This application proposes to investigate the genetic and biochemical basis of production of the antitubercular antibiotic, rifamycin (RIF), in Amycolatopsis mediterranei. as a prelude to determining whether rifamycin analogs can be produced by modification of the production genes. The long-range goals are first to develop a way to clone and express the rifamycin production genes in another actinomycete host and then to explore ways to manufacture new structural congeners of rifamycin by genetic manipulation of the biosynthetic, regulatory and resistance genes. The outcome of this research eventually could provide antitubercular and anti-HIV drugs active against rifamycin-resistant organisms, which are a rapidly worsening problem in human medicine. The overall goal during the first grant period is to clone and define the biosynthetic gene cluster encoding the formation of rifamycin so that the following questions can be explored: What is the relationship of the genes responsible for providing the unique polyketide starter unit to the genes encoding the polyketide synthase (PKS) complex, what is their relationship to the genes conferring resistance to rifamycin upon the producing organism, and can modification of these genes produce novel forms of rifamycin with activity against rifamycin-resistant organisms? To achieve this goal an integrated effort of two laboratories, located at Stanford University and the University of Wisconsin-Madison, working in collaboration with a third group at the University of Washington, will focus on the following tasks. (l) To use the 3-amino-5-hydroxybenzoic acid synthase gene already cloned from A. mediterranei as a starting point to identify by shotgun sequencing potentially relevant genes in the adjacent regions of DNA. (2) To probe an available set of cosmid clones containing the above DNA with consensus probes from the erythromycin PKS genes to identify and map the genes encoding the rifamycin PKS. (3) To probe the same set of cosmid clones with a consensus probe for the RNA polymerase Beta-subunit gene to determine whether such a gene is part of the biosynthetic gene cluster. (4) To characterize the cloned genes by sequence analysis to a level allowing the assignment of probable functions of each open reading frame. (5) To demonstrate functions of the cloned genes by expression in a heterologous host followed by screening for a resistance phenotype or formation of a partial or complete polyketide structure. (6) To express mutated rif PKS genes in a heterologous host to determine if this approach can led to the production of modified forms of rifamycin. (7) To search for alternative rifamycin resistance determinants, other than the one involving modification of RNA polymerase subunit Beta, and to characterize these at the genetic level.
