yield attaining 85.5 % of theoretical yield. The butyrate selectivity, defined as the weight ratio of butyrate to acetate, finally reached 143.
In the third part of this study, we proposed a new production platform of n-butanol. This was carried out by bioconversion of butyrate to n-butanol. The endogenous atoDA and clotridial adhEII were overexprssed in E. coli. After removal of undesired pathways, the engineered E. coli strain (the butyrate-conversion strain) was able to produce n-butanol from supplemented butyrate. The production titer of n-butanol was 6.9 g/L with 92 % of the theoretical yield. Finally, two E. coli strains, the butyrate-producing strain from the second part and the butyrate-conversion strain, were co-cultured in the presence of glucose without butyrate supplementation. It led to the production of n-butanol of 5.5 g/L, accounting for 69 % of the theoretical yield.
In the final part of this study, a new strategy to achieve the redox balance was proposed to improve n-butanol from glucose in E. coli. The n-butanol producer strain from the first part of this study was utilized to achieve this goal. As a consequence, the n-butanol titer reached 6.2 g/L with 76 % of the theoretical yield for the strain with its central metabolism being manipulated.
In conclusion, our proposed strategies in this study are promising. It may open a new avenue in biotechnology for effective production of butyrate and n-butanol in E. coli.

Keywords: Escherichia coli, Butyric acid, n-butanol.

Contents
Chapter 1. Introduction……………………………………………………………...1
1.1. A brief account of biofuels…………………………………………1
1.2. Briefly introduction of butyric acid……………………………….2
1.3. History of A.B.E fermentation for n-butanol production………..3
1.4. Production of biobutanol by non-native host……………………..4
1.4.1. Production of n-butanol by E. coli………………………...4
1.4.2. Production of n-butanol by Lactobacillus brevis………….5
1.4.3. Production of n-butanol by Saccharomyces cerevisiae…...5
1.4.4. Production of n-butanol by Pseudomonas putida………...6
1.4.5. Production of n-butanol by Bacillus subtilis……………...7
1.5. References…………………………………………….......................8
Chapter 2. Genomic engineering of Escherichia coli by the phage attachment
site-based integration system with mutant loxP sites………………..22
2.1. Introduction………………………………………………………..23
2.2. Materials and methods……………………………………………25
2.2.1. Strains and culturing condition…………………………...25
2.2.2. Plasmid construction…………………………………….26
2.2.3. Cloning of heterologous genes……………………………26
2.2.4. Creation of the phage attB site……………………………28
2.2.5. Chromosomal integration of heterologous genes………..29
2.2.6. Marker and replicon rescue…….………………………30
2.2.7. Analytical methods………….……………………………..30
2.3. Results and discussion………………………………………….....31
2.3.1. Insertion of genes by integration vectors………………...31
2.3.2. Genomic insertion of multiple genes…………………….32
2.3.3. Creation of additional attB sites for gene insertion……33
2.3.4. n-Butanol production by the engineered strain………….35
2.4. Conclusions………………………………………………………..36
2.5. References…………………………………………………………38
Chapter 3. Metabolic Engineering of Escherichia coli for Production of Butyric
Acid……………………………………………………………………..57
3.1. Introduction……………………………………………………….58
3.2. Materials and methods……………………………………………60
3.2.1 Gene Deletion and Insertion……………………………….60
3.2.2 Enhanced Expression of Endogenous Genes……………..61
3.2.3 Bacterial Culturing………………………………………....62
3.2.4 Analytical Methods…………………………………………62
3.3. Result……………………………………………………………..63
3.3.1 Reconstruction of the Butyrate-Synthesis Pathway……...63
3.3.2 Production of Butyrate in Engineered E. coli…………….64
3.3.3 Effect of Acetate on Butyrate Production………………...64
3.3.4 Butyrate Production by Glucose Feeding………………...65
3.4. Discussion………………………………………………………….66
3.5. References…………………………………………………………70
Chapter 4. Potential production platform of n-butanol in Escherichia
coli……………………………………………………………………84
4.1. Introduction……………………………………………………….85
4.2. Materials and methods……………………………………………86
4.2.1 Bacterial culturing………………………………………….86
4.2.2 Gene manipulation and Strain construction……………...87
4.2.3 Analytical method…………………………………………..88
4.3. Results.……………………………………………………………..88
4.3.1 Production of n-butanol from butyrate…………………...88
4.3.2Effect of butyrate supplementation on n-butanol production
…………...………………………………………………...90
4.3.3 n-Butanol production by glucose and butyrate feeding….90
4.3.4 n-Butanol production by bacterial co-culturing………….91
4.4. Conclusions………………………………………………………..93
4.5. References…………………………………………………………95
Chapter 5. Genomic Engineering of Escherachia coli for production of
n-Butanol.............................................................................................112
5.1. Introduction……………………………………………………..113
5.2. Materials and methods………………………………………….114
5.2.1 Bacterial culturing ……………………………………….114
5.2.2 Plasmid construction …………………………………….114
5.2.3 Genomic insertion and gene deletion……………………117
5.2.4 Analytical method………………………………………...118
5.2.5 Enzyme activity assay……………………………………119
5.3. Results……………………………………………………………120
5.3.1 n-Butanol production in recombinant E.coli………….120
5.3.2 Increase NADH by pyruvate dehydrogenase……..…..121
5.3.3 Increase NADH via pentose phosphate pathway….…..122
5.4. Discussion………………………………………………………..123
5.5. References……………………………………………………….125
Chapter 6. CONCLUDING REMARKS……………………………………...140
6.1. Summary of our study………………………………………...140
6.2. Future perspectives……………………………………………141





LIST OF FIGURES
Figure 1.1: The synthetic pathway of n-butanol in Clostridium………………….17
Figure 1.2: The production of n-butanol via threonine biosynthetic pathway in E.
coli………………………………………………………………………18
Figure 1.3: The biosynthetic pathway of n-butanol in E.coli…………………19 Figure 1.4: Schematic of biosynthesis of n-butanol via β-oxidation cycle in
E.coli…………………………………………………………………..20
Figure 1.5: The biosynthetic pathway of n-butanol in Lactobacillus brevis……..21
Figure 2.1: Schematic illustration of the procedure for genomic integration of
passenger genes and creation of attB sites………………………………
Figure 2.1: (A) Physical maps of integration vectors and attB-template vector...46
Figure 2.1: (B) Genomic insertion of passenger genes using integration vectors.47
Figure 2.1: (C) The general protocol for creation of attB sites…………………...48
Figure 2.1: (D) Genomic insertion of passenger genes using integration vectors at
new created attB site………………………………………………49
Figure 2.2: The biosynthetic pathway of PHB and n-butanol in E. coli…………51
Figure 2.3: Identification of PHB production in strains by Nile red…………….52
Figure 2.4: Analysis of the inserted DNA by agarose gel electrophoresis………….
Figure 2.4: (A) The inserted PλPL–ter gene of integrants was verified………….53
Figure 2.4: (B) The inserted ɸ80attB-LE*-gen-RE* cassette at the adhE gene of
integrants was verified……………………………………………53
Figure 2.5: Fermentation profiles of recombinant E. coli strains…………………..
Figure 2.5: (A) Growth curve. Cell biomass in terms of g DCW per L was
recorded………………………………………………………….55
Figure 2.5: (B) Fermentation profile curve………………………………………..55
Figure 2.5: (C) Production of organic compounds………………………………..56
Figure 3.1: The metabolic pathway leading to butyrate from glucose in E. coli…
Figure 3.1: (A) The intercellular acetate system…………………………………..78
Figure 3.1: (B) The extracellular acetate system………………………………….78
Figure 3.2: Production of butyrate in the engineered E. coli strains…………….80
Figure 3.3: Effect of acetate on the butyrate production in the engineered E. coli
strain……………………………………………………………………81
Figure 3.4: Time course production of butyrate in E. coli by glucose feeding…….
Figure 3.4: (A) Extra feeding of 6 g/L glucose…………………………………….82
Figure 3.4: (B) Extra feeding of 8 g/L glucose…………………………………….82
Figure 3.4: (C) Extra feeding of 10 g/L glucose…………………………………83
Figure 4.1: The synthetic pathway of n-butanol in E. coli………………………102
Figure 4.1: (A) The synthetic pathway of n-butanol in strain BuT-3E………102
Figure 4.1: (B) The synthetic pathway of n-butanol in strain BuT-3EA……102
Figure 4.2: Production of n-butanol in strain BuT-3E………………………….103
Figure 4.3: Effect of supplemented butyrate on the n-butanol production in
engineered E. coli……………………………………………………….
Figure 4.3: (A) The fermentation production for strain BuT-3E………………105
Figure 4.3: (B) Glucose consumption and biomass for strain BuT-3E for strain
BuT-3E ………………………………………………………….105
Figure 4.3: (C) The fermentation profile for strain BuT-3EA………………….106
Figure 4.4: Effect of substrate feeding on the n-butanol production in engineered
E. coli……………………………………………………………………
Figure 4.4: (A) The fermentation production profile……………………………107
Figure 4.4: (B) Glucose and butyrate consumption profile……………………..107
Figure 4.5: The schematic illustration of coupled metabolic pathways for two
engineered E. coli strains…………………………………………109
Figure 4.6: (A) Strains BuT-3E and BuT-8L-ato with various E/L ratios were
grown in shake flasks……………………………………………110
Figure 4.6: (B) Similar experiment was conducted with the E/L ratio at 1:2. After
fermentation for 16 h, strain BuT-3E accounting for OD550 of 2 in
the culture volume was additionally added to the cell
culture……………………………………………………………..110
Figure 5.1: The metabolic pathway leading to n-butanol from glucose in E. coli
………………………………………………………………………134
Figure 5.2: Production of n-butanol in engineered E. coli………………………135
Figure 5.3: The effect of PDH complex on the n-butanol production…………136
Figure 5.4: (A) Production of n-butanol in strain But-10……………………….137
Figure 5.4: (B) Production of n-butanol in strain But-12……………………….137
Figure 5.5: Production of n-butanol in pgi-null strain…………………………..139


LIST OF TABLES
Table 2.1: Strains, plasmids and primers list used………………………………..43
Table 3.1: Strains, plasmids and primers list used………………………………..75
Table 4.1: Strains, plasmids and primers list used………………………………..99
Table 5.1: Strains, plasmids and primers list used………………………………129
Table 5.2: Summarized productivity, theortical yield, intracellular NADH level
and enzyme activity. …………………………………………………..133
Appendix A………………………………………………………………………...142
Appendix B…………………………………………………………………………143

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