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

dc.contributor.advisorLiu, Hung-wen, 1952-en
dc.creatorMelançon, Charles Evans, 1975-en
dc.date.accessioned2008-08-28T23:11:08Zen
dc.date.available2008-08-28T23:11:08Zen
dc.date.issued2006en
dc.description.abstractNatural 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.
dc.description.departmentChemistry and Biochemistryen
dc.description.departmentBiochemistryen
dc.format.mediumelectronicen
dc.identifierb66062780en
dc.identifier.oclc164319332en
dc.identifier.urihttp://hdl.handle.net/2152/2829en
dc.language.isoengen
dc.rightsCopyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works.en
dc.subject.lcshDeoxy sugarsen
dc.subject.lcshMacrolide antibioticsen
dc.subject.lcshBiosynthesisen
dc.subject.lcshActinomycetalesen
dc.titleInvestigation and engineering of macrolide antibiotic sugar biosynthesis and glycosylation pathways of actinomycetesen
dc.type.genreThesisen
thesis.degree.departmentBiochemistryen
thesis.degree.disciplineBiochemistryen
thesis.degree.grantorThe University of Texas at Austinen
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophyen

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