Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Devido aos incentivos governamentais, os quais impulsionam a produÃÃo de biodiesel, tal combustÃvel tem sido produzido em larga escala. Entretanto, o referido crescimento tem se revelado exacerbado, fazendo surgir um preocupante fator: o destino do glicerol excedente da produÃÃo de biodiesel. Tal inquietaÃÃo se mostra clara ao se revelar um importante dado: para cada tonelada de biodiesel obtido, sÃo gerados 100 Kg de glicerol, o que provoca efeitos adversos à economia do biodiesel. Neste contexto, teme-se que o excesso de glicerina produzida, a qual provoca um elevado nÃvel de poluiÃÃo, possa ser descartada de maneira irresponsÃvel no meio ambiente. Sendo assim, tÃm-se desenvolvido pesquisas destinadas à busca de alternativas para a utilizaÃÃo do volume excedente de glicerol. A bioconversÃo de glicerol por via fermentativa à uma alternativa que agrega um valor significativo para a produtividade da indÃstria de biodiesel. O glicerol pode ser utilizado por inÃmeros micro-organismos, em processos metabÃlicos, como fonte de carbono. Leveduras como as da espÃcie Yarrowia lipolytica, quando cultivadas em meio com limitaÃÃo de nitrogÃnio, sÃo capazes de produzir quantidades significantes de Ãcido cÃtrico a partir do glicerol. O Ãcido cÃtrico Ã, atualmente, um dos mais importantes Ãcidos orgÃnicos produzidos por via fermentativa. Devido Ãs suas caracterÃsticas, tem sido amplamente utilizado na indÃstria de alimentos e bebidas e tambÃm como aditivo em detergentes, produtos farmacÃuticos, cosmÃticos e de higiene pessoal. Desta forma, o presente trabalho teve por objetivo avaliar, atravÃs de fermentaÃÃo, rotas de bioconversÃo do glicerol residual da produÃÃo de biodiesel com elevados nÃveis de impurezas, para obtenÃÃo de Ãcido cÃtrico. Para isso, foram utilizadas, inicialmente, duas cepas de leveduras potencialmente produtoras do Ãcido: Yarrowia lipolytica NRRL YB 323 e Yarrowia lipolytica NRRL YB 423. Utilizando a Metodologia de Planejamento Experimental e AnÃlise da SuperfÃcie de Resposta, foram investigadas as concentraÃÃes iniciais de fonte de carbono e fontes de nitrogÃnio orgÃnico (extrato de levedura) e inorgÃnico (sulfato de amÃnio) em frascos agitados. Os resultados revelaram que a concentraÃÃo inicial ideal de glicerol residual do biodiesel como fonte de carbono, dentro da faixa estudada, foi de 20g/L. Quanto Ãs fontes de nitrogÃnio, pÃde-se constatar que estas nÃo apresentaram notÃvel influÃncia para a produÃÃo do Ãcido. TambÃm verificou-se que a adiÃÃo de Tiamina ao meio nÃo promoveu o aumento na quantidade de Ãcido cÃtrico acumulado. A levedura Yarrowia lipolytica NRRL YB 423 se revelou mais eficaz na produÃÃo do Ãcido. Foram realizados ensaios em fermentador de bancada para avaliar- se a melhor concentraÃÃo de oxigÃnio dissolvido no meio. Viu-se que as concentraÃÃes mais elevadas de oxigÃnio dissolvido no meio fermentativo favorecem a produÃÃo de Ãcido cÃtrico. Para nÃveis de oxigÃnio de 50%, observou-se um menor rendimento, enquanto que, para 70%, a produÃÃo de Ãcido cÃtrico foi favorecida. O rendimento percentual final para a produÃÃo de Ãcido cÃtrico obtido a partir de 20g/L de glicerol residual do biodiesel adicionado inicialmente ao meio foi de 24,80% em trÃs dias de fermentaÃÃo. / Due to government financial incentives, which boost the production of biodiesel, there has been a large scale production of this fuel. However, this growth has proved to be exaggerated, rising a worrying factor: the destination of the glycerol excess from biodiesel production. Such concern is clearly shown to prove an important fact: for every ton of biodiesel produced, 100 kg of glycerol are generated, which leads to adverse effects on the biodiesel economy. In this context, it is feared that the over-produced glycerine, which causes a high level of pollution, can be discarded irresponsibly into the environment. So, researches have been being developed, aiming to find other alternatives for the use of the extra volume of glycerol. The bioconversion of glycerol by fermentation is good option that adds significant value to the productivity of the biodiesel industry. Glycerol can be used by several microorganisms in metabolic processes, as a carbon source. Some yeasts species, such as Yarrowia lipolytica, when grown on a media with a limited source of nitrogen, are able to produce significant amounts of citric acid from glycerol. Citric acid is currently one of the most important organic acids produced by fermentation. Due to its characteristics, it has been widely used in food and beverage industry and also as an additive for detergents, pharmaceuticals, cosmetics and toiletries. Thus, this study aimed to evaluate, through fermentation, bioconversion routes of residual glycerol from biodiesel production with high levels of impurities, in order to obtain citric acid. For this, two potential acid-producing yeast strains (Yarrowia lipolytica NRRL YB 323 and Yarrowia lipolytica NRRL YB 423) were initially used. Using the Methodology of Experimental Design and Response Surface Analysis, it was investigated the initial concentrations of carbon sources as well as organic (yeast extract) and inorganic (ammonium sulfate) nitrogen sources in shake flasks. The results obtained showed that the optimal initial concentration of glycerol from waste biodiesel as carbon source, within the studied range, was 20 g/L. As to the nitrogen sources, they were proved having no remarkable influence on the acid production. It was also found that thiamine addition to the media did not promote the increase on the amount of the previously accumulated citric acid. Yarrowia lipolytica NRRL YB 423 was proved more effective on the acid production. The tests which were carried out in the fermenter aimed to evaluate the optimal concentration of dissolved oxygen in the media. It was observed that highest concentrations of dissolved oxygen in fermentation media, promotes the production of citric acid. For levels of 50% oxygen, there was a lower yield, while for 70%, citric acid production was favored. The final percentage yield for the production of citric acid obtained from 20 g/L of residual glycerol from the biodiesel initially added to the media was 24.80%, at the end of three days of fermentation.

The thesis deals with the study of the different yeast strains from the point of view of extracellular lipolytic enzyme production. First part of this work consisting of appropriate yeasts was developed within study interships in Slovak Academy of Sciences, department of Glycomics in Bratislava. From ten given strains three yeasts such as Pseudozyma fusiformata, Meyerozyma guilliermondii, Yarrowia lipolytica were chosen, these strains showed the highest lipolytic activity and cell growth on basal medium with Tween 80. These yeasts were used for optimization of cultivation conditions and characterization of lipolytic enzymes. The yeasts were cultivated on media with different carbon sources, which appeared to be a most suitable medium the basal medium with Tween. Tween acted as and inducer of lipase production. The substrate specificity was determined using three p-nitrophenylester substrates with varying sizes of the fatty acid site chains. The results showed that tested lipases are probably triacylglycerol-acyl-hydrolases which has the highest activity towards in the water insoluble substrates with medium long chains. The pH optimum and temperature optimum were measured. The results showed that the tested lipases had the highest activity in neutral and mild acid region around 30°C. By measuring of thermal stability has been demonstrated that extracellular lipases are relatively thermostable enzymes. Afterwards the storage stability was measured for 5 weeks when supernatant was kept in fridge at 4°C and in freezing box at -20°C. The results showed that in both cases tested lipases exhibited high storage stability which allows to store the samples without loss of activity for a longer time. Finally, the results of lipolytic and proteolytic activity, cell growth and pH of the medium of yeast Y. lipolytica were compared between the batch cultivation in L-tubes with the continual cultivation in the bioreactor. The highest lipases production was achieved in bioreactor due to the setting conditions of the continual proces to regulate the production and enzymatic stability.

Durant les cultures cellulaires en bioréacteur, la physiologie des micro-organismes et les paramètres physico-chimiques (alimentations en gaz et en substrat, agitation, température, pH, pression) interagissent très fortement. La spécificité des bioréactions microbiennes, en relation avec les couplages irréductibles entre les transferts de chaleur, de matière et de quantité de mouvement, réside dans la complexité (milieu triphasique) et la dynamique (bioréaction autocatalysé) de ces systèmes. L’objectif de ce travail est de progresser dans la compréhension et le contrôle dynamique des intéractions entre les aspects biologiques et les aspects physiques à différentes échelles (macro, micro et moléculaire) pour conduire la réaction biologique vers l’objectif défini (production de biomasse, de métabolites intra ou extra cellulaires, …) et l’optimiser. Les cellules (concentration, forme, dimension, physiologie, …) affectent fortement les propriétés physico-chimiques des moûts et par conséquent, les performances des bioprocédés (vitesses spécifiques, rendements, productivité). Le comportement rhéologique particulier du moût est souvent utilisé pour comprendre l’impact de la biomasse microbienne sur le rendement et les performances du bioprocédé.Dans ce travail, des cultures axéniques, définies comme des cultures pures de microorganismes unicellulaires procaryote et eucaryote, sont considérées. Notre approche s’appuie sur des mesures physiques et physico-chimiques en ligne et hors ligne réalisées sur un bioréacteur instrumenté, mesures qui sont mises en place de façon à respecter les conditions imposées par les contraintes biologiques propres aux microorganismes et à la stratégie de culture choisie. Des cultures d’Escherichia coli et d’Yarrowia. lipolytica, à taux de croissance controlé par l’apport de substrat, ont été réalisées dans une gamme de concentration allant de 0.1 à 100 g l-1. Le bilan qui peut être dressé pour ce travail, tant sur les aspects scientifiques que technologiques, est le suivant :- conception et réalisation d’un outil d’investigation original construit sur la base d’un bioréacteur (20 l) et pourvu d’une boucle de recirculation instrumentée pour la mesure,- identification hydrodynamique (courbes de frottement) de conduites calibrées permettant la viscosimétrie en ligne durant une culture cellulaire, - conception, développement et validation d’un code, LoCoPREL, permettant simultanément le contrôle de la culture cellulaire suivant une stratégie définie, la gestion de séquences de débit dans la boucle de dérivation et l’acquisition des données issues de l’instrumentation spécifique employée,- comparaison des mesures réalisées en ligne à débit constant ou selon des séquences de débit,- mise en évidence du comportement non newtonien des moûts et d’écarts entre les mesures en ligne et hors ligne,- analyse des mesures physiques réalisées en ligne et hors ligne, en lien avec les performances de la culture / During cell cultures in bioreactors, micro-organism physiology closely interacts with physico-chemical parameters such as gas and feed flowrates, mixing, temperature, pH, pressure. The specificity of microbial bioreactions in relation with irreductible couplings between heat and mass transfers and fluid mechanics, led into complex (three-phase medium) and dynamic (auto-biocatalytics reaction) systems. Our scientific approach aims to investigate, understand and control dynamic interactions between physical and biological systems at different scales (macro, micro and molecular) for molecules, strains and/or bioprocess innovation. Cells (concentration, shape, dimension, physiology…) strongly affect physico-chemical properties of broth. Then the modification of these characteristics interacts with bioprocess performances (specific rates, yields…) with an improvement or, more frequently, a decrease of yields. Among these properties, rheological behaviour is a strategy widely used to understand the impact of cells and the derivation of bioprocess performances.In this manuscript, axenic cultures, defined as cultures of a pure and unicellular Prokaryote and Eukaryote microorganisms in bioreactors, are considered. Our approach is based on physical and physico-chemical on-line and off-line measurements in respect with accurate and stringent conditions imposed by cell culture strategy. Escherichia coli and Yarrowia lipolytica cultures were investigated with a control of growth rate by carbon feed in the range from 0.1 up to 100 g l-1. Our scientific and technical actions and results led:- to design and realize an original pilot based on a bioreactor (20 l) with a derivation loop including a specific on-line rheometric device as well as additional physical and biological measurements,- to identify, from a hydrodynamic standpoint, the generalized friction curves of calibrated ducts enabling on-line viscosimetry during cell cultures,- to conceive and validate a homemade software, named LoCoPREL, enabling simultaneously to control cell cultures under defined strategy and to manage flow sequences within the derivation loop,- to discuss and compare on-line physical measurements under constant flow rate and various sequence strategy related to investigated shear-rates,- to highlight about the non-newtonian rheological behaviour of broths and the gap between on-line and off-line measurements,- to analyse on-line and off-line physical measurements in the light of biological performances during fed-batch cultures (mass balance, specific rate, yield).

Les lipases sont des enzymes présentant un grand intérêt industriel. L’intérêt de ces enzymes a conduit à caractériser ces enzymes, à mieux comprendre leur mécanisme réactionnel et leur cinétique, et à établir des méthodes efficaces de production en système d’expression homologue et hétérologue. Plus récemment, l’ingénierie enzymatique permet d’améliorer les caractéristiques des enzymes. Ce thèse s’est fixé deux objectifs principaux: premièrement, la purification et la fonctionnalisation d’acides gras poly-insaturés de type Omega-3 (PUFAs), et spécialement l’acide cis-4, 7, 10, 13, 16, 19-docosahexaénoique (DHA) et deuxièmement la production de lipides structurés (SL). Un premier objectif fut de produire une molécule pharmaceutique, le nicotinyl DHA ester. Le co-substrat du DHA est le nicotinol, un alcool qui après absorption, il est rapidement converti en acide nicotinique (Vitamine B3). La trans-esterification enzymatique entre l’ester éthylique du DHA et le nicotinol a été optimisée dans le but de synthétiser un ester présentant les propriétés cumulatives des deux réactants. Après la sélection de l’enzyme optimale (lipase immobilisée de Candida antarctica; Novozyme 435) et le choix du milieu réactionnel (milieu sans solvant), le procédé a été optimisé. Une conversion supérieure à 97 % a été obtenu en 4 heures avec 45 g.L-1 d’enzyme. Dans ces conditions, une productivité de 4.2 g de produit .h-1.g d’enzyme-1 a été obtenue. Ce projet nécessite une haute pureté en DHA. Un procédé de purification enzymatique a été choisi. Les lipases sont capables de discriminer entre les acides gras en fonction de la longueur de chaine et du degré d’insaturation. Les lipases agissent par résolution cinétique, en réagissant plus efficacement avec les acides gras saturés et mono-insaturés qu’avec les PUFAs résistants. La lipase YLL2 de Yarrowia lipolytica apparait comme un bon candidat car elle est homologue à une des lipases les plus efficaces, la lipase de Thermomyces lanuginosus. YLL2 a permis d’obtenir une discrimination très efficace. Les raisons de la sélectivité de l’enzyme ont été identifiées : il s’agit du positionnement de la double liaison la plus proche de la fonction carboxylique. La concentration en DHA la plus élevée a été obtenue avec YLL2 (73%) avec un pourcentage de récupération du DHA-EE de 89%. YLL2 est par conséquent l’enzyme décrite la plus efficace pour la purification du DHA.La mutagénèse ciblée dans le site actif de YLL2 a été utilisée pour améliorer la sélectivité de cette enzyme. L’analyse de la structure 3D et les alignements avec des lipases homologues a permis de choisir les cibles de mutagénèse dirigée. Les acides aminés cibles ont été changés de manière à restreindre ou élargir le site actif. De ce premier screening de variantes deux positions ont permis d’améliorer la spécificité de l’enzyme, les positions I100 et V235. Finalement la saturation de ces 2 positions a été réalisée. Le dernier objectif de la thèse était la production de SL par acidolysis enzymatique entre l'huile d'olive vierge et les acides caprylic ou capric utilisant la lipase YLL2 immobilisé. Le SL obtenu devrait être riche en acide oléique à la position sn-2 tandis que les C8:0 et C10:0 devraient être principalement estérifiés aux positions sn-1,3. YLL2 immobilisé sur Accurel 1000 a été testé dans un système sans solvant. La réaction d’acidolysis d'huile d'olive avec C8:0 ou C10:0 a été optimisée avec la méthodologie de surface de réponse (RSM). / Lipases are enzymes with applications extended to a wide variety of industries. The variety of lipases applications led to increased research to characterize them and better understand their kinetics and reaction mechanisms and to establish methods for lipase production in homologous and heterologous expression systems. Lately enzymatic engineering allowed the improvement of lipase characteristics. This thesis project studies the use of lipases for two main objectives: lipase-catalyzed purification and functionalization of Omega-3 polyunsaturated fatty acids (PUFAs), especially cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid (DHA) and production of structured lipids (SL). DHA was used for the synthesis of a pharmaceutical molecule, the nicotinyl DHA ester. The co-substrate of the reaction was nicotinol, an alcohol from the group B pro-vitamin, which after absorption is rapidly converted into nicotinic acid (Vitamin B3). The enzymatic trans-esterification of DHA ethyl esters with nicotinol was optimised to synthesise an ester presenting the cumulative properties of the two reactants. After enzyme (immobilized lipase from Candida antarctica; Novozym 435) and reaction medium (solvent-free system) selection, the process was optimised. A conversion to nicotinyl-DHA superior to 97 % was obtained in 4 hours using 45 g.L-1 of enzyme. With a productivity of 4.2 g of product .h-1.g of enzyme-1.This project requires DHA of high purity. Enzymatic purification was chosen for the production of DHA concentrates. Lipases can discriminate between fatty acids in function of their chain length and saturation degree. Lipases react more efficiently with the bulk of saturated and mono-unsaturated fatty acids than with the PUFAs. The objective was the discovery of more specific enzymes for DHA purification. The lipase Lip2 from Yarrowia lipolytica (YLL2) appears as a good candidate since it is homologous to one of the most efficient lipase, the lipase from Thermomyces lanuginosus. YLL2 enables a high discrimination to be obtained, enzyme selectivity being principally due to the positioning of the double-bond the closest from the carboxylic group. The highest concentration of DHA was obtained with YLL2 (73%) with a recovery percentage of DHA-EE of 89%. YLL2 is the most efficient described lipase for DHA purification.Site directed mutagenesis was used to improve YLL2 from Y. lipolytica. Using its three dimensional structure and alignment with homologous lipases, targets for site directed mutagenesis were chosen. Chosen amino acids were substituted by two amino acids of different sizes. From the screening of variants two positions with promising specificities where chosen, positions I100 and V235. Finally saturation of both positions and the analysis of their performances in the selected reactions were carried out. The last objective was the production of SL by enzymatic acidolysis between virgin olive oil and caprylic or capric acids using immobilized Lip2 from Y. lipolytica. The SL obtained should be rich in oleic acid at the sn-2 position while C8:0 and C10:0 should be mainly esterified at the sn-1,3 positions. Lip2 from Y. lipolytica immobilized on Accurel MP 1000 was tested in a solvent-free system. The acidolysis reaction of olive oil with C8:0 or C10:0 was optimized by response surface methodology (RSM)

La compréhension de la fonction des protéines et des systèmes biologiques passe par une connaissance fine des mécanismes moléculaires sous-jacents. La cristallographie et la résonance magnétique nucléaire permettent d'appréhender ces mécanismes au niveau atomique en fournissant des informations sur la structure et sur la dynamique des macromolécules biologiques. Nous nous sommes ainsi intéressés à deux protéines, la lipase lip2 de la levure Yarrowia lipolytica et la protéine membranaire OmpA de la bactérie Klebsiella pneumoniae. Nous avons recherché des conditions d'expression de la protéine lip2 marquée uniformément ou spécifiquement sur une boucle (appelée " lid ") afin d'en étudier la dynamique. Des conditions de marquage uniforme à l'azote 15 de lip2 recombinante dans Yarrowia lipolytica ont été mises au point, mais le marquage acide aminé spécifique n'a pu être réalisé à cause de phénomènes de dilution isotopique trop importants dans cette levure. Nous avons résolu par cristallographie aux rayons X la structure du domaine C-terminal de la protéine OmpA et étudié sa dynamique en solution par RMN (techniques de relaxation 15N). Nous avons caractérisé la dynamique de son domaine N-terminal membranaire reconstitué en liposomes par RMN du solide : en utilisant la rotation à l'angle magique à 60kHz et à la détection 1H sur un spectromètre 1 GHz, nous avons pu attribuer une majorité des résonances du tonneau ? et établir un profil de paramètre d'ordre des vecteurs NH. Des expériences de protéolyse ménagée ont révélé par ailleurs un site de coupure unique à la trypsine au sein de la boucle extracellulaire L3. Enfin, une première caractérisation de la protéine complète exprimée dans la membrane externe d'Escherichia coli a été entreprise par RMN du solide sur membranes externes natives. / Understanding the function of proteins and biological systems requires an accurate knowledge of the underlying molecular mechanisms. Crystallography and nuclear magnetic resonance provide a detailed description of these mechanisms, with an atomic resolution, by providing data on both structures and motions. We investigated two proteins, the lip2 lipase from the yeast Yarrowia lipolytica and the membrane protein OmpA from the bacteria Klebsiella pneumoniae. We tried to produce lip2 with uniform and amino-acid specific stable isotope labelling on its functional loop (the lid) for NMR experiments. The homologous recombinant expression in Yarrowia lipolytica turned out to be the most efficient for uniform labelling but failed for specific labelling due to extensive isotope scrambling. We solved the structure of OmpA C-terminal domain by X-ray crystallography, and analyzed its dynamics in solution by NMR (15N relaxation techniques). We characterized its transmembrane N-terminal domain in proteoliposomes by solid state NMR: using state of the art ultra-fast MAS (60 kHz), 1H detection and a 1 GHz spectrometer, we could assign most ?-barrel resonances and establish a NH order parameter profile. In a complementary approach, we used proteolysis to reveal a unique trypsin cleavage site on the extracellular loop 3. Finally, a first characterization of the full-length protein expressed in the outer membrane of Escherichia coli was initiated by solid state NMR on intact outer membranes.

L’accumulation de lipides chez une espèce levurienne Yarrowia lipolytica souche sauvage a été caractérisée par l’analyse dynamique et systémique des différents états métaboliques identifiés lors des cultures sous conditions environnementales parfaitement maitrisées, à hautes densités cellulaires selon deux stratégies bien distinctes. En premier lieu sur substrat osidique avec le phosphore comme élément inducteur de l’accumulation de lipides, stratégie originale pour déclencher l’accumulation de lipides chez cette souche. Et deuxièmement sur co-susbtrats glucose et oléate et sans aucune limitation nutritionnelle.Ces stratégies de conduites ont permis de dégager les points suivants :- La limitation phosphore déclenche une accumulation en lipides mais aussi en polysaccharides de réserves mobilisables mais non transitoire contrairement à la limitation azote.- La teneur en phosphore de la biomasse catalytique est très variable. De ce fait, le taux de croissance de la biomasse catalytique n’est pas contrôlable par le débit en phosphore.- Le phosphore joue un rôle dans la régulation de l’entrée de glucose dans la cellule, et permet d’éviter la production de citrate lorsque les voies de production de biomasse et de lipides sont en débordement sur une large gamme de rapport C/P (de 0 à 8000 Cmole.mole-1).- La capacité maximale d’accumulation en réserves carbonées chez Y. lipolytica wT est identique quelle que soit la méthode d’accumulation (limitation azote, limitation phosphore, co-substrats glucose / oléate) et est égale à 0,5 Cmole/CmoleX-1. Il existe donc un phénomène de régulation de la levure encore inconnu et limitant l’accumulation en réserves carbonées chez cette souche.Ces résultats ont permis d’identifier des points clés dans l’accumulation en réserves carbonées de cette espèce levurienne et de proposer un mode de conduite original faisant l’objet d’un dépôt de brevet / Lipid accumulation by the yeast Yarrowia lipolytica wT was characterized by dynamic and systemic analysis of different metabolic states in a microbial culture under fully controlled environmental conditions with high cell concentration and under two different strategies:Glucose as the substrate and phosphorus limitation as an inducer of lipid accumulation, an original strategy for lipid accumulation in Y. lipolytica wT.A co-substrate strategy with glucose and oleic acid and without any nutritional limitation.These strategies allowed showing the following points:- Phosphorus limitation triggers a lipid accumulation and a non-transient accumulation of reserve polysaccharide that can be consumed by biomass when necessary, contrary to nitrogen limitation- Phosphorus rate in catalytic biomass shows great variations. Catalytic growth rate cannot be governed by phosphorus input. - Phosphorus has a role in regulating cellular glucose uptake and allows avoiding citric acid production due to overflow of carbon input over a large range of C/P ratios (0 to 8000 Cmol.mol-1)- Maximum capacity of reserve carbon accumulation in Y. lipolytica wT is similar for any culture strategy tested (under nitrogen limitation, phosphorus limitation or with glucose and oleic acid co-substrates) and is equal to 0,5 Cmol/CmolX-1. There is an unknown phenomenon of carbon regulation limiting reserve carbon accumulation in Y. lipolytica wT. Results allowed identifying key points in reserve carbon accumulation in this particular yeast strain and suggesting an original process, claim of a patent

Made available in DSpace on 2017-06-01T18:20:40Z (GMT). No. of bitstreams: 1 edson_rodrigues_vieira.pdf: 14210743 bytes, checksum: c69ef6f544af01646043361a15e15d51 (MD5) Previous issue date: 2013-05-27 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The microbial physiology is a promising area of biotechnology for the synthesis of compounds of high value. The micro-organisms have advantages because of their shorter generation facilities to physiological and genetic changes and the wide variety of metabolic processes. The aim of this study was to degrade oils and fats from lipases produced by Yarrovia lipolytica. Factorial designs were used to investigate the effects of variables in three stages: fermentation for lipase production, bioproduct formulation for stabilization the catalytic action of lipases and treatment of whey for degradation of fats and oils. In the presence of a vegetable oil residue at 3 % and ammonium chloride at 0,2 g.L-1, during 48 h at 28 oC, lipases were produced under submerged cultivation. The cell-free liquid metabolic was stability with sodium sorbate at 0.5 %, glycerol at 5 %, RENEX-95 at 10 %. This bioproduct presented 177 UI.mL-1 of the lipase activity during 30 days of storage at room temperature (27 - 30 °C); it showed optimum pH at 5.0 and 7.0, optimum temperature of 50 oC and thermal stability for 120 min with retention of 100 % of the enzyme activity at 50 oC and pH 5,0. The application of the bioproduct at 7 % during 12 h degraded 98 % of oils and fats present in cheese whey. The lipases produced by Y. lipolytica and formulated with stability agents have potential to be applied in the degradation of oils and fats. / A fisiologia microbiana é uma área promissora da biotecnologia para a síntese de compostos de elevado valor agregado. Os micro-organismos apresentam vantagens devido ao menor período de geração, às facilidades de modificações fisiológicas e genéticas e à grande diversidade de processos metabólicos. O objetivo deste trabalho foi degradar óleos e gorduras de efluente lácteo por lipases produzidas por Yarrovia lipolytica. Planejamentos fatoriais foram utilizados para investigar os efeitos de variáveis em três etapas: em fermentação para produção de lipases, em formulação de bioproduto com atividade lipásica para estabilização da ação catalítica da enzima e em tratamento de soro de queijo para degradação de óleos e gorduras. Na presença de resíduo de refinaria de óleo vegetal a 3 % e cloreto de amônio a 0,2 g.L-1 com 48 h a 28 oC, lipases foram produzidas sob cultivo submerso. O líquido metabólico livre de células com atividade lipásica foi estabilizado com sorbato de sódio a 0,5 %, glicerol a 5 % e RENEX-95 a 10 %. Esse bioproduto apresentou 177 UI.mL-1 da atividade enzimática durante 30 dias sob armazenamento à temperatura ambiente (27 - 30 oC); pH ótimo 5,0 e 7,0, temperatura ótima de 50 oC e estabilidade térmica durante 120 min com retenção de 100 % da atividade enzimática a 50 oC e pH 5,0. A aplicação de 7 % do bioproduto durante 12 h de tratamento de efluente lácteo degradou 98 % dos óleos e gorduras presentes no soro de queijo. As lipases produzidas por Y. lipolytica e formuladas com substâncias estabilizadoras tem potencial para serem aplicadas na degradação de óleos e gorduras.

Une population mondiale croissante et l’épuisement des ressources fossiles a conduit à une augmentation de la demande alimentaire et énergétique. Si les plantes oléagineuses sont majoritairement exploitées pour leurs fruits et leurs graines riches en huiles dans le secteur agroalimentaire, elles sont également valorisées comme alternative aux produits pétrosourcés (biolubrifiants, biocarburants). La production de lipides et d’acides gras inhabituels a rapidement suscité un intérêt envers les organismes unicellulaires : les levures. L’objectif de ce travail consiste à étudier deux modèles biologiques : la graine de lin (Linum usitatissimum), dont l’huile est constituée à 57% d’oméga-3, et la levure oléagineuse Yarrowia lipolytica, exploitée comme châssis biotechnologique. L’approche utilisée pour appréhender le métabolisme lipidique est la fluxomique. De plus, la conception d’un modèle prédictif reposant sur un marquage isotopique (MFA) ou la contrainte (FBA) permet une analyse dynamique du métabolisme. L’étude comparative de trois lignées de lin (teneurs en huile et oméga-3 différentes) a permis une meilleure compréhension des mécanismes menant à l’accumulation des lipides (jusqu’à 44,2 g.100g-1 MS). Ainsi, nous avons pu montrer que l’assimilation du saccharose et la remobilisation de l’amidon sont essentiels à la synthèse des précurseurs et du NADPH nécessaires à la synthèse des AG. Une forte implication de la glycolyse cytosolique et de la voie des pentoses phosphate plastidiale a pu être notée, tandis que la synthèse des protéines et de la paroi cellulaire a été une étape plutôt limitante. De plus, la PDAT semblerait être une enzyme essentielle à l’incorporation d’acides gras polyinsaturés dans les TAG. L’étude de trois souches de Yarrowia lipolytica a également permis d’appréhender le métabolisme de la levure. L’assimilation d’une source de carbone alternative au glucose (glycérol) a entraîné une redirection métabolique majeure vers la néoglucogénèse. Le flux majoritaire pour la synthèse des TAG emprunte la glycolyse et une partie du cycle de Krebs, afin de convertir le citrate en acétyl-CoA. L’optimisation de la voie Kennedy (GPD1 et DGA2) a permis une amélioration du contenu en lipides : +72% par rapport à une souche optimisée pour la synthèse des acides gras inhabituels (expression du gène LRO1, codant pour une PDAT). Les principales voies compétitives sont la synthèse de glucides de réserve et la sécrétion de citrate, réprimée ici grâce à une assimilation de glucose modérée. La PDAT est là encore impliquée dans l’accumulation des acides gras inhabituels. / Growing world population and depletion of fossil resources have led to an increasing food and energy demand. While oleaginous plants are mostly cultivated for their fruits or their seeds in food industry, they are also valued in as an alternative to petrochemicals (biolubricant, biofuels). The production of lipids and unusual fatty acids increased the interest for unicellular organisms: yeasts. The aim of this work is to study two biological models: flax seed (Linum usitatissimum), whose oil is made up of 57% omega-3, and yeast Yarrowia lipolytica, exploited as a biotechnological chassis. The approach used to understand lipid metabolism is fluxomics. In addition, the development of a predictive model based on isotopic labelling (MFA) or constraint-based one (FBA) allows a dynamic analysis of the metabolism. The comparative study of three flax lines (with different oil and omega-3 levels) provided a better understanding of the mechanisms leading to lipid accumulation (up to 44.2 g.100 gDW-1). Therefore, we have been able to show that sucrose assimilation and starch remobilization are essential for fatty acid precursors and cofactors synthesis. Strong involvements of cytosolic glycolysis (G3P, acetyl-CoA) and pentose phosphate pathway (NADPH) have been noted, while protein and cell wall synthesis are limiting steps. In addition, PDAT would be a central enzyme for the incorporation of PUFA into TAGs. The study of three Yarrowia lipolytica strains also helped us to better understand yeast metabolism. The assimilation of an alternative carbone source to glucose, glycerol, led to a major metabolic redirection towards gluconeogenesis. The TAG synthesis flux especially uses glycolysis and a part of TCA cycle to convert citrate into acetyl-CoA. Kennedy pathway optimizations (GPD1 and DGA2 gene overexpression) allowed a lipid content improvement: +72% compared to a strain optimized for the synthesis of unusual fatty acids (LRO1 gene expression, encoding for a PDAT enzyme). The main competitive pathways are carbohydrate synthesis (glycogen) and citrate secretion (here repressed thanks to slow glucose assimilation. PDAT (LRO1 gene) also led to unusual fatty acid accumulation.

Thesis (M.Sc. (Microbiology)) --University of Limpopo, 2011 / Pterocarpus angolensis seed lectin (PAL), a 28 kDa non glycosylated protein, was initially successfully cloned and expressed in E. coli for ease of high protein production. It was discovered, however, as in similar studies that the recombinant PAL yield in E. coli is low and localized intracellularly. This makes extraction even more difficult because most of the protein is lost either when the cell undergoes lysing or when there is incomplete extraction. As a result of the low yields in E. coli, expression vectors were constructed for pal expression in S. cerevisiae, Y. lipolytica and A. niger. Colony PCR of S. cerevisiae transformants confirmed the presence of pal gene whilst sequencing revealed a 66% homology to native PAL. Expression of recombinant PAL in S. cerevisiae, which was expected to be intracellular, was doubtfully unsuccessful since no signal was detected following Western blot analysis. A pBARMTE1-pal expression vector was successfully constructed and could be used for expression studies in Aspergillus niger, however, it was not used in this study. A pal gene whose codons were optimized for Y. lipolytica was synthesized and successfully cloned and expressed in Y. lipolytica. Gene sequence alignment of native pal and the codon optimized pal showed 81% homology whilst the amino acid alignment showed 100% homology. A 31 kDa, recombinant PAL was successfully expressed in Y. lipolytica. The recombinant PAL was approximately 3 kDa larger than native PAL. It was established that this is due to glycosylation of the recombinant PAL. This recombinant protein was found to be more thermostable than native PAL since it demonstrated haemagglutination activity after 10 minutes of exposure in a boiling water bath and only lost activity after 2 hours of exposure to boiling. This study succeeded in producing a more stable extracellular recombinant PAL which demonstrated biochemical activity that was largely similar to that of native PAL but only differed in carbohydrate specificity and haemagglutinating strengths. / Flemish Interuniversity Council (VLIR-UOS)-Own Initiative Project,the SARBIO- South African Regional Co-operation in Biochemistry, Molecular Biology and Biotechnology, the (CSIR) Council for Scientific and Industrial Research,the (NRF) National Research Foundation,(TBI) The Biovac Institute Foundation, and the (SIDA) Swedish International Agency

La levure Yarrowia lipolytica est une levure oléagineuse capable de croître sur les substrats hydrophobes et les composés en C2 comme seul source de carbonne. La première partie de notre étude a permis de déterminer la localisation des protéines Lro1p et Dga1p impliquées dans la dernière étape de la synthèse des triglycérides. Ces protéines sont localisées dans la membrane cytoplasmique et à la surface des corps lipidique pour Lro1p et à la surface des corps lipidique pour Lro1p et à la surface des corps lipidiques pour dga1p. La deuxième partie de cette étude a permis d'avoir une idée plus précise du fonctionnement du cycle du glyoxylate chez la levure Y. lipolytica. Le premier objectif de cette deuxième partie de notre étude était de comprendre le fonctionnement du gène de la malate déshydrogénase chez cette levure. Contrairement à la levure S. cerevisiae qui possède trois gènes codant pour une malate déshydrogénaze, Y. lipolytica ne possède que deux gènes. Le premier gène YALI0D16753g code une malate déshydrogénase mitochondriale et le second gène YALI0E14190g présente une particularité d'épissage alternatif. En effet, le gène YALI0E14190g, en fonction de l'épissage, code une malate déshydrogénase cytoplasmique (séquence C-terminale PAN) ou une malate déshydrogénase adressée aux peroxysomes (séquence C-terminale AKI). Dans une troisième partie, nous nous sommes intéressés aux autres gènes du cycle du glyoxylate. La disruption du gène ICL1 a entrainé une incapacité de croissance du mutant sur acide oléique et sur les composés en C2 (éthanol, acétate). Néanmoins la suppression MLS et CIT2 n'a pas eu d'impact lors de la croissance sur les milieux nécessitant l'implication cycle du glyoxylate.