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Ph. D. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Hydrocarbon pollution is becoming a growing environmental concern in South Africa and globally. This inadvertently supports the need to identify enzymes for their targeted degradation. The search for novel biocatalysts such as monooxygenases, alcohol dehydrogenases and aldehyde dehydrogenases, have relied on conventional culture-based techniques but this allows sourcing of the biomolecules from only 1-10 % of the microbial population leaving the majority of the biomolecules unaccounted for in 90-99 % of the microbial community. The implementation of a metagenomics approach, a culture-independent technique, ensures that more or less than 100 % of the microbial community is assessed. This increases the chance of finding novel enzymes with superior physico-chemical and catalytic traits. Hydrocarbon polluted soils present a rich environment with an adapted microbial diversity. It was thus extrapolated that it could be a potential source of novel monooxygenases, alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH) involved in hydrocarbon degradation pathways. Therefore, the aim of the study was to extract metagenomic DNA from hydrocarbon contaminated soils and construct a metagenomic fosmid library and screen the library for monooxygenases, alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH). Accordingly, the fosmid library was constructed from metagenome of hydrocarbon-contaminated soil. Then the library was functionally screened using hexadecane, octadecene and cyclohexane as substrates and fifteen positive clones were selected. The fosmid constructs of the positive clones were sequenced using PacBio next generation sequencing platform. The sequences were de novo assembled and analysed using CLC Genomic Workbench. The open reading frames (ORF) of the contigs were identified by blasting the contigs against uniport database. Accordingly, four novel genes namely amo-vut1, aol-vut3, dhy-sc-vut5 and dhy-g-vut7 that showed close similarity with our target enzymes were further analysed in silico and codon-optimized as per Escherichia coli codon preference. The codon adjusted sequences were synthesised and cloned into pET30a(+) expression vector. However, it is worth noting that expression of amo-vut1 was not successful since it was later identified to be a multi-pass member protein, which made it insoluble despite the use of detergent to the effect. There is a need to meticulously genetically engineer amo-vut1 to remove the signal and other membrane-bound peptides while maintaining its activity. Yet the other three constructs were successfully transformed and expressed in E. coli BL21 (DE3). The enzymes were purified and characterized and cocktail for hydrolysis of hexanol was succesfully engineered based on AOL-VUT3, DHY-SC-VUT5 and DHY-G-VUT7. Therefore, novel enzymes were mined from metagenome of fossil-oil contaminated soil and effective hydrocarbon-degrading enzyme cocktails containing their combination were successfully engineered.

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Natural products are secondary metabolites produced by plants and microorganisms that often possess medicinal properties and are implicated in organismal defense. Drawbacks to utilizing natural products in the pharmaceutical industry are difficulties with isolation from biological sources and low yields that can lack stereospecificity from synthetic sources. It is paramount to solve these issues and to develop novel natural products to combat the growing antimicrobial resistance crisis, which was responsible for ~5 million deaths in 2019 alone. One approach is utilizing enzymes to synthesize existing natural products to improve the yields and stereospecificity issue. This dissertation is focused on the biochemical characterization of three enzymes-ZvFMO, OxaD, and CreE-that are implicated in the detoxification of natural products used for organismal defense or participate in the biosynthesis of novel natural products. Each of these enzymes belong to the flavin-dependent monooxygenase (FMO) family, which catalyze the oxygenation of a substrate, generating an oxidized product. ZvFMO, from the insect food crop pest, Zonocerus variegatus, was determined to catalyze a highly uncoupled oxygenation reaction of the nitrogen or sulfur atom of various substrates. OxaD, from Penicillium oxalicum F30, catalyzes novel sequential oxidation reactions of the indole nitrogen of roquefortine C. CreE, from Streptomyces cremeus, also catalyzes sequential nitrogen oxidation reactions to convert L-aspartate to nitrosuccinate en route to biosynthesis of cremeomycin. For each enzyme, the steady-state kinetics have been determined using an oxygen consumption assay and the rapid-reaction kinetics were measured using anaerobic time-resolved spectroscopy. All three enzymes feature a fast flavin reduction step and a slow flavin dehydration step. The oxygenation chemistry of each enzyme was found to proceed through a highly reactive oxygenating species, the C4a-hydroperoxyflavin. Site-directed mutagenesis efforts led to the identification of key active site residues involved in flavin motion and substrate binding, revealing important information about the active site architecture for enzyme engineering applications and drug discovery efforts. / Doctor of Philosophy / Natural products are compounds that are produced by many plants, fungi, and bacteria that have potent medicinal properties and can be used to defend the organism against pests. Unfortunately, using these compounds widely in the pharmaceutical industry is difficult because it is hard to isolate the compound of interest from the organism that produces it and attempts to produce it chemically can result in low yields. Additionally, the overuse of the current natural products, which are most of the antibiotics on the market today, has led to an extreme increase in the resistance of bacteria, fungi, and parasites to the natural product-based drug. Therefore, it is essential that a method is developed to produce novel natural products at high yields to combat the antimicrobial resistance crisis. One method is by using enzymes to generate the natural products of interest. Enzymes are biological catalysts that speed up reactions by ensuring that less energy is required to transition from a reactant to a product and are highly efficient. This dissertation focuses on the characterization of three enzymes that could aid in our understanding of natural product chemistry. All three enzymes insert an oxygen atom on a nitrogen of their respective reactant. The first enzyme ZvFMO, is from an insect and its reactivity causes the insect to become resistant to the natural product-based plant defense mechanism, demonstrating that ZvFMO is a great candidate for inhibitor design. OxaD is the second enzyme and is involved in producing natural products that have antimicrobial and anticancer properties. The last enzyme, CreE, is involved in generating the natural product, cremeomycin, which possesses potent antimicrobial and anticancer properties as well. The reactions of OxaD and CreE positions these enzymes as candidates to produce novel natural products and other efforts to expand their reactivity. The rates of each reaction step have been determined in this work. Key amino acids that contribute to the reaction chemistry and the uptake of the reactant have been identified, laying a solid foundation for drug discovery efforts.

Les coumarines et furocoumarines sont des phytoalexines synthétisées par certaines familles de plantes (ex : Rutacées dont font partie les agrumes), pour se défendre contre les bioagresseurs. Les furocoumarines peuvent être toxiques pour l’homme, lorsqu’elles sont combinées à certains médicaments : c’est l’effet pomelo. Aujourd’hui, la plupart des cytochromes P450 impliqués dans la synthèse des furocoumarines chez les Apiacées, ont déjà été caractérisés. En revanche, malgré l’importance économique des agrumes, nous en savons très peu sur la voie de biosynthèse des coumarines et furocoumarines chez ces plantes. Dans ce travail, nous avons créé, optimisé et validé une méthode d’analyse en chromatographie liquide à ultra haute performance couplée à un spectromètre de masse (UPLC-MS), pour identifier et quantifier 28 coumarines et furocoumarines dans la peau et la pulpe d’agrumes. Cette méthode nous a permis de chémotyper 62 variétés d’agrumes, distinguées par leur faible ou forte capacité de production de ces composés. En parallèle, un travail de bioinformatique sur des banques publiques d’ADN génomique d’agrumes, a permis d’identifier sept gènes présentant de fortes homologies de séquences avec ceux intervenant dans la synthèse des furocoumarines chez Pastinaca sativa (CYP71) et chez Arabidopsis thaliana (CYP82). Une analyse quantitative de leur niveau d’expression chez des agrumes, a montré que quatre d’entre eux étaient plus fortement exprimés chez les fruits fortement producteurs de coumarines et furocoumarines. Le clonage de ces gènes et leur expression hétérologue chez la levure, a révélé la fonction de CYP82D64 de pomelo et de Combava, qui hydroxyle la xanthotoxine pour donner la 5-hydroxy-xanthotoxine. La synthèse des coumarines et furocoumarines chez les agrumes, ainsi mieux appréhendée, nous a permis de proposer un schéma de sélection variétale visant à abaisser les taux de ces composés chez les Citrus. Nous avons aussi montré l’évolution convergente des CYP71 et CYP82 dans leur synthèse chez les Apiacées et les Rutacées respectivement. La découverte du premier cytochrome P450 de Citrus intervenant dans la production de ces composés, ouvre de nouvelles perspectives quant à l’élucidation de leur voie de biosynthèse chez les agrumes / Coumarins and furanocoumarins are phytoalexines synthesized by some plant families (e.g. Rutaceæ that include citrus), to defend themselves against bioaggressors. Furanocoumarins can be toxic for humans, when combined with some drugs: this is the grapefruit juice effect. Nowadays, most of the cytochrome P450s involved in the furanocoumarin synthesis in Apiaceæ, have already been characterized. However, despite the economical importance of citrus, a little is known about the coumarins and furanocoumarins pathway in these plants. In this work, we created, optimized and validated an analytical method by ultra high performance liquid chromatography coupled with mass spectrometry (UPLC-MS), to identify and quantitate 28 coumarins and furanocoumarins in citrus peel and pulp. This method allowed us to chemotype 62 citrus varieties, distinguished by their low or high capacity to produce these compounds. In parallel, a bioinformatic work on public banks of genomic DNA from citrus, allowed to identify seven genes with high sequence homologies with those involved in the synthesis of furanocoumarins in Pastinaca sativa (CYP71) and in Arabidopsis thaliana (CYP82). A quantitative analysis of their expression level in citrus showed that four of them were more expressed in high coumarins and furanocoumarins producing fruits. The cloning of these genes and their heterologous expression in yeast, revealed the function of grapefruit and Combava CYP82D64, which catalyzes the hydroxylation of xanthotoxin in 5-hydroxy-xanthotoxin. The synthesis of coumarins and furanocoumarins in citrus, then better apprehended, allowed us to propose a breeding scheme aiming at decreasing the levels of these compounds in Citrus. We also showed the convergent evolution of CYP71 and CYP82 in their synthesis in Apiaceæ and in Rutaceæ respectively. The discovery of the first cytochrome P450 from Citrus involved in the production of these compounds, opens up new prospects for the elucidation of their biosynthetic pathway in citrus

Les furanocoumarines (FCs) sont des métabolites secondaires principalement synthétisés chez quatre familles botaniques et dérivent de la voie de biosynthèse des phénylpropanoïdes. Ces phytoalexines interviennent dans les processus de défense de la plante et présentent un fort potentiel thérapeutique. Des travaux réalisés dans les années 1960 sur des cultures cellulaires en parallèle de l’utilisation de précurseurs radiomarqués ont permis de démontrer que de nombreuses enzymes impliquées dans cette voie appartenaient à la famille des cytochromes P450 (P450s). Seules deux d’entre elles avaient pu être identifiées d’un point de vue moléculaire au début de ce travail de thèse. Afin de générer des informations concernant le génome de plantes productrices de FCs, nous avons fait séquencer les ARNm extraits de feuilles de Pastinaca sativa, de Ruta graveolens et de Cullen cinereum. L’analyse in silico de ces trois banques de données a permis d’identifier près de 800 fragments d’ADNc codants pour des P450s. Des travaux antérieurs réalisés au laboratoire et l’analyse comparative des transcriptomes de ces 3 plantes nous ont amenés à nous focaliser sur la sous-famille CYP71AZ au travers d’une étude fine de CYP71AZ3 et CYP71AZ4. La caractérisation fonctionnelle de ces enzymes a été réalisée dans un système d’expression hétérologue eucaryote : Saccharomyces cerevisiae. Les résultats obtenus ont permis de montrer que CYP71AZ4 avait une spécificité de substrat assez large puisqu’elle pouvait métaboliser au moins une FC et 4 coumarines. L’analyse et la comparaison des constantes cinétiques pour chacun de ces substrats indiquent néanmoins que le psoralène est le substrat préférentiel. La caractérisation fonctionnelle de CYP71AZ3 a mis en évidence que cette enzyme pouvait hydroxyler l’esculétine, une coumarine, mais ne jouait aucun rôle dans la synthèse de FCs. Ces travaux mettent en évidence la diversité fonctionnelle au sein d’une même sous-famille enzymatique et permettent d’émettre des hypothèses nouvelles quant à l’apparition de cette voie de biosynthèse chez les Apiacées d’une part, et chez les autres familles botaniques d’autre part / Furanocoumarins (FCs) are secondary metabolites mainly synthetized in four botanical families deriving from the phenylpropanoid biosynthetic pathway. These phytoalexins are involved in plant defense mechanisms and present strong therapeutic potential. Early studies in the 1960s based on cell cultures and the use of radiolabeled precursors have shown that many enzymes involved in this pathway belong to the cytochrome P450 family (P450s). Only two of them had been identified from a molecular point of view at the beginning of this thesis. In order to generate information regarding the genome of plants producing FCs, we sequenced the mRNA extracted from leaves of Pastinaca sativa, Ruta graveolens, and Cullen cinereum. In silico analysis of these three libraries identified nearly 800 cDNA fragments encoding for P450s. Previous studies in the laboratory and comparative transcriptome analysis of these three plants have led us to focus on the subfamily CYP71AZ through a detailed study of CYP71AZ3 and CYP71AZ4. Functional characterization of these enzymes was performed in an eukaryote heterologous expression system: Saccharomyces cerevisiae. The results showed that CYP71AZ4 had a broad substrate specificity enough as it could metabolize one FC and 4 coumarins. The analysis and comparison of the kinetic constants for each of these substrates indicate, however, that the preferred substrate is psoralen. The functional characterization of CYP71AZ3 showed that this enzyme could hydroxylate esculetin, a coumarin, but played no role in the synthesis of FCs. This study highlights the functional diversity within a single enzyme subfamily and allows to issue new hypotheses about the emergence of this biosynthetic pathway in Apiaceae on one hand, and among other botanical families on the other hand

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