Investigation and engineering of macrolide antibiotic sugar biosynthesis and glycosylation pathways of actinomycetes

Natural products are an important source of bioactive lead compounds used in drug development. The diverse sugar moieties found in natural product structures are often critical to their bioactivity. Therefore, advances in our understanding of natural product sugar biosynthesis and glycosyltransfer and in our ability to synthesize natural product derivatives through manipulation of the biosynthetic machinery are important and can impact the treatment of human diseases. The work described in this dissertation focuses on the functional elucidation of enzymes involved in biosynthesis and glycosyltransfer of the deoxysugar D-mycaminose, which is a structural component of the macrolide antibiotic tylosin, and on the use of genes encoding the biosynthesis and attachment of D-mycaminose, D-desosamine, and other deoxysugars for the engineered production of macrolide derivatives with altered sugar structures. First, the functions of TylM3 as the activator protein for the glycosyltransferase TylM2 in tylosin biosynthesis in Streptomyces fradiae, and the function of the homologous protein MydC as the activator protein for the glycosyltransferase MycB in mycinamicin biosynthesis in Micromonospora griseorubida were elucidated by expression of combinations of their encoding genes in engineered Streptomyces venezuelae hosts. These studies also showed that these glycosyltransferases have relaxed substrate specificity. During this work, a failed attempt to reconstitute the mycaminose biosynthetic pathway in an S. venezuelae mutant resulted in the discovery of a novel hexose 3,4-ketoisomerase, Tyl1a, which is involved in formation of TDP-D-mycaminose. Discovery of Tyl1a allowed reconstitution of the mycaminose pathway in S. venezuelae, and demonstration that Tyl1a alone could convert the desosamine pathway to a mycaminose biosynthesizing pathway. This work resulted in synthesis of several glycosylated macrolide derivatives. The enzymatic activity of purified recombinant Tyl1a was characterized in vitro by 1 H NMR product analysis and steady state kinetics, and the substrate specificity of Tyl1a was found to be relaxed. Finally, three S. venezuelae mutants expressing hybrid deoxysugar biosynthetic pathways were constructed, one of which resulted in formation of non-natural deoxysugar-bearing macrolides. This work has provided important functional information on the sugar biosynthesis enzymes and glycosyltransferases studied, and has illustrated the feasibility of constructing complex engineered deoxysugar biosynthesis pathways in the macrolide producer S. venezuelae.