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.

1. Clark, R.J., et al., Indigo, woad, and Tyrian Purple: important vat dyes from antiquity to the present. 1993. 17(4): p. 191-199.
2. Beeson, K.H., Indigo Production in the Eighteenth Century. The Hispanic American Historical Review, 1964. 44(2): p. 214-218.
3. Gillam, E.M.J., et al., Oxidation of Indole by Cytochrome P450 Enzymes. Biochemistry, 2000. 39(45): p. 13817-13824.
4. McKee, J.R. and M.J.J.o.C.E. Zanger, A microscale synthesis of indigo: Vat dyeing. 1991. 68(10): p. A242.
5. Ghaly R Ananthashankar, A.E., Production, Characterization and Treatment of Textile Effluents: A Critical Review. Vol. 05. 2013.
6. Chemists go green to make better blue jeans. 2018 [cited 2018 September 25]; Available from: https://www.nature.com/articles/d41586-018-00103-8.
7. Han, G.H., et al., Enhanced indirubin production in recombinant Escherichia coli harboring a flavin-containing monooxygenase gene by cysteine supplementation. J Biotechnol, 2012. 164(2): p. 179-87.
8. Bla, et al., Indirubin and Indirubin Derivatives for Counteracting Proliferative Diseases %J Evidence-Based Complementary and Alternative Medicine. 2015. 2015: p. 12.
9. Hoessel, R., et al., Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases. Nature Cell Biology, 1999. 1: p. 60.
10. Zhang, X., et al., Production of indirubin from tryptophan by recombinant Escherichia coli containing naphthalene dioxygenase genes from Comamonas sp. MQ. Appl Biochem Biotechnol, 2014. 172(6): p. 3194-206.
11. Seldes, A.M., et al., Blue pigments in South American painting (1610-1780). 1999: p. 100-123.
12. Warzecha, H., et al., Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures. Plant Biotechnol J, 2007. 5(1): p. 185-91.
13. Lim, H.K., et al., Characterization of a forest soil metagenome clone that confers indirubin and indigo production on Escherichia coli. Appl Environ Microbiol, 2005. 71(12): p. 7768-77.
14. Choi, H.S., et al., A novel flavin-containing monooxygenase from Methylophaga sp strain SK1 and its indigo synthesis in Escherichia coli. Biochem Biophys Res Commun, 2003. 306(4): p. 930-6.
15. Newton, W.A. and E.E. Snell, Formation and Interrelationships of Tryptophanase and Tryptophan Synthetases in Escherichia Coli. J Bacteriol, 1965. 89: p. 355-64.
16. Maugard, T., et al., Beta-glucosidase-catalyzed hydrolysis of indican from leaves of Polygonum tinctorium. Biotechnol Prog, 2002. 18(5): p. 1104-8.
17. Berry, A., et al., Application of metabolic engineering to improve both the production and use of biotech indigo. J Ind Microbiol Biotechnol, 2002. 28(3): p. 127-33.
18. Meyer, A., et al., Hydroxylation of indole by laboratory-evolved 2-hydroxybiphenyl 3-monooxygenase. J Biol Chem, 2002. 277(37): p. 34161-7.
19. Han, X., W. Wang, and X. Xiao, [Microbial biosynthesis and biotransformation of indigo and indigo-like pigments]. Sheng Wu Gong Cheng Xue Bao, 2008. 24(6): p. 921-6.
20. Guengerich, F.P., Cytochrome p450 enzymes in the generation of commercial products. Nature Reviews Drug Discovery, 2002. 1: p. 359.
21. Lu, Y. and L. Mei, Co-expression of P450 BM3 and glucose dehydrogenase by recombinant Escherichia coli and its application in an NADPH-dependent indigo production system. J Ind Microbiol Biotechnol, 2007. 34(3): p. 247-53.
22. Coon, M.J., Multiple oxidants and multiple mechanisms in cytochrome P450 catalysis. Biochem Biophys Res Commun, 2003. 312(1): p. 163-8.
23. Cashman, J.R., Some distinctions between flavin-containing and cytochrome P450 monooxygenases. Biochemical and Biophysical Research Communications, 2005. 338(1): p. 599-604.
24. van Berkel, W.J.H., N.M. Kamerbeek, and M.W. Fraaije, Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts. Journal of Biotechnology, 2006. 124(4): p. 670-689.
25. Ameria, S.P., et al., Characterization of a flavin-containing monooxygenase from Corynebacterium glutamicum and its application to production of indigo and indirubin. Biotechnol Lett, 2015. 37(8): p. 1637-44.
26. Han, G.H., et al., Optimization of bio-indigo production by recombinant E. coli harboring fmo gene. 2008. 42(7): p. 617-623.
27. Alper, H. and G. Stephanopoulos, Global transcription machinery engineering: a new approach for improving cellular phenotype. Metab Eng, 2007. 9(3): p. 258-67.
28. Burgess, R.R. and L. Anthony, How sigma docks to RNA polymerase and what sigma does. Curr Opin Microbiol, 2001. 4(2): p. 126-31.
29. Yu, H., et al., A high-throughput screen for hyaluronic acid accumulation in recombinant Escherichia coli transformed by libraries of engineered sigma factors. Biotechnol Bioeng, 2008. 101(4): p. 788-96.
30. Kitagawa, M., et al., Complete set of ORF clones of Escherichia coli ASKA library (A Complete S et of E. coli K-12 ORF A rchive): Unique Resources for Biological Research. 2005. 12(5): p. 291-299.
31. Riley, M., et al., Escherichia coli K-12: a cooperatively developed annotation snapshot--2005. Nucleic Acids Res, 2006. 34(1): p. 1-9.
32. Rajagopala, S.V., et al., The Escherichia coli K-12 ORFeome: a resource for comparative molecular microbiology. BMC Genomics, 2010. 11: p. 470.
33. Reyes, L.H., M.P. Almario, and K.C. Kao, Genomic Library Screens for Genes Involved in n-Butanol Tolerance in Escherichia coli. Plos One, 2011. 6(3).
34. Li, H.M., J. Chen, and Y.H. Li, Enhanced activity of yqhD oxidoreductase in synthesis of 1,3-propanediol by error-prone PCR. Progress in Natural Science-Materials International, 2008. 18(12): p. 1519-1524.
35. Rui, L., K.F. Reardon, and T.K. Wood, Protein engineering of toluene ortho-monooxygenase of Burkholderia cepacia G4 for regiospecific hydroxylation of indole to form various indigoid compounds. Appl Microbiol Biotechnol, 2005. 66(4): p. 422-9.
36. Gibson, D.G., et al., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods, 2009. 6(5): p. 343-5.
37. Datsenko, K.A. and B.L. Wanner, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A., 2000. 97(12): p. 6640-5.
38. Lessard, J.C., Chapter Eleven - Growth Media for E. coli, in Methods in Enzymology, J. Lorsch, Editor. 2013, Academic Press. p. 181-189.
39. Austin, S. and K. Nordstrom, Partition-mediated incompatibility of bacterial plasmids. Cell, 1990. 60(3): p. 351-4.
40. Velappan, N., et al., Plasmid incompatibility: more compatible than previously thought? Protein Engineering, Design and Selection, 2007. 20(7): p. 309-313.
41. Keseler, I.M., et al., The EcoCyc database: reflecting new knowledge about Escherichia coli K-12. Nucleic Acids Research, 2017. 45(D1): p. D543-D550.
42. Sezonov, G., D. Joseleau-Petit, and R. D'Ari, Escherichia coli physiology in Luria-Bertani broth. Journal of Bacteriology, 2007. 189(23): p. 8746-8749.
43. Favrot, L., J.S. Blanchard, and O. Vergnolle, Bacterial GCN5-Related N-Acetyltransferases: From Resistance to Regulation. Biochemistry, 2016. 55(7): p. 989-1002.
44. Venkat, S., et al., Characterizing lysine acetylation of Escherichia coli type II citrate synthase. Febs Journal, 2019. 286(14): p. 2799-2808.
45. Sargent, F., et al., Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit - Identification of a soluble precursor of the small subunit in a hypB mutant. European Journal of Biochemistry, 1998. 255(3): p. 746-754.
46. Dubini, A., et al., How bacteria get energy from hydrogen: a genetic analysis of periplasmic hydrogen oxidation in Escherichia coli. International Journal of Hydrogen Energy, 2002. 27(11-12): p. 1413-1420.
47. Bagramyan, K. and A. Trchounian, Structural and functional features of formate hydrogen lyase, an enzyme of mixed-acid fermentation from Escherichia coli. Biochemistry-Moscow, 2003. 68(11): p. 1159-1170.
48. Szymanski, M.R., M.J. Jezewska, and W. Bujalowski, Interactions of the Escherichia coli Primosomal PriB Protein with the Single-stranded DNA. Stoichiometries, Intrinsic Affinities, Cooperativities, and Base Specificities. Journal of Molecular Biology, 2010. 398(1): p. 8-25.
49. Koo, J.T., J. Choe, and S.L. Moseley, HrpA, a DEAH-box RNA helicase, is involved in mRNA processing of a fimbrial operon in Escherichia coli. Molecular Microbiology, 2004. 52(6): p. 1813-1826.
50. Hacker, J., Role of Fimbrial Adhesins in the Pathogenesis of Escherichia-Coli Infections. Canadian Journal of Microbiology, 1992. 38(7): p. 720-727.
51. Stohl, E.A. and H.S. Seifert, The recX gene potentiates homologous recombination in Neisseria gonorrhoeae. Molecular Microbiology, 2001. 40(6): p. 1301-1310.
52. Kawamura-Sato, K., et al., Role of multiple efflux pumps in Escherichia coli in indole expulsion. Fems Microbiology Letters, 1999. 179(2): p. 345-352.
53. Dombroski, A.J., et al., Polypeptides containing highly conserved regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA. Cell, 1992. 70(3): p. 501-12.