The wide-spread use of antibiotics has created tremendous evolutionary pressure on microbes to develop resistance. With the appearance of both methicillin resistant Staphylococcus aureus and vancomycin resistant Enterococci, there is a critical need for new antibiotics and/or analogues of existing antibiotics to overcome resistant strains. Many antibiotics have sugars essential for activity. Kahne and coworkers have shown that changing these sugars provide modified antibiotic activities. Chemical methods for glycosylation are inefficient and do not enable rapid access to derivatives due to protecting group manipulation, building block synthesis and purification of complex mixtures. Enzymatic approaches, in which known antibiotic glycosyltransferases (Gtfs) are used to make novel glycoconjugates, are efficient but limited in scope because most antibiotic Gtfs have restricted substrate selectivities. Evolved glycosyltransferases (Gtfs) could be made to put non-native carbohydrates on natural product aglycones and this strategy would benefit greatly by a facile screen or selection for antibiotic activity against pathogenic bacteria. This proposal uses two complementary phage-based strategies to evolve Gtfs for the production of novel antibiotics. The first strategy, termed a zone of clearing assay, screens genes encoded via lambda phage for their ability to produce novel antibiotics reported on a lawn of both Escherichia coli and a Gram-positive reporter strain. This strategy can be used to select for evolved Gtfs capable of utilizing substrates provided in the media to produce bioactive analogues of existing antibiotics. This strategy can also be used to screen environmental DNA libraries for the combinatorial biosynthesis of non-carbohdyrate containing antibiotic. The second strategy, M13-phage display, uses Gtfs displayed on the surface of M13 phage and selection of an active Gtf is accomplished by capturing of a biotinylated product attached to the phage surface. These two strategies provide complimentary options for producing novel bioactive compounds to help in the fight against bacterial resistance. ? ? ?
| Swoboda, Jonathan G; Campbell, Jennifer; Meredith, Timothy C et al. (2010) Wall teichoic acid function, biosynthesis, and inhibition. Chembiochem 11:35-45 |
| Lee, Kyungae; Campbell, Jennifer; Swoboda, Jonathan G et al. (2010) Development of improved inhibitors of wall teichoic acid biosynthesis with potent activity against Staphylococcus aureus. Bioorg Med Chem Lett 20:1767-70 |
