title: Enhanced indirubin production in recombinant Escherichia coli harboring a flavin-containing monooxygenase gene by cysteine supplementation.
abstract: In our previous study, a batch fermentation of recombinant Escherichia coli DH5α cells harboring the fmo gene from Methylophaga aminisulfidivorans MP(T) produced indirubin (5.0mg/L) and indigo (920mg/L) in a 5L fermenter containing tryptophan medium (2g/L tryptophan, 5g/L yeast extract, 10g/L NaCl). In this study, it was found that indirubin production greatly increased when 0.36g/L cysteine was added to the tryptophan medium, although cysteine inhibited the growth of the recombinant E. coli harboring the fmo gene. However, the addition of cysteine did not inhibit the expression level and activity of FMO in the cell. Indigo was synthesized by the dimerization of two 3-hydroxyindole molecules under the non-enzymatic reaction. Cysteine influenced the regioselectivity of FMO and enhanced the synthesis of 2-hydroxyindole instead of 3-hydroxyindole, which might function to increase indirubin production. The optimal culture conditions for indirubin production in tryptophan medium were determined from the response surface methodology analysis: 2g/L tryptophan, 5g/L yeast extract, 10g/L NaCl, 0.36g/L (3mM) cysteine, pH 8.0 at 35°C. Under these conditions, the recombinant E. coli cells were capable of producing 223.6mg/L of indirubin from 2g/L of tryptophan. The intracellular accumulation of the indirubin crystals might stress the cell, which may be a main reason for the poor growth of the recombinant E. coli pBlue 1.7.
authors.affiliation: Department of Environmental Engineering, Chosun University, Gwangju, Republic of Korea.
authors.forename: Gui Hwan
authors.initials: GH
authors.lastname: Han
authors.forename: Geun Ho
authors.initials: GH
authors.lastname: Gim
authors.forename: Wonduck
authors.initials: W
authors.lastname: Kim
authors.forename: Sun Il
authors.initials: SI
authors.lastname: Seo
authors.forename: Si Wouk
authors.initials: SW
authors.lastname: Kim
mesh_terms: D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D007211:Indoles / Q000032:analysis / Q000378:metabolism; D053858:Metabolic Networks and Pathways / Q000187:drug effects / Q000502:physiology; D008854:Microscopy, Electron; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D019163:Reducing Agents / Q000494:pharmacology; D014364:Tryptophan / Q000378:metabolism
chemical_list: D007211:Indoles; D011994:Recombinant Proteins; D019163:Reducing Agents; D014364:Tryptophan; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D003545:Cysteine; D010100:Oxygen; C027185:indirubin