Engineering Novel Polyketide Antibiotics by Altering the Glycosylation Pattern Through Combinatorial Biosynthesis and Isolation of the Gilvocarcin Biosynthetic Gene Cluster

Résumé du livre

Microorganisms have long been recognized as a valuable source for useful bioactive compounds. In particular, filamentous soil bacteria of the genus Streptomyces are known for their ability to produce large numbers of structurally diverse polyketide compounds, including commercial important antibiotics and antitumor drugs, e.g. erythromycin, tetracycline, mithramycin, and doxorubicin. An important structural characteristics of many polyketide drugs is the presence of deoxysugars attached to the polyketide aglycon. These sugars are important for the molecular interaction of a drug with its target site and are often essential for biological activity. It would therefore be of great value to be able to manipulate the deoxysugar moieties of polyketides in order to alter their biological properties. As part of our attempt to obtain new bioactive compound through the recombination of selected genes from different biosynthetic pathways, a strategy that is known as combinatorial biosynthesis, we have focused our attention on post-polyketide modifying enxymes, in particular on glycosyltransferases and deoxysugar modifying enxymes. Our overall goal is the design of new polyketide drugs by genetically manipulating their glycosylation pattern. A successful implementation of this approach requires the availability of suitable enzymes as well as knowledge about their specific functions and substrate flexibilities. The work presented in this dissertation addresses all of these aspects. The search for genes with new catalytic functions is the focus of specific aims 1 and 2 in which genes of the gilvocarcin biosynthetic pathway were identified. This sets the stage for a future application of these genes in combinatorial experiments. Specific aim 3 describes the assignment of specific functions to three methyltransferases involved in permethyl-L-rhamnose biosynthesis from the elloramycin producer S. olivaceus. In addition, valuable information about the substrate flexibility of hese methyltransferases were obtained. In specific aims 4 and 5, new hybrid polyketides were generated by combining either deoxysugar genes from the oleandomycin and erythromycin pathways, or by the heterologous expression of glycosyltransferases from the biosynthesis of landomycin A in mutant strains of the urdamycin producer S. fradiae.