Polyketides are a class of natural products that exhibit remarkable structural and functionally diversity and are highly sought after due to their medicinally important activities. For many decades now, polyketide synthases (PKSs), the mega-enzymes responsible for biosynthesis of polyketides have been the focus of extensive investigation to make new polyketides by polyketide engineering strategies. While there are many established methods to investigate polyketide enzymes and biosynthesis mechanisms, they have substantial shortcomings that have limited the extent of success with polyketide engineering efforts. This thesis focuses on developing simple, flexible yet powerful tools for examining polyketide biosynthesis by overcoming some deficiencies in currently used techniques. Reactive heterocylces have been designed for direct labeling of key polyketide synthase enzymes to provide a direct insight into its functions and mechanisms. First β-lactones and then β-lactams have been used as small molecule probes to perform site-specific labeling of acyl carrier proteins and further used for mechanistic interrogation of key steps in polyketide biosynthesis. The utility of these probes has been demonstrated by comparison to traditional probes and has been successfully applied to examine substrate selectivity of keto synthases, key enzymes in polyketide biosynthesis. The applications of the tools described in this manuscript only scratch the surface of their capabilities and are expected to significantly aid in the study of new and existing PKS systems leading to improved understanding of how these extraordinary biosynthetic machines function.

No description available.

Bacillus amyloliquefaciens FZB42 besitzt einen beeindruckenden Effekt zur Verbesserung des Pflanzenwachstums. Um die Mechanismen, vor allem auf molekularer Ebene, zu verstehen, wurde das komplette Genom von FZB42 in dieser Arbeit sequenziert. Abwesenheit von der weit verbreiteten Phagen-verwandten Genen im Genom von B. subtilis 168, der in enger Verwandtschaft zum FZB42 steht, ist ein besonderes Merkmal. Dagegen enthält das Genom von FZB42 viele DNA-Inseln, in denen unikale Gene in FZB42 als Cluster gefunden wurden. Viele Gene, die möglicherweise zur Pflanzenwachstumsförderung beitragen, wurden in dieser Arbeit identifiziert. B. amyloliquefaciens FZB42 ist natürlich kompetent. Das kompetente Stadium in FZB42 kommt früher als in B. subtilis 168, nämlich während der späten exponentiellen Wachstumsphase. Das FZB42-Genom enthält den kompletten Satz von Genen, die für die Entwicklung der genetischen Kompetenz nötig sind. Ausgenommen von Gene für Quorum-Sensing-System ist die Mehrzahl der Kompetenz-Gene von FZB42 sehr ähnlich zu denen in B. subtilis 168. Das FZB42 Genom birgt ein enormes Potential zur Produktion von sekundären Metaboliten. Genetische Manipulationen wurden durchgeführt, um die Funktionen der trans-AT Domänen und der Modifikationsdomänen in den PKS-Gen-Clustern zu erklären. Mit Ausnahme von fünf Gen-Clustern in B. subtilis 168 (Surfactin, Fengycin, Bacillibactin, Bacillaene und Bacilysin), sind Bacillomycin D, Difficidin, Macrolactin und ein hypothetisches Tripeptid einzigartig im Genom der FZB42. FZB42 kann kein bekanntes ribosomal synthetisiertes Bacteriocin produzieren kann. Gleichzeitig beinhaltet sein Genom ein Gen-Cluster, das wahrscheinlich für die Produktion eines neuartigen Bacteriocins verantwortlich ist. Die eindrucksvolle genetische Kapazität zur Herstellung von antagonistischen sekundären Metaboliten ermöglicht es FZB42, nicht nur erfolgreich neben konkurrierenden Organismen innerhalb seiner natürlichen Umgebung zu überleben, sondern auch Pflanzen gegen pathogene Bakterien und Pilze zu schützen. / Bacillus amyloliquefaciens FZB42 has an impressive effect to improve plant growth. In order to understand the mechanisms, especially at the molecular biological level, the whole genome of FZB42 was sequenced in this work. The absence of extended phage insertions which are typical for the closely related B. subtilis 168 genome is a particular feature. On the other hand, several DNA islands where unique genes in FZB42 were found clustered. Many candidate genes that may contribute to the plant growth promotion were identified in this works. B. amyloliquefaciens FZB42 is naturally competent. FZB42 exhibited its maximal competence earlier than B. subtilis, during late exponential growth. Not surprisingly, the FZB42 genome harbors the complete set of genes necessary for development of genetic competence. The majority of competence genes are highly homologous to their counterparts in B. subtilis 168, excluded from genes for the quorum-sensing system. The FZB42 genome harbors enormous potential for producing secondary metabolites. Genetic manipulation was carried out to investigate the trans-AT domains and some modification domains in the pks gene clusters. With the exception of five gene clusters in B. subtilis 168 (Surfactin, Fengycin, Bacillibactin, Bacillaene and Bacilysin), Bacillomycin D, Difficidin, Macrolactin and a hypothetical tripeptide are unique in the genome of the FZB42. A remarkable feature of the FZB42 genome is that it does not produce any known ribosomally synthesized bacteriocin, whereas a gene cluster probably responsible for production of a new bacteriocin was identified in this work. The impressive genetic capacity to produce antagonistic acting secondary metabolites not only enables FZB42 to cope successfully with competing organisms within its natural environment, but also to protect plants from pathogenic bacteria and fungi.

No description available.

Phytocannabinoids are the active substances responsible for the medicinal and psychotropic effects of Cannabis sativa. Although the bioactivity of cannabis and its preparations have been known for millennia, several steps in the biosynthetic pathway leading to phytocannabinoids remain unclear. Phytocannabinoids are prenylated resorcylic acids which are formed in specialized plant organs called glandular trichomes. Following the analysis of a pre-generated cannabis trichome cDNA library, a type III polyketide synthase (tetraketide synthase; TKS) was identified and assayed, yielding three major compounds, hexanoyl triacetic acid lactone (HTAL), pentyl diacetic acid lactone (PDAL), and olivetol, yet no resorcylic acid was detected. This lack of resorcylic acid in enzyme assays has instigated the characterization of TKS and a search for putative cyclases in the cannabis trichome cDNA library, and involved protein pulldown, co-immunoprecipitation, and co-assay experiments. These experiments led to the discovery of a novel polyketide cyclase protein named olivetolic acid cyclase (OAC) responsible for the proper cyclization of a polyketide intermediate produced by TKS. This thesis shows that TKS assays conducted with OAC produce olivetolic acid (OA), an intermediate required during the biosynthesis of cannabinoids. The TKS/OAC spatial relationship was also investigated following the creation of fluorescent fusion proteins which show that the enzymes co-localized in vivo when viewed with confocal microscopy. Furthermore, yeast two-hybrid assays using TKS and OAC were performed to establish whether the enzymes physically interact. Finally, an attempt to determine the responsible amino acids involved in OAC’s mechanism was conducted by comparing the activity of single point OAC mutants with the wild-type OAC. Based on the available data, mechanisms for the production of HTAL, PDAL, olivetol, and OA are proposed.

In addition to its canonical role in aminoacylation, threonyl-tRNA synthetase (TARS) possesses pro-angiogenic activity that is susceptible to the TARS-specific antibiotic borrelidin. However, the therapeutic benefit of borrelidin is offset by its strong toxicity to living cells. The removal of a single methylene group from the parent borrelidin generates BC194, a modified compound with significantly reduced toxicity but comparable anti-angiogenic potential. Biochemical analyses revealed that the difference in toxicities was due to borrelidin's stimulation of amino acid starvation at ten-fold lower concentrations than BC194. However, both compounds were found to inhibit in vitro and in vivo models of angiogenesis at sub-toxic concentrations, suggesting a similar mechanism that is distinct from the toxic responses. Crystal structures of TARS in complex with each compound indicated that the decreased contacts in the BC194 structure may render it more susceptible to competition with the canonical substrates and permit sufficient aminoacylation activity over a wider concentration of inhibitor. Conversely, both borrelidin and BC194 induce identical conformational changes in TARS, providing a rationale for their comparable effects on angiogenesis. The mechanisms of TARS and borrelidin-based compounds on angiogenesis were subsequently tested using zebrafish and cell-based models. These data revealed ectopic branching, non-functional vessels, and increased cell-cell contracts following BC194-treatment or knockdown of TARS expression, suggesting a role for the enzyme in the maturation and guidance of nascent vasculature. Using various TARS constructs this function was found to be dependent on two interactions or activities associated with the TARS enzyme that are distinct from its canonical aminoacylation activity. Furthermore, observations that TARS may influence VEGF expression and purinergic signaling suggest the possibility for a receptor-mediated response. Taken together, the results presented here demonstrate a clear role for TARS in angiogenesis, independent of its primary function in translation. Although the exact molecular mechanisms through which TARS and borrelidin regulate this activity remain to be determined, these data provide a foundation for future investigations of TARS's function in vascular biology and its use as a target for angiogenesis-based therapy.

Polyketides are a group of bioactive natural products synthesized by bacteria, fungi and plants with various acyl-CoA precursors, such as malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA. A sufficient supply of these precursors is a prerequisite for the high level production of polyketide products. A thorough understanding of relative roles of various metabolic pathways involved in precursor supply makes increased production by genetical manipulation, and thus rational strain improvement, a reality. Monensin A is a polyketide antibiotic assembled from one ethylmalonyl-CoA, seven methylmalonyl-CoA and five malonyl-CoA molecules by Streptomyces cinnamonensis. In the present work, the origin of these biosynthetic precursors was investigated using an industrially mutagenized monensin producer and industrial fermentation conditions. A hitherto disregarded metabolic pathway was discovered to play a significant role in providing methylmalonyl-CoA for monensin biosynthesis by gene disruption, isotope-labeling of monensin and analysis of in vivo acyl-CoA pools. This pathway starts from biosynthesis of butyryl-CoA from two molecules of acetyl-CoA, and goes through the intermediate of isobutyryl-CoA, and finally produces methylmalonyl-CoA by direct oxidation of the pro-S methyl group of isobutyryl-CoA.Industrial fermentation of the industrially mutagenized monensin producer yields significantly more monensin than the routine laboratory fermentation. This suggested the presence of abundant in vivo malonyl-CoA and methylmalonyl-CoA in this process and presented an opportunity to utilize it as a biological system for the high-titer production of heterologous polyketides derived from malonyl-CoA and/or methylmalonyl-CoA. The tetracenomycin C polyketide synthase (PKS) synthesizes tetracenomycin C, a polyketide with ten molecules of malonyl-CoA. In this work, the tetracenomycin C PKS gene cluster was introduced into two industrially mutagenized strains of Streptomyces cinnamonensis. Unprecedented multi-gram/liter of tetracenomycin production was observed in the resulting two strains, indicating the high potential of industrially mutagenized monensin production strains as efficient hosts for the production of malonyl-CoA-derived polyketides. For additional improvement in tetracenomycin yield, we attempted to increase malonyl-CoA supply to tetracenomycin C PKS by genetically manipulating metabolic pathways affecting production of malonyl-CoA and eliminating competition from monensin PKS for malonyl-CoA. However, only decreased tetracenomycin production was observed, demonstrating that the regulation of malonyl-CoA-related metabolic pathways is a complex process.

O espaço intergênico (IGS) juntamente com suas subunidades flanqueadoras (cpcB) e (cpcA) do operon do ficocianina foi usado para identificar linhagens de cianobactérias. Dentro do domínio Bacteria somente as cianobactérias possuem o operon da ficocianina e a região cpcBA-IGS é suficientemente variável para diferenciar linhagens desses microrganismos. No presente estudo 25 linhagens de cianobactérias isoladas de diversos locais brasileiros foram caracterizadas usando a seqüência cpcBA-IGS. DNA genômico foi extraído das ordens Chroococcales (oito linhagens), Oscillatoriales (duas linhagens), Nostocales (onze linhagens) e Stigonematales (quatro linhagens). Os oligonucleotídeos iniciadores PCβF/PCαR, específicos para a seqüência cpcBA-IGS, foram usados para amplificar fragmentos de DNA de aproximadamente 685 pb. Os produtos da PCR foram clonados, seqüenciados e as seqüências foram comparadas pela análise BLAST. Todas as seqüências de Microcystis e também as seqüências de Radiocystis fernadoi SPC736, Planktothrix mougeotii SPC788, Geitlerinema splendidum SPC923, Microchaete investiens CENA64 e Gloeotrichia UFV-B2 mostraram identidades com seqüências do GenBank. Entretanto, nenhuma identidade foi encontrada para as seqüências restantes. As relações filogenéticas das seqüências de cpcBA-IGS foram investigadas junto com outras seqüências de cianobactéria do Genbank usando a análise “Neighbour Joining". A topologia da árvore foi congruente com outras árvores de cianobactérias, com exceção de todas as seqüências sem identidades no GenBank, as quais formaram um agrupamento separado. Os dados das seqüências de cpcBA-IGS analisadas confirmam que as cianobactéria heterocitadas formam um grupo monofilético. Estudos anteriores realizados com linhagens de cianobactéria mostraram que estes microrganismos são uma fonte rica de produtos naturais. No presente estudo conduzido com 59 linhagens de cianobactérias, sendo a maioria isolada de ambientes brasileiros, isto foi confirmado. Para alcançar esse objetivo, dois conjuntos de iniciadores degenerados foram usados para produzir seqüências amplificadas por PCR das sintetases de peptídeos não-ribossômicos (NRPSs), e de sintases policetídeos (PKSs) modulares, as quais são enzimas multifuncionais envolvidas na produção de produtos naturais. O sistema híbrido NRPS/PKS também foi amplificado por PCR usando uma combinação de iniciadores de NRPS e de PKS. Essa abordagem molecular mostrou a presença de genes de NRPS e de PKS em 93% e 81% linhagens de cianobactérias, respectivamente. Genes de NRPS/PKS foram encontrados em 87% das cianobactérias examinadas. Numa tentativa de atribuir funções a oito fragmentos de PKS identificados por PCR, estas seqüências foram clonadas, seqüenciadas e analisadas filogeneticamente. As seqüências de PKSs da Microcystis aeruginosa NPCD1 e Fischerella CENA62 mostraram correlação com a síntese de sideróforo e de microcistina, respectivamente. Todas as 59 linhagens foram analisadas para a produção do microcistinas e 20 linhagens apresentaram resultados positivos. Para a maioria das linhagens potencialmente produtoras de microcistinas os produtos de PCR esperados de NRPS, PKS e NRPS/PKS foram amplificados. A produção de sideróforos foi testada em 28 linhagens e somente cinco produziram resultados positivos. Em três linhagens produtoras de sideróforos todos os três sistemas moleculares analisados estavam presentes. Estes resultados serão altamente valiosos na exploração futura de cada peptídeo dessas cianobactérias e para a elucidação da bioatividade de tais produtos naturais. / The intergenic spacer (IGS) together with its flanking subunits  (cpcB) and  (cpcA) of the phycocyanin operon has been used to identify cyanobacterial strains. Within the Bacteria domain only cyanobacteria present phycocyanin operon and the cpcBA-IGS region is variable enough to differentiate strains of these microorganisms. In the present study 25 cyanobacterial strains isolated from several Brazilian locations were characterized using the cpcBA-IGS sequence. Genomic DNA was extracted from the orders Chroococcales (eight strains), Oscillatoriales (two strains), Nostocales (eleven strains) and Stigonematales (four strains). The primers PCβF/PCαR targeting the cpcBA-IGS sequence were used to amplify DNA fragments of approximately 685 bp. The PCR products were cloned, sequenced and the sequences were compared by BLAST analysis. All Microcystis sequences and also sequences from Radiocystis fernadoi SPC736, Planktothrix mougeotii SPC788, Geitlerinema splendidum SPC923, Microchaete investiens CENA64 and Gloeotrichia UFV-B2 showed identities with sequences from GenBank. However, no identities were found for the remaining sequences. Phylogenetic relationships of the cpcBA-IGS sequences were investigated together with other cyanobacterial sequences from Genbank using the Neighbour Joining analysis. The tree topology was congruent with previous cyanobacterial trees, except for all sequences with no identities in the GenBank, which formed a separated cluster. The cpcBA-IGS sequences analysis data confirm that heterocyte-forming cyanobacteria are a monophyletic group. Previous studies carried out with cyanobacterial strains showed that these microorganisms are a rich source of natural products. This has been confirmed in the present study conducted with 59 cyanobacterial strains, with the majority of them isolated from Brazilian environment. To reach this goal, two sets of degenerate primers were used to generate PCR amplification sequences of nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), which are multifunctional enzymes implicated in natural products production. Also, NRPS/PKS hybrid system was PCR amplified by using a combination of NRPS and PKS primers. This molecular approach revealed the presence of NRPS and PKS genes in 93% and 81% cyanobacterial strains, respectively. NRPS/PKS genes were found in 87% of cyanobacteria examined. In an attempt to attribute functions to eight PCR identified PKS fragments, these sequences were cloned, sequenced and phylogenetically analyzed. PKSs sequences of Microcystis aeruginosa NPCD1 and Fischerella CENA62 showed correlation with the synthesis of siderophore and microcystin, respectively. All 59 strains were analyzed for microcystin production and 20 strains presented positive results. For the majority of potentially producing-microcystin strains expected PCR products of NRPS, PKS and NRPS/PKS were amplified. The siderophores production was tested in 28 strains and only five gave positive results. In three producing-siderophore strains all three molecular systems analyzed were present. These results will be highly valuable for further exploring each of these cyanobacterial peptides and for elucidating the bioactivity of such natural products.

Madeirolide A (1) is a structurally novel polyketide natural product first isolated from the deep-sea sponge Leiodermatium sp. by Wright in 2009. Initial biological investigations of madeirolide A revealed potent inhibition of the fungal pathogen Candida albicans but failed to determine any appreciable cytotoxicity when tested against a limited range of cancer cell lines. The unusual bioactivity of madeirolide A coupled with uncertainty over the true stereostructure of the natural product makes it a compelling target for synthesis. This thesis discloses synthetic efforts towards the total synthesis of madeirolide A with an emphasis on the construction of the all-cis C21 - C27 eastern tetrahydropyran. Chapters 1 and 2 provide an introduction to the importance of natural products in drug discovery and outline the context of this project with details of the isolation and biological activity of madeirolide A. Previous synthetic efforts are also reviewed including those from within the group which formed the basis of the present studies. Chapter 3 describes the synthesis of a fully elaborated C1 - C11 fragment, building upon previously published work in the group. Specifically, it details the successful completion of a modified approach designed to avoid some of the major challenges previously encountered such as undesired migration of protecting groups and challenges in selectively installing an (E)-vinyl iodide. Chapter 4 discusses ongoing efforts towards the challenging C12 - C27 fragment of madeirolide A. The stereocontrolled synthesis of several linear C19 - C27 precursors is outlined, followed by details of screening reactions conducted to affect the desired oxy- Michael cyclisation. Additionally, extensive computational studies have been undertaken in an attempt to rationalise the frustrating lack of reactivity observed with the goal of developing a substrate suitably elaborated to cyclise. Finally, the asymmetric synthesis of the C13 - C17 subfragment is outlined, which will provide eventual access to the eastern tetrahydrofuran. Chapter 5 summarises the synthetic work carried out thus far and explores potential strategies for the future completion of the natural product with a focus on alternative disconnections of the eastern tetrahydropyran.

Heterocycles play an important role in many biological processes and are widespread among natural products. Oxazole-containing natural products possess a broad range of bioactivities and are of great interest in the pharmaceutical and agrochemical industries. Herein, the biosynthetic routes to the oxazole-containing phthoxazolins and the bis(benzoxaozle) AJI9561, were investigated. Phthoxazolins A-D are a group of oxazole trienes produced by a polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway in Streptomyces sp. KO-7888 and Streptomyces sp. OM-5714. The phthoxazolin pathway was used as a model to study 5-oxazole and primary amide formation in PKS-NRPS pathways. An unusually large gene cluster for phthoxazolin biosynthesis was identified from the complete genome sequence of the producer strains and various gene deletions were performed to define the minimal gene cluster. PhoxP was proposed to encode an ATP-dependent cyclodehydratase for 5-oxazole formation on an enzyme-bound N-formylglycylacyl-intermediate, and its deletion abolished phthoxazolin production. In vitro reconstitution of the early steps of phthoxazolin biosynthesis was attempted to validate the role of PhoxP, but was unsuccessful. Furthermore, Orf3515, a putative flavin-dependent monooxygenase coded by a remote gene, was proposed to hydroxylate glycine-extended polyketide-peptide chain(s) at the α-position to yield phthoxazolins with the primary amide moiety. On the other hand, an in vitro approach was employed to establish the enzymatic logic of the biosynthesis of AJI9561, a bis(benzoxazole) antibiotic isolated from Streptomyces sp. AJ9561. The AJI9561 pathway was reconstituted using the precursors 3-hydroxyanthranilic acid and 6-methylsalicylic acid and five purified enzymes previously identified from the pathway as key enzymes for benzoxazole formation, including two adenylation enzymes for precursor activation, an acyl carrier protein (ACP), a 3-oxoacyl-ACP synthase and an amidohydrolase-like cyclase. Intermediates and shunt products isolated from enzymatic reactions containing different enzyme and precursor combinations were assessed for their competence for various steps of AJI9561 biosynthesis. Further bioinformatic analysis and in silico modelling of the amidohydrolase-like cyclase shed light on the oxazole cyclisation that represents a novel catalytic function of the amidohydrolase superfamily.