靛藍是一種有機化合物，具有鮮明的藍色，而靛玉紅是深粉紅色，常用於傳統中藥已有數百年歷史。靛藍和靛玉紅之微生物合成可用帶加氧酶質體的大腸桿菌生產靛藍和靛玉紅。鑑於靛玉紅的醫學益處及更高之附加價值，許多生產目標是增加靛玉紅之合成比率。目前最大障礙是加氧酶區域選擇性，極大可能生產靛藍而不是靛玉紅。本論文目的是透過基因庫，檢驗對改變靛藍和靛玉紅產率或提高靛藍的生產之整體影響。在測試加氧酶和宿主大腸桿菌之不同組合後，產生靛藍和靛玉紅最高產量之菌株為XL-1 + NDO（PP）和XL-1 + FMO（CG）。本研究的下一部分是研究基因庫對靛藍和靛玉紅合成的影響。通過易出錯之PCR創建了一個突變rpoD基因庫，具有低、中和高之錯誤率。我們將ASKA基因庫和一個突變之rpoD基因庫轉化我們的重組菌株，並於瓊脂平板上篩選出比對照菌株變成越藍色或粉紅之菌株。篩選出的菌株未顯示出區域選擇性的變化，但我們能夠鑑定出幾隻菌株，它們比對照菌株產出更多之靛藍多達50％。在我們最後一次改變加氧酶區域選擇性之嘗試中，通過對谷氨酸棒桿菌的fmo基因進行了易出錯PCR隨機誘變。儘管沒有突變菌株顯示出區域選擇性之變化，但兩株菌株產出比對照組菌株高達兩倍之靛藍（分別為176.1 mg / l和218.7 mg / l對108.5 mg / l）。對這些突變菌株進行基因定序，以了解它們在大腸桿菌靛藍生物合成之影響。

Indigo is an organic compound with a vibrant blue color while indirubin is deep pink color used in traditional Chinese medicine for hundreds of years. The microbial synthesis of indigo and indirubin is possible with recombinant E. coli strains harboring an oxygenase plasmid to produce indigo and indirubin. Given the medical benefits and higher cost of indirubin, the goal of many is to increase indirubin synthesis. The roadblock that is met is the oxygenase’s regioselectivity heavily favors producing indigo instead of indirubin. The aim of this thesis is to examine the effect of global perturbation by gene library to alter the indigo and indirubin production ratio or improve indigo production. After testing different combinations of oxygenases and host E. coli, the highest indigo and indirubin producing strains were XL-1 + NDO(PP) and XL-1 + FMO(CG). The next part of this study is to examine the effects of a gene library on the synthesis of indigo and indirubin. A mutant rpoD library was created through error-prone PCR with low, medium and high error rates. We transformed the ASKA library and a mutant rpoD library into our recombinant strains and screened for colonies that turned more blue or pink compared to the control strain on agar plates. No strains showed evidence of regioselectivity change after screening but we were able to identify several strains that produced up to 50% more indigo compared to the control strain. In our last attempt to alter regioselectivity of the oxygenase, random mutagenesis through error-prone PCR was conducted on the fmo gene from Corynebacterium glutamicum. Although no mutant strains showed change in regioselectivity, two strains in particular were shown to produce up to two times more indigo compared to the control strain (176.1 mg/l and 218.7 mg/l versus 108.5 mg/l respectively). These mutant strains were sequenced to elucidate their role in improving indigo biosynthesis in E. coli.

Table of Contents
摘要 i
Abstract ii
Acknowledgements iii
I. Literature Review 1
1.1 Introduction to Indigo and Indirubin 1
1.2 Microbial biosynthesis of indigo and indirubin 2
1.3 Microbial oxygenases demonstrating biosynthesis of indigo and indirubin 3
1.3.1 Naphthalene dioxygenase 3
1.3.2 P450 monooxygenase 4
1.3.3 Flavin-containing monooxygenase 4
2.1 Effect of global perturbation by gene library on indigo and indirubin production 5
2.1.1 Sigma factor library 6
2.1.2 ASKA library 6
2.1.3 Random mutagenesis on enzyme to enhance activity or change regioselectivity 7
II. Motivation and Strategies 8
1. Changing the indigo and indirubin production ratio 8
2. Screening for best producer of indigo and indirubin 8
3. Effect of gene library on regioselectivity of oxygenase 8
4. Effect of gene library on indigo and indirubin production without exogenous tryptophan in medium 9
5. Error-prone PCR on oxygenase enzyme to screen for higher indigo or indirubin production 9
III. Materials and Methods 11
1. Chemicals and reagents 11
2. DNA manipulation 11
3. Mutant library construction by error-prone PCR 11
4. Culture medium and conditions 12
5. Screening for potential colonies of interest with ASKA library 14
6. Screening for potential colonies of interest with rpoD mutant gene libraries 15
7. Screening for potential colonies of interest with FMO(CG) mutant gene library 16
8. Determining number of colony forming units (CFU) for medium comparison test 16
9. Chemical analysis 17
10. Strains and primers table 17
IV. Results and Discussion 22
1. Screening for best combination of oxygenase and host E. coli 22
2. Confirming cysteine effect on indigo and indirubin production 24
3. Indigo and indirubin production comparison in different medium 25
4. Screening for single colonies of interest with ASKA library - part one 27
5. XL-1 + NDO(PP) production comparison of high vs medium copy plasmid 28
6.1 Screening for single colonies of interest with ASKA library - part two 29
6.2 Indigo and Indirubin production of strains with ASKA library in medium with/without tryptophan 31
6.3 Sequence results and confirmation of ASKA gene in recombinant E. coli strains 34
7.1 Screening for single colonies of interest with mutant sigma factor(rpoD*) library 36
7.2 Indigo and indirubin production of strains with mutant sigma factor (rpoD*) library in medium with/without tryptophan 38
8.1 Screening for single colonies of interest with mutant FMO(CG)* library 40
8.2 Indigo and indirubin production with mutant FMO(CG)* library 41
V. Conclusion 42
1. Bioprospecting best indigo and indirubin producing strain 42
2. ASKA and sigma factor library to alter regioselectivity or improve oxygenase 42
3. Random mutagenesis on oxygenase to alter regioselectivity or improve efficiency 43
VI. References 44

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