Many cancers have long been known to display an absolute requirement for the amino acid methionine (L-Met). Studies have shown that in the absence of L-Met, sensitive neoplasms experience cell cycle arrest and perish. Without the metabolic deviations that characterize L-Met auxotrophs, normal cells are able to grow on precursors such as homocysteine and tolerate periods of L-Met starvation. The differential requirement for this amino acid between normal and tumor cells has been exploited through enzymatic serum degradation of L-Met by a bacterial methionine-γ-lyase (MGL). Though MGL was able to deplete L-Met to therapeutically useful levels in animal models and exert a significant cytotoxic effect on malignant cell lines in vitro and on tumor xenografts in vivo, the clinical implementation of this enzyme is hampered by its short serum half-life and potential for catastrophic immune response. In the chapters that follow, we describe the engineering of a novel human methionine degrading enzyme (hMGL) that overcomes the limitations of the bacterial therapeutic. We have shown that hMGL is capable of degrading methionine at a therapeutically useful rate and inducing extensive cell killing in a variety of neoplasms. This enzyme is expected to have low immunogenicity in patients and a high therapeutic index. We have developed a high throughput screen for methionine degrading activity that we can utilize to further engineer the enzyme based on the results of additional preclinical development. We have found that hMGL is also capable of degrading cystine to operate as a dual amino acid depletion treatment that is expected to be more potent than methionine depletion alone. Due to the wide array of neoplasms sensitive to methionine and cystine starvation, the engineered enzyme holds a great deal of promise as a unique and powerful cancer therapeutic. / text

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The use of enzymes, nature´s own catalysts, both isolated or as whole cells to perform chemical transformations is called biocatalysis. As a complement to classical chemical catalysis, biocatalysis can be an environmentally friendly and more economical option in the production and synthesis of chemicals. Research on the application of amine transaminases in synthesis of chiral amines have exploded over the last two decades and interest from the industry is increasing. Amine transaminases are promising catalysts due to their ability to perform reductive amination of ketones with excellent enantioselectivity. For a process to be efficient, high substrate specificity of the applied enzyme is an important factor. A variant of Chromobacterium violaceum amine transaminase that was obtained through rational design has an increased specific activity toward (S)-1-phenylethylamine and a set of 4´-substituted acetophenones. This result makes this variant a promising catalyst for the asymmetric synthesis of similar amines. Amine transaminase catalyzed asymmetric synthesis of amines generally suffers from unfavorable equilibrium. Two methods that include spontaneous tautomerization and biocatalytic amidation for equilibrium displacement have therefore been developed. Efficient assays and screening methods are demanded for the discovery and development of novel amine transaminases. For this purpose, a sensitive fluorescence-based assay that holds promise as a high-throughput screening method was developed. One of the major obstacles for application of enzymes in industrial processes is the instability of the enzyme toward harsh conditions. The stability of Chromobacterium violaceum amine transaminase was investigated and improved using co-solvents and other additives. Co-lyophilization with surfactants was also applied to improve the performance of the same enzyme in organic solvents. / <p>QC 20161017</p>

Les organophosphorés (OPs) sont des neurotoxiques. Leur décontamination est difficile et coûteuse. L’utilisation d’enzymes capables de les détoxifier est une solution élégante. Les enzymes bactériennes sont peu stables et chères à produire. Nous avons identifié des enzymes très résistantes, capable de biodégrader lentement ces composés. Nous avons alors développé une stratégie permettant d'améliorer l'activité de l'enzyme très stable en nous basant sur les enzymes les plus actives. Celle-ci fut améliorée de plus de 1000 fois envers ces insecticides. Enfin, nous avons analysé l'origine de ces améliorations et mis en évidence des concepts émergents dans l'évolution des enzymes. / Organophosphorus compounds are neurotoxic. Their decontamination is difficult and cost prohibitive. An appealing solution resides in the use of enzymes capable of degrading such compounds. Bacterial enzymes are poorly stable and expensive. We identified highly resistant enzymes capable of slowly biodegrading these compounds. We have developped a strategy allowing to increase the activity of our enzyme by using the similarities with the highly active enzymes. The activity was increased by a factor of 1000 against OPs. We analyzed the origins of these ameliorations and showed emergent concepts in enzyme evolution.

Biocatalysis represents an attractive route to the production of various compounds which are difficult or impossible to synthesise and isolate using traditional chemical synthesis. In particular, the production of chiral molecules is a function ideally suited to biocatalysis, due to the natural stereospecificity of enzymes. The synthesis of such chiral molecules is essential in the production of pharmaceuticals, additives for the food and drinks industry and the creation of specialist polymers. CYP105AS1, isolated from Amycolatopsis orientalis, is a cytochrome P450 enzyme which produces the inactive 6-epi-pravastatin of the blockbuster anti-cholesterol drug pravastatin. Previous directed evolution efforts have engineered this enzyme to produce a five-point mutant, known as P450prava, which partially reversed the stereospecificity of the enzyme to produce a majority pravastatin product mixture. This thesis details work to use structure-led engineering approaches to redesign the active site of P450prava to introduce stringent stereospecificity. A combinatorial approach of manual and computational rational design was pursued, leading to the creation of a novel T95F/V180M double mutant of P450prava. This double mutant was found to have successfully eliminated the unwanted 6-epi-pravastatin enantiomer from the product mix, leaving a pure pravastatin product. P450prava was also shown to bind and hydroxylate other statin substrate molecules, demonstrating its versatility in the production of drug metabolites and other high-value oxyfunctionalised molecules. This property, along with its proven tolerance of significant active site engineering efforts, demonstrates the viability of the P450prava as a platform for the creation of novel biocatalysts for the production of various hydroxylated products from diverse substrate molecules.

This study provides insight into the structure/function relationship between desaturases and acetylenases, and indicates amino acid residues within acetylenases which influence reaction outcome. <i>Oleate desaturases</i> belong to a family of enzymes capable of introducing cis double bonds between C12 - C13 in oleate esters. Acetylenases are a subset of oleate desaturase enzymes which introduce a triple bond in the C12 - C13 position of linoleate. To better understand which amino acids could be responsible for differentiating the activity of acetylenases from typical desaturases, a total of 50 protein sequences were used to compare the two classes of enzymes resulting in the identification of 11 amino acid residues which are conserved within either separate family but differ between the two groups of enzymes. These identified amino acid residues were then singularly altered by site-directed mutagenesis to test their role in fatty acid modification. Specifically, the wild type acetylenase, Crep1 from <i>Crepis alpina</i>, and a number of point mutants have been expressed in <i>Saccharomyces cerevisiae</i>, followed by fatty acid analysis of the resulting cultures. Results indicate the importance of 4 amino acid residues within Crep1 (Y150, F259, H266, and V304) with regards to desaturase and acetylenase chemoselectivity, stereoselectivity, and/or substrate recognition. The F259L mutation affected the acetylenase by converting it to an atypical FAD2 capable of producing both cis and trans isomers. The V304I mutation resulted in the conversion of Crep1 into a stereoselective FAD2, where only the cis isomers of 16:2 and 18:2 were produced. The Y150F mutation led to a loss of acetylenase activity without affecting the inherent desaturase activity of Crep1. The H266Q mutation appears to affect substrate selection causing an inability to bind substrate (16:1-9c and/or 18:1-9c) in a cisoid conformation, resulting in an increased accumulation of trans product. The changes in enzyme activity detected in cultures expressing Crep1 mutants demonstrate the profound effect that exchanging as little as one amino acid can have on an enzyme properties. Enzymes retain some conservation of amino acids necessary for activity, such as those involved in metal ion binding, whereas subtle changes can affect overall enzyme function and catalysis.

This study provides insight into the structure/function relationship between desaturases and acetylenases, and indicates amino acid residues within acetylenases which influence reaction outcome. <i>Oleate desaturases</i> belong to a family of enzymes capable of introducing cis double bonds between C12 - C13 in oleate esters. Acetylenases are a subset of oleate desaturase enzymes which introduce a triple bond in the C12 - C13 position of linoleate. To better understand which amino acids could be responsible for differentiating the activity of acetylenases from typical desaturases, a total of 50 protein sequences were used to compare the two classes of enzymes resulting in the identification of 11 amino acid residues which are conserved within either separate family but differ between the two groups of enzymes. These identified amino acid residues were then singularly altered by site-directed mutagenesis to test their role in fatty acid modification. Specifically, the wild type acetylenase, Crep1 from <i>Crepis alpina</i>, and a number of point mutants have been expressed in <i>Saccharomyces cerevisiae</i>, followed by fatty acid analysis of the resulting cultures. Results indicate the importance of 4 amino acid residues within Crep1 (Y150, F259, H266, and V304) with regards to desaturase and acetylenase chemoselectivity, stereoselectivity, and/or substrate recognition. The F259L mutation affected the acetylenase by converting it to an atypical FAD2 capable of producing both cis and trans isomers. The V304I mutation resulted in the conversion of Crep1 into a stereoselective FAD2, where only the cis isomers of 16:2 and 18:2 were produced. The Y150F mutation led to a loss of acetylenase activity without affecting the inherent desaturase activity of Crep1. The H266Q mutation appears to affect substrate selection causing an inability to bind substrate (16:1-9c and/or 18:1-9c) in a cisoid conformation, resulting in an increased accumulation of trans product. The changes in enzyme activity detected in cultures expressing Crep1 mutants demonstrate the profound effect that exchanging as little as one amino acid can have on an enzyme properties. Enzymes retain some conservation of amino acids necessary for activity, such as those involved in metal ion binding, whereas subtle changes can affect overall enzyme function and catalysis.

The search for alternatives to oil-based monomers has sparked interest for scientists to focus on the use of renewable resources for energy production, for the synthesis of polymeric materials and in other areas. With the use of renewable resources, scientists face new challenges to first isolate interesting molecules and then to process them. Enzymes are nature’s own powerful catalysts and display a variety of activities. They regulate important functions in life. They can also be used for chemical synthesis due to their efficiency, selectivity and mild reaction conditions. The selectivity of the enzyme allows specific reactions enabling the design of building blocks for polymers. In the work presented here, a lipase (Candida antarctica lipase B (CalB)) was used to produce building blocks for polymers. An efficient route was developed to selectively process epoxy-functional fatty acids into resins with a variety of functional groups (maleimide, oxetane, thiol, methacrylate). These oligoester structures, based on epoxy fatty acids from birch bark and vegetable oils, could be selectively cured to form thermosets with tailored properties. The specificity of an esterase with acyl transfer activity from Mycobacterium smegmatis (MsAcT) was altered by rational design. The produced variants increased the substrate scope and were then used to synthesize amides in water, where the wild type showed no conversion. A synthetic procedure was developed to form mixed dicarboxylic esters by selectively reacting only one side of divinyl adipate in order to introduce additional functional groups. / <p>QC 20170823</p>

<p>Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose.</p><p>Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns.</p><p>Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.</p>

L’ingénierie métabolique utilise des techniques de clonage pour moduler directement les voies métaboliques des microorganismes dans le but de produire des molécules d’intérêts. Précédemment envisagée pour surproduire des métabolites endogènes, l’ingénierie métabolique est aussi considérée maintenant comme une approche prometteuse pour la biosynthèse de composés non naturels par l'expression de voies métaboliques synthétiques. Cependant, malgré leur évolution au cours de millions d’années, les enzymes sont cependant peu ou pas adaptées aux nouvelles fonctions catalytiques requises par ce métabolisme synthétique. Le but de cette thèse est donc d’améliorer deux enzymes qui sont requises pour la construction et le fonctionnement de voies artificielles conduisant à la biosynthèse de molécules d’intérêts, en particulier le (L)-2,4-dihydroxybutyrate et le 1,3-propanediol, en appliquant des concepts d'ingénieries microbienne et enzymatique. / Metabolic engineering, defined as the rational engineering of organisms towards production goals, has greatly evolved since its conception over three decades ago. Once applied to overproduce cell endogenous metabolites, it is now a promising approach also for the biosynthesis of non-natural compounds through the expression of synthetic metabolic pathways. Improved over billions of years by evolution, enzymes are however less adapted to new catalytic functions as required by synthetic metabolism. The present work was aimed at the construction and optimization of artificial routes for the biosynthesis of two industrially relevant commodity chemicals (L-2,4-dihydroxybutyrate and 1,3-propanediol) through the application of concepts of enzyme rational design, directed evolution and microbial engineering.