Abstract
This thesis is divided into six chapters. Chapter one provides both an introduction to the biosynthesis of polyketide metabolites, and includes a detailed review of the accumulated evidence for the formation of the 'tetracycline' antibiotics. Chapter two describes a study of various tetracycline-producing streptomycetes in submerged fermentation. The formation of the antibiotics in relation to various fermentation parameters has been explored, and methods for their determination studied, prior to an investigation of their biogenesis through the application of autoradiography. In chapter three, the development of a washed cell autoradiographic technique for the observation of de novo oxytetracycline biosynthesis is described. Incubation with radioaetively-labelled precursors led to the accumulation of numerous metabolites, in addition to the antibiotic. Two components derived from[1-[14]C] acetate were identified as the amino acids, aspartate and glutamate. Qualitatively similar autoradiograms were obtained from other tetracycline-producing organisms. Chapter four describes the extension of the autoradiographic survey to include non-tetracycline elaborating streptomycetes, in an effort to evaluate the usefulness of the technique as a taxonomic probe for common metabolites. In strains preferentially forming polyketide secondary metabolites, glutamate was identified as a major acetate-derived component. However, three reported beta-lactam producing streptomycetes did not show this trait. Chapter five describes a [13]C nmr study of the origins of the tetracene nucleus of oxytetracycline. Feeding studies with[1-[13]C]- and [1,2-[13]C[2]]acetate, have confirmed an exclusive polyketide origin of the tetracyclic carbon skeleton, and suggested the mode of cyclisation of an hypothetical linear polyketide intermediate. Subsequent incorporation of[1,2,3-[13]C[3]]malonate has confirmed the existence of a mechanism for the incorporation of an intact malonate primer unit (carbons 1,2 and the carboxamide) into oxytetracycline. Feeding studies with [1-[13]C,18O]acetate have shown the acetogenic origins of the oxygen substituents at carbons 1,3,10,11,and 12 of oxytetracycline, whilst incorporation of 13 [1-[13]C,2-CD[3]]acetate has implied retention of detectable levels of deuterium at carbons 7 and 9 only. The experimental section may be found in chapter six, whilst an appendix is included at the end of the report, giving full details of media composition necessary for the growth of the organisms studied.