Polyketides are a large group of structurally diverse natural products that have shown a variety of biological activities. These molecules are synthesized by polyketide synthases (PKSs). PKSs are classified into three types based on their sequence, primary structure, and catalytic mechanism. Because of the bioactivities of polyketide natural products, this study is focused on the engineering of PKS pathways for efficient production of useful bioactive molecules or structural modification to create new molecules for drug development. One goal of this research is to create an efficient method to produce pharmaceutically important molecules. Seven biosynthetic genes from plants and bacteria were used to establish a variety of complete biosynthetic pathways in Escherichia coli to make valuable plant natural products, including four phenylpropanoid acids, three bioactive natural stilbenoids, and three natural curcuminoids. A curcumin analog dicafferolmethane was synthesized by removing a methyltransferase from the curcumin biosynthetic pathway. Furthermore, introduction of a fungal flavin-dependent halogenase into the resveratrol biosynthetic pathway yielded a novel chlorinated molecule 2-chloro-resveratrol. This demonstrated that biosynthetic enzymes from different sources can be recombined like legos to make various plant natural products, which is more efficient (2-3 days) than traditional extraction from plants (months to years). Phenylalanine ammonia-lyase (PAL) is a key enzyme involved in the first biosynthetic step of some plant phenylpropanoids. Based on the biosynthetic pathway of curcuminoids, a novel and efficient visible reporter assay was established for screening of phenylalanine ammonia-lyase (PAL) efficiency in Escherichia coli. The other goal of this research is to characterize and engineer natural product biosynthetic pathways for new bioactive molecules. The biosynthetic gene cluster of the antibacterial compound dutomycin was discovered from Streptomyces minoensis NRRL B-5482 through genome sequencing. Confirmation of the involvement of this gene cluster in dutomycin biosynthesis and creation of a series of new molecules were successfully conducted by rationally modifying the biosynthetic pathway. More importantly, a new demethylated analog of dutomycin was found to have much higher antibacterial activity against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

Karenia brevis is the major harmful algal bloom-forming species in the Gulf of Mexico, and produces neurotoxins, known as brevetoxins, that cause large fish kills, neurotoxic shellfish poisoning, and human respiratory distress. Brevetoxins are polyethers that bind voltage-sensitive sodium channels, opening them for prolonged periods of time. Clonal cultures of K. brevis exhibit unique brevetoxin profiles, which not only differ from one another, but also change when subjected to different environmental conditions. The brevetoxin structures were elucidated 30 years ago without any breakthroughs for the biosynthetic pathway. These unique ladder-like polyethers have 10 (PbTx-1) or 11 (PbTx-2) rings, indicating that they are synthesized as secondary metabolites by polyketide synthases. The extensive size of the genome and the lack of histones and nucleosomes combined with the additional regulatory step of a trans-splicing spliced leader sequence make normal molecular techniques ineffective in determining the genes involved in toxin synthesis. The goal of this project is to identify a potential link between toxin, gene, and function. One objective is to take the next step towards identifying the genes associated with the synthesis and regulation of brevetoxins and to help elucidate the hypothesized gene clusters of multi-protein enzymatic complexes involved in brevetoxin production, one for each backbone. The second objective is to make an effort to determine the in vivo function of the costly brevetoxins by identifying possible ion channels, which could be osmotically regulated by the toxins. Genes for polyketide synthases (PKS) were identified in K. brevis, obtained from Expressed Sequence Tag (EST) libraries. In this work, reverse transcription polymerase chain reactions (RT-PCR) were used to generate pools of complementary DNA (cDNA), which was used in real-time quantitative polymerase chain reactions (qPCR) to give relative amounts of PKS transcripts. K. brevis clones have shown a significant increase in toxin production after a rapid shift from high salinity to low salinity, indicating a regulation of brevetoxin synthesis. To gain a better understanding of regulation of toxin production during algal blooms, we compared the toxin levels under different conditions to the transcript levels of PKS genes, as determined by quantitative RT-PCR. In a separate line of investigation, an in silico analysis of the EST library was performed to identify ion channel genes expressed by K. brevis, which may be the in vivo binding site of brevetoxin. The information generated from this project will help to elucidate the effects of environmental variations on toxin production and the biological function of toxin production -- valuable information for the shellfish industries and public health.

Fungal polyketides are a diverse class of natural products that possess many pharmacological properties, including anticancer properties. These properties are evident in the resorcylic acid lactones, a family of polyketides, including zearalenone and radicicol, which shows potent inhibition of tumour cell growth. The key step in the biosynthesis of these lactones is macrocyclization of a linear carboxylic acid into the macrolactone. This reaction is catalyzed by a polyketide synthase (PKS) thioesterase enzyme. Bacterial PKS thioesterases (TEs) have been extensively studied and their substrate specificity has been characterized in vitro. They are highly substrate selective for the macrocyclization reaction. Since Fungal PKS TEs show little sequence homology to bacterial TEs, we have begun investigating their substrate specificity. In particular we are examining the ability of fungal TEs to macrocyclize compounds with varying ring sizes, stereogenic configuration, and nucleophiles. Herein we present the synthesis of a number of diverse TE substrates and the in vitro macrocyclization results for the TEs from zearalenone and radicicol biosynthetic pathway with these substrates.

The Sigma-54 Promoter Database contains computationally predicted sigma-54 promoters from over 60 prokaryotic species. Organisms from all major phyla were analysed and results were made available online at http://www.sigma54.ca. This database is particularly unique due to its inclusion of intragenic regions, grouping of data by COG and COG category, and the ability to summarize results either by phylum or database-wide. ClusterMine360 (http://www.clustermine360.ca/) is a database of microbial polyketide and nonribosomal peptide gene clusters. It takes advantage of crowd-sourcing by allowing members of the community to make contributions while automation is used to help achieve high data consistency and quality. The database currently has over 200 gene clusters from over 185 compound families. It also features a unique sequence repository containing over 10,000 PKS/NRPS domains. The sequences are filterable and downloadable as individual or multiple sequence FASTA files. This database will be a useful resource for members of the PKS/NRPS research community enabling them to keep up with the growing number of sequenced gene clusters and rapidly mine these clusters for functional information.

Fungal polyketides are a diverse class of natural products that possess many pharmacological properties, including anticancer properties. These properties are evident in the resorcylic acid lactones, a family of polyketides, including zearalenone and radicicol, which shows potent inhibition of tumour cell growth. The key step in the biosynthesis of these lactones is macrocyclization of a linear carboxylic acid into the macrolactone. This reaction is catalyzed by a polyketide synthase (PKS) thioesterase enzyme. Bacterial PKS thioesterases (TEs) have been extensively studied and their substrate specificity has been characterized in vitro. They are highly substrate selective for the macrocyclization reaction. Since Fungal PKS TEs show little sequence homology to bacterial TEs, we have begun investigating their substrate specificity. In particular we are examining the ability of fungal TEs to macrocyclize compounds with varying ring sizes, stereogenic configuration, and nucleophiles. Herein we present the synthesis of a number of diverse TE substrates and the in vitro macrocyclization results for the TEs from zearalenone and radicicol biosynthetic pathway with these substrates.

Drug discovery is reliant on new developments in natural product chemistry as well as advances in chemical synthesis. The interconnectivity and interdependence of natural and synthetic investigation in drug discovery is evident. The chemical exploration reported herein elaborates the relationship between natural product chemistry and chemical synthesis. Of particular interest are chemicals from organisms residing in less accessible environments, particularly Antarctica and endophytic microbial communities. Degradation via reductive ozonolysis of palmerolide A, a macrocyclic polyketide isolated from the Antarctic tunicate Synoicum adareanum, and subsequent synthetic preparation of the resulting polyols (1,2,6-hexanetriol and 1,2,3,6-hexanetetraol) led to a revision in the absolute configuration of the bioactive natural product (7R, 10R, 11R to 7S, 10S, 11S). A partial synthesis of palmerolide A (C3-14) was completed using Grubb’s 2nd generation catalyst to couple fragments formed using the previously developed methodology from the degradation study. Isolation of indole-pyrimidine containing alkaloids meridianins A, B, C, and E from the Antarctic tunicate Synoicum sp. prompted a synthetic investigation of psammopemmin A, a related alkaloid from the Antarctic sponge Psammopemma sp. resulting in reassignment of the structure of psammopemmin A to that of meridianin A. Both meridianin A and psammopemmin A were synthesized through a Suzuki coupling of the same 4-indolol nucleophile to the apposite pyrimidine electrophile. Several synthetic 3-pyrimidylindole analogs were also prepared and investigated for central nervous system, antimalarial, and cytotoxic activity. Chemical investigation of extracts from mangrove fungal endophytes that displayed antimalarial properties in vitro resulted in the isolation of several potent but cytotoxic and cytostatic compounds: cytochalasin D, roridin E, and 12,13-deoxyroridin E.

The production of new drugs as potential pharmaceutical targets is arguably one of the most important avenues of medicine, as existing diseases not only require treatment, but it is also certain that new diseases will appear in the future which will need treatment. Indeed, existing medicines such as antibiotics and immunosuppressants maintain their current activities in their respective realms, yet the molecular and stereochemical complexity of these compounds cause a burden on organic synthetic chemists that may prohibit the high yields required to manufacture a drug. The enzyme systems that naturally manufacture these compounds are incredibly efficient in doing so, and also do not use environmentally harmful solvents, chiral auxiliaries, or metals that are utilized in the current syntheses of these compounds; therefore utilizing these enzymes' machinery for the biocatalysis of new medicinally-relevant compounds, as researchers have in the past, is undeniably a rewarding endeavor. In order to harness these systems' biocatalytic potential, we must understand the processes which they operate. This work focuses on ketoreductase domains, since they are responsible for setting most of the stereocenters found within these complex secondary metabolites. We have supplied a library of substrates to multiple ketoreductases to test their limits of stereospecificity and found that, for the most part, they maintain their natural product stereospecificity seen in nature. We were even able to convert a previously nonstereospecific ketoreductase to a stereospecific catalyst. We have also developed a new technique to follow ketoreductase catalysis in real-time, which can also differentiate between which diastereomeric product is being produced. Finally, we have elucidated the structure of a ketoreductase that reduces non-canonically at the [alpha]- and [beta]- position, and functionally characterized its activities on shortened substrate analogs. With the knowledge gained from this dissertation we hope that the use of ketoreductases as biocatalysts in the biosynthesis of new natural product-based medicines is a much nearer reality than before. / text

Saccharopolyspora spinosa is a rare actinomycete that synthesizes the secondary metabolite spinosyn A, which is an active ingredient in several important commercial insecticides. Spinosyn aglycone formation occurs via a type I polyketide synthase. After release of the polyketide chain from the synthase, various tailoring enzymes modify the aglycone core. These unique enzyme transformations result in unusual structural characteristics found in spinosyn A. The enzymes SpnG, SpnP, SpnF and SpnL each perform a key reaction during post-PKS processing. The work presented in this dissertation focuses on the structural determination and analysis of SpnG, SpnP, SpnF and SpnL. SpnG, which naturally catalyzes the 9-OH rhamnosylation of spinosyn, is capable of adding diverse sugars to the spinosyn aglycone from TDP-hexoses, such as TDP-glucose. However, the substitution of UDP-glucose for TDP-glucose as the donor substrate is known to result in a >60,000-fold reduction in k [subscript cat]. The structure of SpnG at 1.65 Å resolution, the 1.86 Å resolution structure of SpnG bound to TDP, and the 1.70 Å resolution structure of SpnG bound to AGL were determined. The SpnG-TDP complex reveals how SpnG employs N202 to discriminate between TDP- and UDP-sugars. The SpnG-AGL complex shows that SpnG binds the acceptor substrate primarily through hydrophobic interactions and implicates H13 as the potential catalytic base. A model for how rhamnose binds in the active site was constructed to elucidate which features enable SpnG to transfer diverse hexoses. SpnP transfers forosamine from a TDP-D-forosamine donor substrate to a spinosyn pseudoaglycone acceptor substrate. The structures of SpnP and its complex with TDP were determined to 2.50 Å and 3.15 Å resolution, respectively. SpnP possesses a structural feature that has only been previously observed in a related glycosyltransferase, which employs an auxiliary protein that aids in its catalysis. This unique feature may be a used as a predictive motif of glycosyltransferases that interact with an auxiliary protein. SpnF and SpnL are two novel S-adenosyl-L-methionine dependent cyclases. Structural data was utilized in order to gain insight into the unusual cycloaddition catalyzed by the putative Diels-Alderase and Rauhut-Currierase, SpnF and SpnL, respectively. Together these structures provide valuable insights into the unusual mechanisms involved in spinosyn biosynthesis. / text

Total syntheses of the neuroregenerative natural products vinaxanthone and xanthofulvin have been accomplished. The synthetic routes to both molecules utilize a highly regioselective furan Diels-Alder cycloaddition - aromatization sequence to furnish the catechol fragment present in both natural products. The pentasubstituted catechol was elaborated to a vinylogous amide which was used twice in both syntheses, exploiting the pseudosymmetry found in vinaxanthone and xanthofulvin. This approach enabled the dimerization of 5,6-dehydropolivione forming vinaxanthone, lending significant evidence to a non-enzymatically driven formation of vinaxanthone in Nature. The total synthesis of vinaxanthone was accomplished in nine steps, the shortest synthesis to date, and an additional route was devised to access a set of analogs for biological study. The first total synthesis of xanthofulvin was accomplished in 18 steps and the convergent nature of the synthetic plan allows for analog synthesis. The sets of vinaxanthone and xanthofulvin analogs will be used to examine their inhibition of Semaphorin3A, a protein which inhibits neuronal regeneration, and is the biological target for both molecules.

The Sigma-54 Promoter Database contains computationally predicted sigma-54 promoters from over 60 prokaryotic species. Organisms from all major phyla were analysed and results were made available online at http://www.sigma54.ca. This database is particularly unique due to its inclusion of intragenic regions, grouping of data by COG and COG category, and the ability to summarize results either by phylum or database-wide. ClusterMine360 (http://www.clustermine360.ca/) is a database of microbial polyketide and nonribosomal peptide gene clusters. It takes advantage of crowd-sourcing by allowing members of the community to make contributions while automation is used to help achieve high data consistency and quality. The database currently has over 200 gene clusters from over 185 compound families. It also features a unique sequence repository containing over 10,000 PKS/NRPS domains. The sequences are filterable and downloadable as individual or multiple sequence FASTA files. This database will be a useful resource for members of the PKS/NRPS research community enabling them to keep up with the growing number of sequenced gene clusters and rapidly mine these clusters for functional information.