August 2012 Ph.D. Dissertation Production of indigo and indirubin using recombinant Escherichia coli harboring a novel flavin-containing monooxygenase gene Graduate School of Chosun University Department of Environmental Engineering Gui Hwan Han Production of indigo and indirubin using recombinant Escherichia coli harboring a novel flavin-containing monooxygenase gene Methylophaga aminisulfidivorans MPT 유래 flavin-containing monooxygenase 유전자를 내포시킨 재조합 대장균을 이용한 인디고 및 인디루빈의 생산 August 24 2012 Graduate School of Chosun University Department of Environmental Engineering Gui Hwan Han Production of indigo and indirubin using recombinant Escherichia coli harboring a novel flavin-containing monooxygenase gene Supervisor : Prof. Si Wouk Kim A dissertation submitted to the Department of Environmental Engineering and the Graduate School of Chosun University in fulfillment of the requirements for the degree of Doctor of Philosophy April 2012 Graduate School of Chosun University Department of Environmental Engineering Gui Hwan Han This certifies that the degree of Doctor of Philosophy of Gui Hwan Han is approved by Heon Man Lim Chair of committee Il Soo Bang Committee member Hyung Joon Cha Committee member Jintae Lee Committee member Si Wouk Kim Committee member June 2012 Graduate School of Chosun University Contents Contents ······································································································································ i List of tables ···························································································································· v List of figures ························································································································· vi Abatract ····································································································································· x Chapter I. General introduction ······························································································ 1 1. Dyes and pigments ········································································································· 2 2. Synthetic dyes ·················································································································· 2 3. Natural dyes ····················································································································· 3 4. Indigo and indirubin ······································································································· 4 4-1. Plant indigo ················································································································ 4 4-2. Chemical indigo ········································································································· 6 4-3. Biological indigo ········································································································ 8 4-4. Indirubin ······················································································································ 9 5. A novel bacterial flavin-containing monooxygenase gene from Methylophaga aminisulfidivorans MPT ································································ 11 6. Research objective ··········································································································· 14 Chapter II. Analysis of promoter region of flavin-containing monooxygenase gene (fmo) from M. aminisulfidivorans MPT ····································································· 16 1. Introduction ······················································································································ 17 2. Materials and methods ·································································································· 19 2-1. Strains, media and chemicals ················································································· 19 2-2. Cloning of different upstream length of fmo gene ············································· 19 2-3. 5’-rapid amplification of cDNA ends (5’-RACE) ················································ 21 2-4. Primer extension analysis ························································································ 22 2-5. Western blot analysis ······························································································ 23 - i - 3. Results and discussion ···································································································· 24 3-1. Identification of transcription start site of fmo ORF ·········································· 24 3-2. Relationship between fmo transcripts and indigo production ····························· 29 Chapter III. Optimization of indigo production using recombinant E. coli harboring fmo gene ·························································································· 30 1. Introduction ······················································································································ 31 2. Materials and methods ···································································································· 34 2-1. Materials ···················································································································· 34 2-2. Strains, plasmids, media and fermenters ······························································· 34 2-3. General DNA manipulation ···················································································· 34 2-4. Deletion of plasmid pBlue 2.0 ·············································································· 35 2-5. Selection of hosts for indigo production ······························································ 37 2-6. Statistical procedure ································································································· 37 2-7. Determination of indigo ·························································································· 39 3. Results ···························································································································· 40 3-1. Construction of recombinant plasmid pBlues ······················································· 40 3-2. Indigo production using different bacterial hosts harboring fmo gene ·············· 42 3-3. Set up of model equations ····················································································· 45 3-4. Optimization of medium composition ··································································· 48 3-5. Optimization of temperature and pH ····································································· 48 3-6. Indigo production under optimized conditions ····················································· 52 4. Discussion ······················································································································· 54 - ii - Chapter IV. Mass production of indigo in two types of fermentation systems using recombinant E. coli harboring fmo gene ······················································· 56 1. Introduction ······················································································································ 57 2. Materials and methods ···································································································· 58 2-1. Strains, media and fermenters ················································································ 58 2-2. Batch and repeated-batch fermentation ·································································· 58 2-3. Continuous fermentation ························································································ 59 2-4. Analytical methods ··································································································· 59 3. Results ···························································································································· 61 3-1. Effect of aeration and agitation rate on the profiles of dissolved oxygen, cell growth and indigo production ················································································ 61 3-2. 3,000-L batch and repeated-batch fermentation ···················································· 66 3-3. Continuous fermentation ························································································ 69 3-4. Plasmid stability ······································································································· 72 4. Discussion ························································································································· 73 Chapter V. Production of indirubin using recombinant E. coli harboring fmo gene ···· 77 1. Introduction ······················································································································ 78 2. Materials and methods ···································································································· 81 2-1. Media and chemicals ····························································································· 81 2-2. Cloning, expression and purification of FMO ····················································· 81 2-3. Optimization of medium composition and culture conditions for indirubin production ················································································································· 82 2-4. Fermentation ·············································································································