Chapter 2

Hygromycin A, an antibiotic produced by Streptomyces hygroscopicus, is an inhibitor of the bacterial ribosomal peptidyl transferase. The antibiotic binds to ribosome in a distinct but overlapping manner with other antibiotics, and offers a different template for generation of new agents effective against multidrug-resistant pathogens. Reported herein are the results from a series of stable isotope incorporation studies which have demonstrated the biosynthetic origins of the three distinct structural moieties which compromise hygromycin A. The 31.5 kb hygromycin A biosynthetic gene cluster has been identified, cloned and sequenced. It contains 29 genes whose predicted products can be ascribed roles in hygromycin A biosynthesis, regulation, and resistance. A series of gene deletion studies have been carried out, and provided both intermediates in the biosynthetic pathway and a new analog, 5"-dihydrohygromycin A. The convergent biosynthetic pathway established for hygromycin A offers significant versatility for applying the techniques of combinatorial biosynthesis and mutasynthesis to produce new antibiotics which target the ribosomal peptidyl transferase activity.