(+)-Neopeltolide is a highly potent marine polyketide natural product with activity against multiple cancer cell lines in vitro. The nanomolar range of antifungal and anticancer cytotoxicity in this tetrahydropyran (THP)-containing polyketide, combined with its limited natural supply, has led to several syntheses. In this study, the feasibility of an oxa-Michael conjugate addition route to cis-2,6-THP rings is examined through the efforts toward a total synthesis of the macrocyclic core of (+)-neopeltolide using a highly convergent route. This study is based on the successful preliminary results with a simple 14-member ring model system and the synthesis of the key aldehyde intermediate shown below. The highlighted transformation of this synthesis will be a transannular oxa-conjugate addition to generate the cis-2,6-tetrahydropyran ring system. This route also highlights a highly convergent Wittig coupling to generate the full carbon framework of (+)-neopeltolide. One of the key goals of this project is to compare this synthesis with a chemo-enzymatic total synthesis that relies on chemistry catalyzed by polyketide synthase enzymes in the late stage of the synthesis.

(+)-Neopeltolide is a highly potent marine polyketide natural product with activity against multiple cancer cell lines in vitro. The nanomolar range of antifungal and anticancer cytotoxicity in this tetrahydropyran (THP)-containing polyketide, combined with its limited natural supply, has led to several syntheses. In this study, the feasibility of an oxa-Michael conjugate addition route to cis-2,6-THP rings is examined through the efforts toward a total synthesis of the macrocyclic core of (+)-neopeltolide using a highly convergent route. This study is based on the successful preliminary results with a simple 14-member ring model system and the synthesis of the key aldehyde intermediate shown below. The highlighted transformation of this synthesis will be a transannular oxa-conjugate addition to generate the cis-2,6-tetrahydropyran ring system. This route also highlights a highly convergent Wittig coupling to generate the full carbon framework of (+)-neopeltolide. One of the key goals of this project is to compare this synthesis with a chemo-enzymatic total synthesis that relies on chemistry catalyzed by polyketide synthase enzymes in the late stage of the synthesis.

Macrocyclization is a synthetically challenging step in the total synthesis of natural products. The success of chemical approaches such as the Corey-Nicolaou, Yamaguchi and Keck macrolactonization is heavily based on the confirmation and stereochemistry of the substrate. While there have been some advances in computational modeling, it has been difficult to predict whether the above-mentioned reactions will work. We have begun characterizing polyketide thioesterase catalytic activity and substrate tolerance to find more efficient and dependable routes towards macrolactonization and macrolactamization.

(+)-Neopeltolide is a highly potent marine polyketide natural product with activity against multiple cancer cell lines in vitro. The nanomolar range of antifungal and anticancer cytotoxicity in this tetrahydropyran (THP)-containing polyketide, combined with its limited natural supply, has led to several syntheses. In this study, the feasibility of an oxa-Michael conjugate addition route to cis-2,6-THP rings is examined through the efforts toward a total synthesis of the macrocyclic core of (+)-neopeltolide using a highly convergent route. This study is based on the successful preliminary results with a simple 14-member ring model system and the synthesis of the key aldehyde intermediate shown below. The highlighted transformation of this synthesis will be a transannular oxa-conjugate addition to generate the cis-2,6-tetrahydropyran ring system. This route also highlights a highly convergent Wittig coupling to generate the full carbon framework of (+)-neopeltolide. One of the key goals of this project is to compare this synthesis with a chemo-enzymatic total synthesis that relies on chemistry catalyzed by polyketide synthase enzymes in the late stage of the synthesis.

Macrocyclization is a synthetically challenging step in the total synthesis of natural products. The success of chemical approaches such as the Corey-Nicolaou, Yamaguchi and Keck macrolactonization is heavily based on the confirmation and stereochemistry of the substrate. While there have been some advances in computational modeling, it has been difficult to predict whether the above-mentioned reactions will work. We have begun characterizing polyketide thioesterase catalytic activity and substrate tolerance to find more efficient and dependable routes towards macrolactonization and macrolactamization.

Since the 1970s, a growing body of research has suggested that bacteria play an active role in precipitation. These bacteria are capable of catalyzing the formation of ice at relatively warm temperatures utilizing a specific protein family which aids in the binding of water molecules. However, the overall biodiversity, concentration, and relationship of ice nucleation active (ice+) bacteria with air mass trajectories and precipitation chemistry is not well studied. Precipitation events were collected over 15 months in Blacksburg, VA and ice+ bacteria were isolated from these samples. From these samples, 33,134 total isolates were screened for ice nucleation activity (INA) at -8 °C. A total of 593 of these isolated positively confirmed for INA at the same temperature in subsequent tests. The precipitation events had a mean concentration of 384±147 colony forming units per liter. While the majority of confirmed ice+ bacteria belonged to the gammaproteobacteria, a well-studied class of bacteria, including ice+ species of Pseudomonas, Pantoea, and Xanthomonas, two isolates were identified as Lysinibacillus, a Gram-positive member of the Firmicute phylum. These two isolates represent the first confirmed non-gammaproteobacteria with INA. After further characterization, the two isolates of Lysinibacillus did not appear to use a protein to freeze water. Instead, the Lysinibacillus isolates used a secreted, nanometer-sized molecule that is heat, lysozyme, and proteinase resistant. In an attempt to identify the mechanism responsible for this activity, species type strains were tested for INA and UV mutants were generated to knock out the ice+ phenotype. Based on these results, only members of the species L. parviboronicapiens exhibit INA and the genes responsible for the activity may lie within a type-1 polyketide synthase/non-ribosomal peptide synthase gene cluster. This gene cluster is absent from the genomes of all non-ice+ strains of Lysinibacillus, and contains mutations in five of the nine ice nucleation inactive mutants generated from the rain isolated strain. To better understand the phylogenetic relationship among ice+ Lysinibacillus, a comprehensive reference guide was compiled to provide the most up-to-date information regarding the genus and each of its species. This reference will be available to other researchers investigating Lysinibacillus species or other closely related genera. / PHD / It is a common misconception that water freezes at 0°C (32°F). In clouds, water may remain liquid until -37 to -40°C (-35 to -40°F). At temperatures warmer than this, water molecules must collect around small particles that can help form ice, called ice nuclei. Numerous ice nuclei have been identified, ranging from dirt and dust, to volcanic ash, and even to pollen, fungi, and bacteria. One of these bacteria, Pseudomonas syringae, was identified as an ice nucleus in the 1970’s when it was discovered that it was increasing susceptibility of corn to frost damage. Since then, other Pseudomonas species as well as other bacteria within the same family of bacteria have been shown to have the ability to freeze water at relatively warm temperatures utilizing a specialized protein. Despite numerous studies on how these bacteria can exist in the atmosphere and how they can freeze water, the extent of this freezing ability, the concentration of bacteria in precipitation, and how cloud chemistry affects these bacteria has not been widely studied. In this study, precipitation was collected over the course of 15 months and the bacteria found within the collected precipitation were checked to see if they could act as ice nuclei. We found many of the previously described bacterial ice nuclei in the precipitation samples, but also identified a previously unidentified bacterium capable of freezing water. This bacterium, Lysinibacillus parviboronicapiens, does not use the same method of freezing as the other described bacterial ice nuclei. As such, we set out to determine the method it uses. We have determined that this bacterium utilizes a heat-stable, nanometer-sized particle that is not a protein. To better understand this molecule, representative strains of each species of this genus of bacteria were tested for their ability to freeze water, however, only this species has the ability. To further identify the molecule, UV radiation was used to disrupt the bacteria’s ability to freeze water, and the genes responsible were identified. Based on these results, we have tentatively identified the responsible genes as part of a polyketide synthase gene cluster. This gene cluster is responsible for producing small molecules that provide some survival advantage for the bacteria, in our case, possibly the ability to freeze water. As a final step, and to help serve other researchers, a comprehensive analysis of the entire Lysinibacillus genus has been performed and a reference guide has been generated to help describe and distinguish individual species.

Epicoccum nigrum é um fungo ubíquo conhecido por sua capacidade de produzir vários metabólitos secundários bioativos e pelo seu uso potencial como agente de biocontrole contra vários fitopatógenos. Entre os compostos produzidos por E. nigrum, epicolactona é um policetídeo com uma estrutura bastante complexa. O objetivo desta tese foi identificar e caracterizar genes relacionados à biossíntese da epicolactona em E. nigrum. Três mutantes defectivos para a produção de epicolactona anteriormente gerados por mutagênese aleatória foram analisados. Entretanto, os resultados mostraram que o T-DNA provavelmente estava inserido em regiões regulatórias. Usando ferramentas de bioinformática, seis genes de PKSs foram selecionados para deleção. A deleção do gene PKSi12 mostrou que os seus produtos estão relacionados à atividade antagonista. A análise química permitiu a identificação putativa dos preditos precursores da epicolactona, os quais tiveram sua produção afetada no mutante ΔPKSi12. Uma possível via de biossíntese de epicoccona B e epicoccina por E. nigrum foi proposta. / Epicoccum nigrum is a ubiquitous fungus mainly known for its ability to produce many bioactive secondary metabolites and for its potential use as a biocontrol agent against many phytopathogens. Among the compounds produced by E. nigrum, epicolactone is a polyketide with a very complex structure. The aim of this thesis was to identify and characterize genes related to epicolactone biosynthesis in E. nigrum. Three defective epicolactone mutants previously generated by random mutagenesis were analyzed. However, the results showed that the T-DNA was probably inserted in regulatory regions. Using a genome mining approach, six PKS genes were selected for deletion. The deletion of PKSi12 gene showed that its products are related to E. nigrum antagonistic activity against fungal phytopathogens. The chemical analysis allowed a putative identification of the previously proposed epicolactone precursors, which production was affected in the ΔPKSi12 mutant. A proposed biosynthesis of epicoccone B and epicoccine by E. nigrum was suggested.

Les polykétides sont des composés naturels. Ces composés possèdent des rôles thérapeutiques variés tels que antifongiques, antibiotiques, anticancéreux, immunosuppresseurs ou encore anticholestérolémiques. Par conséquent, la recherche de nouvelles structures possédant des bioactivités diverses se révèlent être intéressante. Une stratégie prometteuse pour créer des nouveaux polykétides est l’ingénierie génétique des enzymes synthétisant ces molécules, les polykétide synthases modulaires (PKS), une approche désignée sous le terme de « biologie synthétique ». Pour ce faire, il faut comprendre de façon détaillée le fonctionnement de ces systèmes multienzymatiques. Plusieurs points restent à éclaircir, dont : i) le contrôle de la stéréochimie du polykétide ; et ii) l’interaction des sous-unités composant la PKS. Lors de ma thèse, j’ai identifié deux kétoréductases (KR) qui, introduites dans un contexte modulaire intrinsèquement non-épimérisant, sont capables d’épimériser le méthyle en Cα de façon efficace. Cependant, la modification de la stéréochimie du polykétide ne dépend pas exclusivement des propriétés intrinsèques de la KR mais aussi du contexte modulaire. J’ai également contribué à la réalisation d’un second projet, pour lequel notre équipe a mis en évidence une nouvelle classe de domaine de docking de PKS de type trans-AT présentant une nouvelle topologie. L’un des DD étudié est une protéine intrinsèquement désordonnée dont le repliement est induit par son partenaire. Nous avons caractérisé l’interface complète entre deux sous-unités de PKS de type trans-AT, révélant une chambre de réaction protégée dans laquelle les chaînes de polykétide peuvent croître / Polyketides are natural products which exhibit a variety of therapeutic activities, including anti-fungal, antibiotic, anticancer, immunosuppressant and anti-cholesterolemic properties. Given their medical and economic importance, there is significant interest in identifying new structures with new biological activities. A promising strategy to create such analogues is to genetically engineer the enzymes responsible for synthesizing these molecules, the modular polyketide synthases (PKSs), an approach referred to as ‘synthetic biology’. However, in order to increase the efficacy of this approach, we must understand in detail how the PKS multienzymes work. A number of issues remain to be clarified, including: i) polyketide stereocontrol, ii) the interaction of the component subunits PKS. During my thesis, I identified two ketoreductase (KR) domains which when introduced into an intrinsically non-epimerizing modular context, were able to efficiently epimerise at the Cα of a model polyketide. I also showed that the modular context in which the KR functions has an influence on the ultimate stereochemical outcome. I also made essential contributions to a second project, in which the group identified a novel family of docking domains (DD) in the trans-AT type of PKS which present a novel topology. One of the two model DDs studied is an intrinsically disordered protein whose folding is induced by its partner. Finally, we were able to visualize a complete intersubunit interface within a trans-AT PKS, revealing a protected reaction center in which polyketide chains can grow.

In the past two decades a new barley threat has emerged as the disease Ramularia leaf spot (RLS) became more prevalent in temperate regions worldwide. This disease, first identified in the late 19th century, is caused by the filamentous fungus Ramularia collo-cygni (Rcc) and can cause substantial yield losses as well as reduce grain quality. RLS typically occurs late in the growing season and characteristic disease symptoms are usually seen after the crop has flowered. Expression of RLS lesions is thought to be associated with the action of fungal secondary metabolism products. The one group of secondary metabolites (SMs) characterised to date from Rcc, the anthraquinone toxins rubellins, are known to cause necrosis to plant tissues in a non-host specific manner. Therefore, it appears that fungal secondary metabolism might be a key component in understanding the interaction between Rcc and its host. In this study, more than 23 core genes involved in the biosynthesis of SMs belonging to the polyketide and non-ribosomal peptide pathways were identified in the genome of Rcc. Putative clusters containing genes with a predicted function relating to secondary metabolism were identified by in silico genome walking in the genetic loci adjacent to Rcc SM core genes. Two gene clusters containing no SM core gene were also identified. Five of the putative SM clusters exhibited similarity to the known fungal SM biochemical pathways involved in gliotoxin, monodictyphenone, ferricrocin, betaenone and chaetoglobosins biosynthesis. Several gene clusters exhibited similarity to SM clusters from fungal species where the SM pathway is uncharacterised. Changes in transcript abundance of selected SM core genes during RLS development in artificially inoculated barley seedlings were tested. Transcript levels were found to be the highest at an early stage of disease development, typically during the asymptomatic and early lesions formation stages and declined over time, suggesting that the associated SMs in Rcc, may not necessarily be involved in symptoms appearance. The in planta mode of action of the non-host specific photoactivated toxin rubellin D was studied in the model plant Arabidopsis thaliana. Rubellin-induced cell death appeared phenotypically reminiscent of programmed cell death (PCD). Full expression of rubellin D-induced cell death required the host salicylic acid (SA) pathway and the host proteasome supporting the PCD response to this fungal SM. However, a clear correlation between toxin sensitivity and disease susceptibility could not be found, suggesting a potential alternative role for rubellin in disease symptom development.

Rhodococcus is a soil bacterium, member of the Actinobacteria, and a close relative of the prolific small molecule producer Streptomyces. Recent interest in Rhodococcus as an under investigated source of possible bioactive secondary metabolites is sparked by the discovery of many polyketide synthase and non-ribosomal peptide synthetase genes of unknown function from sequenced Rhodococcus genomes. Rhodococcus species strain MTM3W5.2 was recently shown to produce a strong inhibitory compound with activity against most strains of Rhodococcus and closely related genera. A goal of this investigation is to discover the gene(s) required to synthesize this inhibitory molecule. The engineered Rhodococcus transposon, pTNR, was used to generate random insertional mutations in the genome of MTM3W5.2. The transposon insertion sites for 8 non-producing mutants were cloned and sequenced. Genes that encode polyketide synthases usually form parts of large biosynthetic gene clusters responsible for the production of small polyketide molecules.