No description available.

Thesis (M.S.)--Oregon State University, 2006. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Hung-wen Liu. Vita. Includes bibliographical references. Available also from UMI company.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

No description available.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2007. / Härtill 4 uppsatser.

Tunicamycin nucleoside antibiotics were the first known to target the formation of peptidoglycan precursor lipid I in bacterial cell wall biosynthesis. They have also been used extensively as inhibitors of protein N-glycosylation in eukaryotes, blocking the biogenesis of early intermediate dolichyl-pyrophosphoryl-N-acetylglucosamine. Despite their unusual structures and useful biological properties, little is known about their biosynthesis. Elucidating the metabolic pathway of tunicamycins and gaining an understanding of the enzymes involved in key bond forming processes would not only be of great academic value in itself, it would also unlock a comprehensive toolbox of biosynthetic machinery for the production of tunicamycin analogues which have the potential to act as novel therapeutic antibiotics or as specific inhibitors of medicinally important NDP-dependent glycosyltransferases. I – Cloning the tunicamycin biosynthetic gene cluster. We report identification of the tunicamycin biosynthetic genes in Streptomyces chartreusis following genome sequencing and a chemically-guided strategy for in silico genome mining that allowed rapid identification and unification of an operon fractured across contigs. Heterologous expression established a likely minimal gene set necessary for antibiotic production, from which a detailed metabolic pathway for tunicamycin biosynthesis is proposed. II – Natural product isolation and degradation. We have developed efficient methods for the isolation of tunicamycins from liquid culture in preparative quantities. A subsequent relay synthesis furnished advanced biosynthetic intermediates for use as precursors in the production of tunicamycin analogues and as substrates for the in vitro characterisation of individual Tun enzymes. III – Functional characterisation of tun gene products. Individual tun gene products were over-expressed and purified from recombinant E. coli hosts, allowing in vitro functional studies to take place. An NMR assay of biosynthetic enzyme TunF showed it acted as a UDP-GlcNAc-4-epimerase. Putative glycosyltransferase TunD showed hydrolytic activity towards substrate UDP-GlcNAc but failed to accept to the expected natural acceptor substrate, providing unexpected insights into the ordering of biosynthetic events in the tunicamycin pathway. Initial studies into the over-expression of the putative sugar N-deacetylase TunE were also described. IV – Towards synthesis of tunicamycin fragments. Investigations into a novel synthesis of D-galactosamine – a structural motif within tunicamycin – led to the unexpected observation of inverted regioselectivity upon RhII-catalysed C-H insertion of a D-mannose-derived sulfamate. This was the first example of N-insertion at the beta- rather than gamma-C-H based on conformation alone and warranted further investigation. The X-ray structure of a key sulfamate precursor offered valuable insights as to the source of this unique selectivity.

Natural product drug discovery has traditionally been the corner stone of medicine having provided cures to many of today's most common diseases. In particular, antibiotics have revolutionised healthcare and extended human lifespan. However, since their introduction into the clinic, resistance to these drugs has arisen. With the number of new antibiotics being discovered in recent years declining, and fewer drugs making it past clinical trials, we have reached the point where antibiotic resistant infections have become common place and a serious threat to health and society. There is now an urgent requirement for the discovery of new antibiotics and in particular those with unexploited mode of action. This thesis details the different areas of natural product drug development from discovery through to analogue generation. In Chapter one, the history of natural products as therapeutics is explored with a particular focus on antibiotics and how resistance arises against these agents. It outlines why the discovery of new antibiotics is so important and new methods used to facilitate this search. Chapter two follows with the development of a screening platform for antibiotic induction, using the model Streptomyces; Streptomyces coleiolor M145. A variety of culture additives are explored for their ability to induce secondary metabolism production. Chapter three then details the sampling and identification of microbes from a pseudo-marine environment and their screening for their ability to produce secondary metabolites with antibiotic properties. The second half of this thesis centres on the non-ribosomal peptide echinomycin. Collaborators Aquapharm supplied the marine derived strain AQP-4895, capable of producing echinomycin. Chapter four details the establishment of AQP-4895 culturing conditions and the shift observed in production profile. Next Chapter five looks at producing echinomycin analogues through precursor directed biosynthesis. A range of halogenated quinoxaline carboxylic acids are synthesised and fed to AQP-4895, and the respective echinomycin analogues monitored by LC-MS. Chapter Six then aims to direct biosynthesis of the halogenated analogues, using mutasynthesis. Due to the lack of genetic data available surrounding the strain, an unusual approach was taken, using iPCR to create a template for homologous recombination.

Tunicamycins are potent antimicrobial agents but are also toxic to mammalian cells, which render them clinically impractical to use to treat infectious diseases. Instead, they have been used extensively as biochemical tools to study the N-linked glycosylation of proteins. However, despite such a routine application, their inhibitory mechanisms are still not clear. The central objective of this thesis was to develop novel tunicamycin analogues that are non-toxic to eukaryotic cells that could serve as potential antimicrobial drug candidates. We hypothesised that if we retain the lipid character of tunicamycin structure and modify the GlcNAc moiety then the antimicrobial activity would be retained but the tunicamycins inhibitory action towards GPT would be abolished, thus diminishing tunicamycins cytotoxicity towards mammalian cells. <b>I - Semi-synthesis of the Tunicamycin Core Scaffolds and Lipid Analogues</b> Semi-synthetic strategies were devised for isolating tunicamycin core scaffolds and for the selective addition of lipid chains at the 10'-N and 2"-N positions of tunicamycin, yielding the first library of novel tunicamycin lipid analogues. <b>II - Biological Evaluations of the Tunicamycin Core Scaffolds and Lipid Analogues</b> For the first time, the antibacterial activity of tunicamycins was shown to be dependent on the presence of a lipid chain. The tunicamycin core scaffolds were shown to lack antibacterial activity and cytotoxicity. More importantly, the library of tunicamycin lipid analogues with lipid chain length from seven to twelve carbons showed titrated antibacterial activity profile. Furthermore, the tunicamycin lipid analogues were not only found to have potent antibacterial and anti-M. tuberculosis activities but were non-cytotoxic compared to tunicamycins. The relative therapeutic index calculated for the tunicamycin lipid analogues was up to several thousand folds more than tunicamycins. <b>III - Investigation of the tunB and tunF Knockout in the tun Gene Cluster</b> The tunB and tunF single knockout mutations were made in the tun gene cluster by PCR-targeting and then heterologously expressed in S. coelicolor. The tunB knockout successfully abolished tunicamycin biosynthesis and showed evidence by MS the first existence of exo-glycal intermediates in sugar biology, further supporting the discovery of TunA as a novel NDP-sugar 5,6-dehydrogenase. <b>IV - Investigation of the TunD and TunE Enzymatic Activities in Tunicamycin Biosynthetic Pathway</b> The recapitulation of TunD glycosyltransferase and TunE deacetylase activities in vitro were attempted. Recombinant TunD was refolded from insoluble TunD inclusion bodies, while TunE was isolated in small quantities. However, no TunD and TunE activities were found using proposed intermediates. The co-translation of the tun gene cluster and the formation of multi-protein complex are proposed to be involved in the tunicamycin biosynthesis.