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.
Rdc2 is the first flavin-dependent halogenase identified from fungi. Based on the reported structure of the bacterial halogenase CmlS, we have built a homology model for Rdc2. The model suggests an open substrate binding site that is capable of binding the natural substrate, monocillin II, and possibly other molecules such as 4-hydroxyisoquinoline (1) and 6-hydroxyisoquinoline (2). In vitro and in vivo halogenation experiments confirmed that 1 and 2 can be halogenated at the position ortho to the hydroxyl group, leading to the synthesis of the chlorinated isoquinolines 1a and 2a, respectively, which further expands the spectrum of identified substrates of Rdc2. This work revealed that Rdc2 is a useful biocatalyst for the synthesis of various halogenated compounds.
Engineered production of fungal anticancer cyclooligomer depsipeptides in Saccharomyces cerevisiae, Metabolic Engineering, http://dx. doi.org/10. 1016/j.ymben.2013.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A comparative evaluation of various biomaterials for their resistance to bacterial colonization and encrustation in infected urine is an important area in urological biomaterials research. This article describes an in vitro dynamic perfusion system that allows four reactors containing 24 1-in. catheter samples (6 per reactor) to be simultaneously perfused at a constant flow rate by synthetic urine. A common urease-producing urinary pathogen, Proteus mirabilis, was maintained at a level of 10(6) colony-forming units/mL for 7 days in the dynamic perfusion reactors. The pH and bacterial population were monitored every 24 h and the percentage of encrustation on latex and hydrogel-coated commercial catheter materials gave reproducible results in three different runs, 15.2 +/- 3.65% and 13.8 +/- 2.58%, respectively. A major issue of inlet clogging due to ascending bacteria or ammonia has been rectified using a dismountable inlet assembly. An incubator coupled with a cooling system allowed accurate temperature maintenance of 37 degrees C in all four reactors. Results from scanning electron microscopy of some latex samples are also presented.
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