title: Efficient production of indigoidine in Escherichia coli.
abstract: Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. In our previous study, an indigoidine synthetase Sc-IndC and an associated helper protein Sc-IndB were identified from Streptomyces chromofuscus ATCC 49982 and successfully expressed in Escherichia coli BAP1 to produce the blue pigment at 3.93 g/l. To further improve the production of indigoidine, in this work, the direct biosynthetic precursor L-glutamine was fed into the fermentation broth of the engineered E. coli strain harboring Sc-IndC and Sc-IndB. The highest titer of indigoidine reached 8.81 ± 0.21 g/l at 1.46 g/l L-glutamine. Given the relatively high price of L-glutamine, a metabolic engineering technique was used to directly enhance the in situ supply of this precursor. A glutamine synthetase gene (glnA) was amplified from E. coli and co-expressed with Sc-indC and Sc-indB in E. coli BAP1, leading to the production of indigoidine at 5.75 ± 0.09 g/l. Because a nitrogen source is required for amino acid biosynthesis, we then tested the effect of different nitrogen-containing salts on the supply of L-glutamine and subsequent indigoidine production. Among the four tested salts including (NH4)2SO4, NH4Cl, (NH4)2HPO4 and KNO3, (NH4)2HPO4 showed the best effect on improving the titer of indigoidine. Different concentrations of (NH4)2HPO4 were added to the fermentation broths of E. coli BAP1/Sc-IndC+Sc-IndB+GlnA, and the titer reached the highest (7.08 ± 0.11 g/l) at 2.5 mM (NH4)2HPO4. This work provides two efficient methods for the production of this promising blue pigment in E. coli.
authors.affiliation: Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT, 84322-4105, USA.
authors.forename: Fuchao
authors.initials: F
authors.lastname: Xu
authors.forename: David
authors.initials: D
authors.lastname: Gage
authors.forename: Jixun
authors.initials: J
authors.lastname: Zhan
mesh_terms: D000643:Ammonium Chloride / Q000737:chemistry; D000645:Ammonium Sulfate / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D060847:Metabolic Engineering; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D009566:Nitrates / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D010881:Piperidones / Q000378:metabolism*; D017680:Potassium Compounds / Q000737:chemistry; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D009566:Nitrates; D010710:Phosphates; D010881:Piperidones; D017680:Potassium Compounds; C024788:ammonium phosphate; D000643:Ammonium Chloride; D005973:Glutamine; C446045:indigoidine; C104933:BaP1 metalloproteinase; D008666:Metalloendopeptidases; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase; C023844:potassium nitrate; D000645:Ammonium Sulfate