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Lichens produce a diverse array of bioactive secondary metabolites, many of which are unique to the organisms. Their potential applications, however, are limited by their finite sources and the slow-growing nature of the organisms in both laboratory and environmental conditions. This thesis set out to investigate polyketide synthase genes in lichens, with the ultimate goal of providing a sustainable source of lichen natural products to support these applications. To expand the diversity of PKS genes that could be detected in lichens, new degenerate primers targeting ketoacylsynthase (KS) domains of specific clades of PKS genes have been developed and tested on various lichen samples. Using these primers, 19 KS domains from various lichens were obtained. Phylogenetic analysis of the KS domains was used to infer the function of the PKS genes based on the predicted PKS domain architecture and chemical analysis by TLC and/or HPLC. KS domains from PKS clades not previously known in lichens were identified; this included the clade III NR (non-reducing)-PKSs, PR (partially reducing)-PKSs and HR (highly reducing)-PKSs. The discovery of clade III NR-PKSs with C-methyltransferase (CMeT) domain and their wide occurrence in lichens was especially significant. Based on the KS domain phylogenetic analysis and compounds detected in the individual lichens, the clade III NR-PKSs were hypothesized to be involved in the biosynthesis of β-orsellinic acid and methylphloroacetopheno ne - the monoaromatic precursors for many lichen coupled phenolic compounds, such as β-orcinol depsides/depsidones and usnic acid. A strategy has been developed to isolate clade III NR-PKSs directly from environmental lichen DNA using clade III NR-type KS amplified from the degenerate primers (NR3KS-F/R) as homologous probes. Another pair of degenerate primers specific to the CMeT domain of NR-PKSs has also been developed to facilitate the cloning and probing of new clade III NR-PKS genes in lichens. A clade III NR-PKS gene (xsepks1) from X. semiviridis was cloned successfully. This is the first report of the isolation of a full-length PKS gene from environmental lichen DNA. The domain architecture of xsepks1 is KS-AT-ACP-CMeT, as expected for a clade III NR-PKS, suggesting that the newly developed clade-specific primers are useful for cloning new clade III NR-PKS genes and that KS domain phylogenetic analysis can predict the functional domains in PKSs. Attempts were made to characterize the function of xsepks1 by heterologous expression in Aspergillus species. Both A. nidulans (transformed with 5´partial xsepks1 including native promoter) and A. oryzae (transformed with full-length xsepks1 under the regulation of starch-inducible amyB promoter) were tested as potential hosts for the expression of lichen PKS genes. Transcriptional analysis showed that A. nidulans could potentially utilize the lichen PKS gene promoter and both fungal hosts could splice the introns of a lichen PKS gene. Several compounds unique to the A. oryzae transformants carrying xsepks1 were detected, but they could not be reproduced in subsequent fermentations even though the gene was transcribed into mRNA. None of the expected products (β-orsellinic acid, methylphloroacetophenone or similar methylated monoaromatic compounds) was detected in A. oryzae transformants, and the function of xsepks1 remains to be determined. The other clade III NR-PKS genes detected in X. semiviridis cou ld also be responsible for the biosynthesis of β-orsellinic acid or methylphloroacetophenone, as precursors of the major secondary metabolites detected in X. semiviridis (i.e. fumarprotocetraric acid, succinprotocetraric acid and usnic acid). Overall, the work in this thesis demonstrated the prospect of using a molecular approach to access the lichen biosynthetic potential without going through the cumbersome culturing stage.

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.

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.

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

Les cyanobactéries sont des procaryotes photosynthétiques ubiquitaires qui produisent un grand nombre de métabolites secondaires, dont des toxines. Parmi ces cyanotoxines, l'anatoxine-a est une neurotoxine puissante qui provoque une mort rapide après ingestion. La mort est causée par asphyxie car ces alcaloïdes sont de puissants agonistes du récepteur nicotinique de l'acétylcholine.L'équipe, au sein de laquelle j'ai effectué ma thèse, étudie la biosynthèse de l'anatoxine-a et de ses dérivés, chez les cyanobactéries. Des travaux précédents de l'équipe ont permis d'identifier le cluster de gènes responsable de la biosynthèse de l'anatoxine-a et de l'homoanatoxine-a, dans le génome de la cyanobactérie Oscillatoria sp. PCC 6506, une souche productrice d'homoanatoxine-a. Une voie de biosynthèse, à partir de la proline a été proposée par l'équipe.J'ai travaillé sur l'étude des dernières étapes de cette voie de biosynthèse, qui met probablement en jeu une polyketide synthase (PKS) AnaG et une thioestérase AnaA. Lors de ces étapes le précurseur de l'homoanatoxine-a est condensé à une unité acétate, puis subirait une méthylation, une hydrolyse et une décarboxylation, pour donner l'homoanatoxine-a. Néanmoins, la PKS AnaG ne possède ni domaine thioestérase ni domaine décarboxylase, et les dernières étapes de la biosynthèse sont donc mal définies. Nous avons décidé d'exprimer différents domaines d'AnaG chez Escherichia coli pour obtenir plus d'informations sur ces étapes. Nous avons également tenté de préparer un analogue du substrat putatif d'AnaG par synthèse chimique.Par ailleurs, nous avons étudié la biosynthèse de la dihydroanatoxine-a chez Cylindrospermum stagnale PCC 7417. / Cyanobacteria are photosynthetic ubiquiterious prokaryotes which produce a high range of secondary metabolites including toxins. Among these cyanotoxins anatoxin-a is a potent neurotoxin which causes the rapid death on ingestion. The death is caused by respiratory failure because these alkaloid are potent agonists of the nicotinic alcetylcholine receptor. The team in which I did my PhD thesis studies the biosynthesis of anatoxin-a and of its derivatives in cyanobacteria. Preceding works by our team have permitted the identification of the cluster of genes that is responsible for the biosynthesis of anatoxin-a and homoanatoxin-a in the cyanobacterium Oscillatoria sp. PCC 6506. A biosynthetic pathway from proline was also proposed by the team. I have worked on the final stages of this biosynthesis pathway which probably involves a polyketide synthase (PKS), AnaG, and a thioesterase, AnaA. During these stages, the homoanatoxin-a precursor is likely condensed to one acetate unit, and then it is subjected to a methylation, a hydrolysis and a decarboxylation , to yield homoanatoxin-a. The PKS AnaG possesses neither a thioesterase domain nor a decarboxylase domain, and the last steps of the biosynthesis are therefore not well defined. We have chosen to express different domains of AnaG in Escherichia coli to obtain more information on these steps. We have also attempted by chemical synthesis to prepare an analog of the substrate of AnaG. With these tools in hand and with the use of mass spectrometry we hope to be able to confirm the biosynthetic pathway we have put forth. We have also studied the biosynthesis of dihydroanatoxin-a in Cylindrospermum stagnale PCC 7417.

This research is focused on investigation and engineering of natural product biosynthetic pathways for efficient production of pharmaceutically important molecules or generation of new bioactive molecules for drug development. Natural products are an important source of therapeutics, such as chromomycin (anti-cancer), emodin (anti-inflammatory and anti-tumor) and sprolaxine (anti-Helicobacter pylori). Metabolic engineering of natural product biosynthetic pathways shows its promise for creating and producing valuable compounds with chemical diversity for drug discovery. One goal of this research is to create highly efficient strains to biosynthesize valuable natural products. The engineered Streptomyces roseiscleroticus strain constructed in this work showed higher titers of chromomycins than previously reported, which was achieved by characterizing and engineering the chromomycin biosynthetic gene cluster. I activated the polyketide biosynthetic pathway by engineering two regulatory genes, and optimized the culture conditions to increase the titer of chromomycins. The production of emodin nowadays mostly relies on conventional plant cultivation and organic solvent extraction, which is time-consuming and cost-ineffective. This work built a biosynthetic platform using industrial strains Saccharomyces cerevisiae and Pichia pastoris with eight genes from fungi and yeast, which affords a more efficient biosynthetic process of emodin. On the other hand, we used Escherichia coli as a platform for heterologous expression of PKSs and engineering of particular biosynthetic pathways to generate chemical diversity in natural products. The type III polyketide synthase (PKS) involved in the biosynthesis of spirolaxine was identified in this research, which is important for complete elucidation of the biosynthetic pathway of this anti-Helicobacter pylori natural product. Heterologous expression of this PKS in E. coli generated five new pharmaceutically valuable alkylresorcinols. Addition of glucose or pyruvate into the fermentation broths of E. coli expressing another type III PKS StTS resulted in a significant change in the product profiles. Five new products are produced and structurally characterized. Therefore, this work provides a new approach to generating new bioactive molecules in E. coli, the most widely used heterologous expression host.

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.