Die Retrotransposonen sind ubiquitäre Komponenten des eukaryotischen Genoms. In der Hefe Yarrowia lipolytica existiert neben anderen Retrotransposonen das Retrotransposon Ylt1 (Yarrowia lipolytica retrotransposon). Dieses Retrotransposon besteht aus den flankierenden "Long Terminal Repeat" (LTR), zeta von jeweils 714 Nukleotiden und interner Region, eta von 8025 Nukleotiden. Deswegen baut die gesamte Sequenz von 9453 Nukleotiden. Der etwa Bereich enthält einen durchgängigen offenen Leserahm (ORF) (826 bis 8687 Nukleotiden, 2621 Aminosäure). Die LTR Sequenzen enthalten nicht nur TATA-Box, sondern auch ein mögliches Terminations-Signal der Transkription (TAGT) sowie ein Polyadenylierungssignal (AATAAA). Eine Primer Bindungsstelle und ein Polypurin-Bereich sind ebenfalls innerhalb der Ylt1 Sequenz enthalten. Dieses Retrotransposon enthält codierenden Bereiche für ein Gag-Strukturprotein (Gag), Protease (PR), Reverse Transkriptase (RT), RNase H (RH) und Integrase (IN). Das Retrotransposons Ylt1 kommt in verschiedenen Stämmen der Hefe Y. lipolytica vor. Die Transpositionsaktivität dieses Retrotransposon war nachweisbar. Die Aktivität des LTR-Promotors ist von der Kohlenstoffquellen abhängig. Acetat und Ethanol bewirken eine Erhöhung der Promotoraktivität auf das 4fache der basalen Aktivität. Nachdem der Ylt1-ORF unter Kontrolle des ICL1-Promotors exprimiert wurde, konnten jeweils etwa 140 kDa (mit dem Anti HA-Antikörper) bzw. ca. 74 kDa (mit Anti GFP-Antikörper) große Proteine aus den zwei DNA Konstrukte nachgewiesen werden. / The retrotransposons are ubiquitous components of eukaryotic genome. The retrotransposon Ylt1 (Yarrowia lipolytica retrotransposon) was detected in the genome of the dimorphic yeast Yarrowia lipolytica. This retrotransposon is 9453 nucleotides in length and contains two identical long terminal repeats (LTR), zeta of 714 nucleotides and internal region, eta of 8025 nucleotides. The LTR sequences contain not only TATA-Box but also predicted termination signal for transcription (TATG) and polyadenylation signal (AATAAA). The both putative primer binding site (PBS) and polypurine tract were found in the retrotransposon Ylt1 sequence. This retrotransposon contains a single open reading frame (ORF) (826 - 8687 nucleotides, 2621 amino acids), which encodes Gag protein (Gag), protease (PR), reverse transcriptase (RT), RNase H (RH) and integrase (IN). The distribution of retrotransposon Ylt1 among Y. lipolytica strains indicated that the full length elements as well as solo LTR are abundant in several strains. The transposition activity of retrotransposon Ylt1 on acetate as a carbon source was also observed. The promoter activity of the LTR sequence depends on the carbon source. Acetate and ethanol activated 4 fold from the basal activity of the LTR promoter. After expression of Ylt1-ORF under the strong inducible ICL1 promoter from two DNA constructs, which contain HA or GFP epitope, a protein about 140 kDa (with anti HA-antibody) or 75 kDa (with anti GFP-antibody) were detected.

Techn. Universiẗat, Diss., 2001--Dresden.

L’astine C est un peptide cyclique assemblé au cours d’un mécanisme nonribosomique par une synthétase dite NRPS (NonRibosomal Peptide Synthetase). Ce peptide nonribosomique possède des propriétés thérapeutiques intéressantes avec notamment des activités anti-tumorale et anti-inflammatoire. Jusqu’ici ce composé était exclusivement extrait à partir des racines d’Aster tataricus, une plante utilisée traditionnellement en médecine japonaise et chinoise. Récemment, une production d’astine C a été mise en évidence chez Cyanodermella asteris, un champignon endophyte de cette plante. Cette découverte ouvre de nouvelles perspectives en matière de production d’astine C, qui reste néanmoins très limitée en raison du faible taux de croissance du champignon endophyte. Au cours de cette étude, deux approches ont été développées parallèlement afin d’augmenter les taux de production de l’astine C. La première stratégie consistait à augmenter les rendements en optimisant la production homologue à partir de C. asteris. Dans cette optique, un système de culture a été établi afin de cultiver le champignon exclusivement sur un support en acier inoxydable. Ce mode de culture a favorisé à la fois le développement de la biomasse fongique et la production du composé d’intérêt. En vue d’optimiser ce procédé, l’impact de plusieurs paramètres de culture (modalité de préparation du support, type d’inoculum, pH de culture, et composition du milieu de culture) sur la production d’astine C a été évalué. Les paramètres de culture optimisés ont permis d’améliorer de nouveau les rendements en astine C, ce qui constitue une première étape dans le développement d’un procédé de production à l’échelle industrielle. En parallèle, des travaux ont été menés afin de développer un système de production hétérologue d’astine C chez la levure. Cette approche n’a pu être considérée qu’après avoir identifié, par le biais d’analyses bioinformatiques, les gènes impliqués dans la voie de biosynthèse de l’astine. Une fois ces gènes identifiés, une revue de la littérature a permis, entre autres, de dresser un bilan des outils moléculaires disponibles pour le clonage des larges séquences nucléiques codant pour les NRPSs, et de sélectionner des hôtes hétérologues appropriés. Des séquences complète ou partielle du gène codant pour l’astine synthétase ont été clonées respectivement chez Saccharomyces cerevisiae et Yarrowia lipolytica. Pour les deux levures considérées, une expression hétérologue a été constatée. Chez S. cerevisiae, la synthèse de la NRPS d’astine n’a pas pu être démontrée. En revanche, pour la première fois, la production d’une structure de type NRPS chez Y. lipolytica a pu être mise en évidence. Bien qu’aucun peptide nonribosomique n’ait été détecté, cette étude a permis de lever une partie des verrous limitant le développement d’un mode de production hétérologue d’astine chez la levure. / Astin C is a cyclic peptide assembled through a nonribosomal mechanism by a NonRibosomal Peptide Synthetase (NRPS). This nonribosomal peptide displays promising therapeutic properties including anti-tumor and anti-inflammatory activities. For decades, this compound was only extracted from the roots of Aster tataricus, a well-known plant in traditionnal japanese and chinese medicine. Recently, Cyanodermella asteris, a fungal endophyte of this plant, was demonstrated to be able to synthesize astin C. This discovery offers new opportunities for the production of this compound of interest. Nonetheless the very low growth rate of this endophytic fungus is an obstacle limiting the astin C production. In this study, two distinct approaches were conjointly considered to upscale the production rate of this compound. The first strategy was related to an optimization of the homologous production from C. asteris. For this purpose, an innovative cultivation system has been developed enabling to grow the fungus exclusively on a stainless steel support. This cultivation condition turned out to be favorable both for the fungal biomass development and for the astin C production. In order to further optimize this process, the effects of several culture parameters (i.e. support pre-treatment procedure, inoculum type, pH, medium composition) on the astin C production rates was investigated. Under optimized conditions, astin C yields were further enhanced, constituting a first step towards the development of an astin C production process at industrial scale. Meanwhile, a study was conducted in order to develop a heterologous expression system for astin C production in yeast. The identification, through bioinformatics analyses, of the genes involved in the astin biosynthesis was a precondition for the development of this approach. Once these genes have been identified, a literature review has enabled to compile the molecular tools applicable for the cloning of NRPS long lenght sequence, and to select a proper host to heterologously express them. The whole sequence or a truncated one have been transfered respectively to Saccharomyces cerevisiae or Yarrowia lipolytica. In boh considered yeasts, a heterologous expression of the foreign sequences was confirmed. In S. cerevisiae, the synthesis of the astin NRPS could not be demonstrated. On the other hand, in Y. lipolytica, for the first time, the production of a NRPS-type structure was detected. Although no nonribosomal peptide was detected, this study enabled to overcome several of the hurdles limiting the development of a astin heterologous production way in yeast.

A levedura Yarrowia lipolytica tem sido muito investigada, especialmente por ser um microrganismo oleaginoso, ou seja, capaz de acumular grandes quantidades de lipídios, o que ocorre majoritariamente em organelas denominadas partículas lipídicas. Estes lipídios apresentam várias potenciais aplicações biotecnológicas, como por exemplo na produção de óleo microbiano (single cell oil) e na produção de biodiesel. Durante este projeto de mestrado, objetivou-se estudar a fisiologia de duas linhagens da levedura Y. lipolytica, sendo uma tradicionalmente estudada pela comunidade científica internacional (linhagem w29) e outra isolada da Baía da Guanabara, no Rio de Janeiro (linhagem IMUFRJ 50682). Foram realizados cultivos em frascos agitados tipo Erlenmeyer com defletores tampados com algodão (volume total 500 mL, volume de meio 100 mL, 28 oC e 200 rotações por minuto), durante os quais foi possível: 1) escolher um meio de cultivo de composição totalmente definida, com tiamina como único fator de crescimento, adequado a estudos de fisiologia quantitativa com esta levedura; 2) verificar que Y. lipolytica não é capaz de crescer com sacarose ou xilose como única fonte de carbono; 3) verificar que Y. lipolytica cresce com velocidade específica de crescimento máxima (Máx) de 0,49 h-1 num meio complexo contendo glicose, extrato de levedura e peptona (meio YPD), 0,31 h-1 em meio definido com glicose como única fonte de carbono e 0,35 h-1 no mesmo meio, mas com glicerol como única fonte de carbono, sem excreção de metabólitos para o meio de cultivo; 4) verificar que ocorreu limitação por oxigênio nos cultivos em frasco agitado, sendo este o motivo pelo qual as células deixaram de crescer exponencialmente; 5) verificar que o uso de ureia, em vez de sulfato de amônio, como fonte de nitrogênio, contribui para uma variação menor do pH durante os cultivos, sem prejuízo ao crescimento da levedura; 6) observar que, ao se restringir a oferta de nitrogênio à levedura (aumento da relação C/N inicial no meio de 12,6 para 126), as células têm sua morfologia alterada e apresentam maior quantidade de partículas lipídicas; 7) determinar uma composição elementar para a biomassa de Y. lipolytica (CH1,98O0,58N0,13), em que os átomos de carbono encontram-se em média mais reduzidos do que na biomassa de leveduras como Saccharomyces cerevisiae e Dekkera bruxellensis. Foram também realizados cultivos em biorreator em batelada (1 L de volume útil, 28 oC, aerobiose plena e pH controlado em 5,0), durante os quais foi possível: a) estabelecer um protocolo de cultivo para Y. lipolytica em biorreator (que envolvem agitação mecânica, aeração e uso de anti-espumante, entre outras diferenças em relação aos cultivos em frasco); b) confirmar os valores dos principais parâmetros fisiológicos apresentados por esta levedura, anteriormente obtidos a partir de cultivos em frasco; c) confirmar que o fator de conversão de substrato a células (Yx/s) é maior para cultivos realizados com glicerol como fonte única de carbono (0,53 g/g para a linhagem IMUFRJ 50682), do que com glicose (0,48 g/g para a mesma linhagem). Finalmente, cultivos realizados em quimiostato com glicerol como fonte de carbono e energia, limitados por amônio (fonte de nitrogênio, relação C/N 126), às vazões específicas de 0,25 h-1 e 0,15 h-1, permitiram observar que o número de partículas lipídicas por célula de Y. lipolytica permaneceu em torno de 2 em ambas as situações e houve uma diminuição no teor de nitrogênio nas células quando a velocidade específica de crescimento diminuiu de 0,25 para 0,15 h-1. / The yeast Yarrowia lipolytica has been intensively investigated, especially for being an oleaginous microorganism, thus possessing the capacity of accumulating high amounts of lipids, which mainly takes place in organeles known as lipid bodies. These lipids present several potential biotechnological appications, as in the production of single cell oil or biodiesel. During this research project, we aimed at investigating the physiology of two Y. lipolytica strains: w29, traditionally investigated by the international scientific community, and IMUFRJ 50682, isolated from the Guanabara Bay in Rio de Janeiro. Shake-flask cultivations in baffled Erlenmeyer flasks (total volume 500 mL, liquid volume 100 mL, 28 oC and 200 rotations per minute) with cotton stoppers were carried out and allowed us to: 1) choose a fully defined cultivation medium, in which tiamine is the sole growth factor, suitable for quantitative physiological studies with this yeast; 2) verify that Y. lipolytica is not capable of growing on sucrose or xylose as the sole carbon source; 3) observe that Y. lipolytica grows with a maximum specific growth rate (MAX) of 0.49 h-1 in a complex medium containing glucose, yeast extract and peptone (YPD medium), 0.31 h-1 in a defined medium with glucose as the sole carbon source, and 0.35 h-1 in the same medium, but with glycerol as the sole C-source, without excreting metabolites to the cultivation medium; 4) verify that oxygen limitation took place during our shakeflask cultivations and that this caused cells to leave the exponential growth phase; 5) verify that urea can substitute ammonium as the sole nitrogen-source for Y. lipolytica, keeping pH variations less pronounced, without compromising cell growth; 6) observe that cells presented an altered morphology and higher amounts of lipid bodies, when less nitrogen was added to the medium (C/N ratio increased from 12.6 to 126); 7) determine an elemental composition for the biomass of Y. lipolytica (CH1,98O0,58N0,13), in which the average carbon atom was more reduced with respect to the biomass of yeasts such as Dekkera bruxellensis and Saccharomyces cerevisiae. Bioreactor cultivations in batch mode (working volume 1 L, 28 oC, full aerobiosis and pH controlled at 5.0) were also carried out, which allowed us to: a) define a protocol for the cultivation of Y. lipolytica in this system (which involves mechanical agitation, aeration and the use of anti-foam, among other differences with respect to shake-flask cultivations); b) confirm the main physiological parameters presented by this yeast, previously obtained from shake-flask cultivations; c) confirm that the biomass yield on substrate (Yx/s) is higher on glycerol than on glucose (0.53 g/g and 0.48 g/g, respectively). Finally, N-limited chemostat cultivations with glycerol as the carbon and energy source and ammonium as the N-source were also performed (dilution rates of 0.25 h-1 and 0.15 h-1, C/N ratio in the medium of 126), allowing us to verify that the number of lipid particles per cell is around 2 under both conditions and that there was a decrease in the N content in the cells when the specific growth rate decreased from 0.25 to 0.15 h-1.

Biodiesel é um biocombustível que consiste na mistura de ésteres monoalquílicos de ácidos graxos de cadeia longa. O processo usual de produção deste combustível é a transesterificação de óleos vegetais com álcoois de cadeia curta. Nesse processo, a matéria prima deve conter baixo conteúdo de ácido graxos livres ( ≤ 1%) e água (≤ 0,5%). Como alternativa ao processo de transesterificação, destaca-se o emprego de matérias-primas de baixo custo, com elevado teor de ácidos graxos livres, para a síntese de ésteres alquílicos através de reações de esterificação. As reações de produção do biodiesel podem ser catalisadas por via química (ácida e básica) ou enzimática. Na catálise enzimática, os biocatalisadores empregados são as lipases, que catalisam a hidrólise e síntese de ésteres e podem ser obtidas a partir de microrganismos, plantas ou tecido animal, sendo as de origem microbiana as mais utilizadas. O objetivo principal deste trabalho foi avaliar o potencial da lipase de Yarrowia lipolytica, uma levedura não convencional, na síntese de ésteres do ácido oleico visando à obtenção de ésteres alquílicos (biodiesel). Foram estudados os efeitos da temperatura (25, 30, 35, 40, 50 e 60oC), do teor enzimático (5, 10, 20, 30 e 40% v/v) e do tipo de álcool (metanol, etanol, n-propanol e n-butanol ) nas reações de esterificação do ácido oleico empregando o extrato enzimático líquido produzido por Yarrowia lipolytica. Os resultados obtidos mostraram que as reações conduzidas a 30oC e com 10% v/v do extrato enzimático apresentaram maior taxa inicial de reação. Também foi avaliada a utilização do extrato enzimático liofilizado (5% m/v) e do PES (produto enzimático sólido) (5% m/v) de Yarrowia lipolytica na reação de esterificação do ácido oleico com n-butanol a 30oC. O maior consumo de ácido oleico ocorreu na reação conduzida com o PES. O efeito da temperatura (25, 30, 35, 40 e 50oC) na síntese de oleato de butila foi, então, investigado nas reações empregando PES como biocatalisador e a maior conversão de ácido oleico foi verificada na temperatura de 40oC / Biodiesel is a biofuel which consists in a mix of monoalkylesters from long chain fatty acids. The usual method of producing this fuel is transesterification of vegetable oils with lower alcohols. In this process, the raw material should have a low content of free fatty acid (≤ 1%) and water(≤ 0.5%). As an alternative to the transesterification process stands out the use of low cost raw materials with high content of free fatty acids for the synthesis of alkyl esters through esterification reactions. Reactions for biodiesel production can be catalyzed chemically (acid and basic) or enzymatic. In enzymatic catalysis biocatalysts employed are lipases which are enzymes that catalyze hydrolysis and ester synthesis and can be obtained from microorganisms, plants and animal tissue and the most widely used is from microbial origin. The main objective of this study was to evaluate the potential of Yarrowia lipolyticas lipase a non-conventional yeast on the synthesis of esters of oleic acid in order to obtain alkyl esters (biodiesel). Effects of temperature (25, 30, 35, 40, 50 and 60oC) enzyme content (5, 10, 20, 30 and 40% v/v) and type of alcohol (methanol, ethanol, n-propanol and n-butanol) were studied in the esterification reactions of oleic acid employing enzymatic liquid produced by Yarrowia lipolytica. The results demonstrated that reactions conducted at 30oC and with 10% v/v of the enzyme extract showed higher initial reaction rate and higher conversion of oleic acid for all alcohols studied. Use of lyophilized enzyme extract (5%w/v) and SEP(solid enzymatic product) (5% w/v) from Yarrowia lipolytica was also evaluated in the esterification reaction of oleic acid with n-butanol at 30oC. The higher consumption of oleic acid has occurred in the reaction conducted with SEP. The effect of temperature (25, 30, 35, 40 and 50oC) in the synthesis of butyloleate was then investigated in reactions employing SEP as biocatalyst, and the higher conversion of oleic acid was observed at 40oC

Biodiesel é um biocombustível que consiste na mistura de ésteres monoalquílicos de ácidos graxos de cadeia longa. O processo usual de produção deste combustível é a transesterificação de óleos vegetais com álcoois de cadeia curta. Nesse processo, a matéria prima deve conter baixo conteúdo de ácido graxos livres ( ≤ 1%) e água (≤ 0,5%). Como alternativa ao processo de transesterificação, destaca-se o emprego de matérias-primas de baixo custo, com elevado teor de ácidos graxos livres, para a síntese de ésteres alquílicos através de reações de esterificação. As reações de produção do biodiesel podem ser catalisadas por via química (ácida e básica) ou enzimática. Na catálise enzimática, os biocatalisadores empregados são as lipases, que catalisam a hidrólise e síntese de ésteres e podem ser obtidas a partir de microrganismos, plantas ou tecido animal, sendo as de origem microbiana as mais utilizadas. O objetivo principal deste trabalho foi avaliar o potencial da lipase de Yarrowia lipolytica, uma levedura não convencional, na síntese de ésteres do ácido oleico visando à obtenção de ésteres alquílicos (biodiesel). Foram estudados os efeitos da temperatura (25, 30, 35, 40, 50 e 60oC), do teor enzimático (5, 10, 20, 30 e 40% v/v) e do tipo de álcool (metanol, etanol, n-propanol e n-butanol ) nas reações de esterificação do ácido oleico empregando o extrato enzimático líquido produzido por Yarrowia lipolytica. Os resultados obtidos mostraram que as reações conduzidas a 30oC e com 10% v/v do extrato enzimático apresentaram maior taxa inicial de reação. Também foi avaliada a utilização do extrato enzimático liofilizado (5% m/v) e do PES (produto enzimático sólido) (5% m/v) de Yarrowia lipolytica na reação de esterificação do ácido oleico com n-butanol a 30oC. O maior consumo de ácido oleico ocorreu na reação conduzida com o PES. O efeito da temperatura (25, 30, 35, 40 e 50oC) na síntese de oleato de butila foi, então, investigado nas reações empregando PES como biocatalisador e a maior conversão de ácido oleico foi verificada na temperatura de 40oC / Biodiesel is a biofuel which consists in a mix of monoalkylesters from long chain fatty acids. The usual method of producing this fuel is transesterification of vegetable oils with lower alcohols. In this process, the raw material should have a low content of free fatty acid (≤ 1%) and water(≤ 0.5%). As an alternative to the transesterification process stands out the use of low cost raw materials with high content of free fatty acids for the synthesis of alkyl esters through esterification reactions. Reactions for biodiesel production can be catalyzed chemically (acid and basic) or enzymatic. In enzymatic catalysis biocatalysts employed are lipases which are enzymes that catalyze hydrolysis and ester synthesis and can be obtained from microorganisms, plants and animal tissue and the most widely used is from microbial origin. The main objective of this study was to evaluate the potential of Yarrowia lipolyticas lipase a non-conventional yeast on the synthesis of esters of oleic acid in order to obtain alkyl esters (biodiesel). Effects of temperature (25, 30, 35, 40, 50 and 60oC) enzyme content (5, 10, 20, 30 and 40% v/v) and type of alcohol (methanol, ethanol, n-propanol and n-butanol) were studied in the esterification reactions of oleic acid employing enzymatic liquid produced by Yarrowia lipolytica. The results demonstrated that reactions conducted at 30oC and with 10% v/v of the enzyme extract showed higher initial reaction rate and higher conversion of oleic acid for all alcohols studied. Use of lyophilized enzyme extract (5%w/v) and SEP(solid enzymatic product) (5% w/v) from Yarrowia lipolytica was also evaluated in the esterification reaction of oleic acid with n-butanol at 30oC. The higher consumption of oleic acid has occurred in the reaction conducted with SEP. The effect of temperature (25, 30, 35, 40 and 50oC) in the synthesis of butyloleate was then investigated in reactions employing SEP as biocatalyst, and the higher conversion of oleic acid was observed at 40oC

A levedura Yarrowia lipolytica tem sido muito investigada, especialmente por ser um microrganismo oleaginoso, ou seja, capaz de acumular grandes quantidades de lipídios, o que ocorre majoritariamente em organelas denominadas partículas lipídicas. Estes lipídios apresentam várias potenciais aplicações biotecnológicas, como por exemplo na produção de óleo microbiano (single cell oil) e na produção de biodiesel. Durante este projeto de mestrado, objetivou-se estudar a fisiologia de duas linhagens da levedura Y. lipolytica, sendo uma tradicionalmente estudada pela comunidade científica internacional (linhagem w29) e outra isolada da Baía da Guanabara, no Rio de Janeiro (linhagem IMUFRJ 50682). Foram realizados cultivos em frascos agitados tipo Erlenmeyer com defletores tampados com algodão (volume total 500 mL, volume de meio 100 mL, 28 oC e 200 rotações por minuto), durante os quais foi possível: 1) escolher um meio de cultivo de composição totalmente definida, com tiamina como único fator de crescimento, adequado a estudos de fisiologia quantitativa com esta levedura; 2) verificar que Y. lipolytica não é capaz de crescer com sacarose ou xilose como única fonte de carbono; 3) verificar que Y. lipolytica cresce com velocidade específica de crescimento máxima (Máx) de 0,49 h-1 num meio complexo contendo glicose, extrato de levedura e peptona (meio YPD), 0,31 h-1 em meio definido com glicose como única fonte de carbono e 0,35 h-1 no mesmo meio, mas com glicerol como única fonte de carbono, sem excreção de metabólitos para o meio de cultivo; 4) verificar que ocorreu limitação por oxigênio nos cultivos em frasco agitado, sendo este o motivo pelo qual as células deixaram de crescer exponencialmente; 5) verificar que o uso de ureia, em vez de sulfato de amônio, como fonte de nitrogênio, contribui para uma variação menor do pH durante os cultivos, sem prejuízo ao crescimento da levedura; 6) observar que, ao se restringir a oferta de nitrogênio à levedura (aumento da relação C/N inicial no meio de 12,6 para 126), as células têm sua morfologia alterada e apresentam maior quantidade de partículas lipídicas; 7) determinar uma composição elementar para a biomassa de Y. lipolytica (CH1,98O0,58N0,13), em que os átomos de carbono encontram-se em média mais reduzidos do que na biomassa de leveduras como Saccharomyces cerevisiae e Dekkera bruxellensis. Foram também realizados cultivos em biorreator em batelada (1 L de volume útil, 28 oC, aerobiose plena e pH controlado em 5,0), durante os quais foi possível: a) estabelecer um protocolo de cultivo para Y. lipolytica em biorreator (que envolvem agitação mecânica, aeração e uso de anti-espumante, entre outras diferenças em relação aos cultivos em frasco); b) confirmar os valores dos principais parâmetros fisiológicos apresentados por esta levedura, anteriormente obtidos a partir de cultivos em frasco; c) confirmar que o fator de conversão de substrato a células (Yx/s) é maior para cultivos realizados com glicerol como fonte única de carbono (0,53 g/g para a linhagem IMUFRJ 50682), do que com glicose (0,48 g/g para a mesma linhagem). Finalmente, cultivos realizados em quimiostato com glicerol como fonte de carbono e energia, limitados por amônio (fonte de nitrogênio, relação C/N 126), às vazões específicas de 0,25 h-1 e 0,15 h-1, permitiram observar que o número de partículas lipídicas por célula de Y. lipolytica permaneceu em torno de 2 em ambas as situações e houve uma diminuição no teor de nitrogênio nas células quando a velocidade específica de crescimento diminuiu de 0,25 para 0,15 h-1. / The yeast Yarrowia lipolytica has been intensively investigated, especially for being an oleaginous microorganism, thus possessing the capacity of accumulating high amounts of lipids, which mainly takes place in organeles known as lipid bodies. These lipids present several potential biotechnological appications, as in the production of single cell oil or biodiesel. During this research project, we aimed at investigating the physiology of two Y. lipolytica strains: w29, traditionally investigated by the international scientific community, and IMUFRJ 50682, isolated from the Guanabara Bay in Rio de Janeiro. Shake-flask cultivations in baffled Erlenmeyer flasks (total volume 500 mL, liquid volume 100 mL, 28 oC and 200 rotations per minute) with cotton stoppers were carried out and allowed us to: 1) choose a fully defined cultivation medium, in which tiamine is the sole growth factor, suitable for quantitative physiological studies with this yeast; 2) verify that Y. lipolytica is not capable of growing on sucrose or xylose as the sole carbon source; 3) observe that Y. lipolytica grows with a maximum specific growth rate (MAX) of 0.49 h-1 in a complex medium containing glucose, yeast extract and peptone (YPD medium), 0.31 h-1 in a defined medium with glucose as the sole carbon source, and 0.35 h-1 in the same medium, but with glycerol as the sole C-source, without excreting metabolites to the cultivation medium; 4) verify that oxygen limitation took place during our shakeflask cultivations and that this caused cells to leave the exponential growth phase; 5) verify that urea can substitute ammonium as the sole nitrogen-source for Y. lipolytica, keeping pH variations less pronounced, without compromising cell growth; 6) observe that cells presented an altered morphology and higher amounts of lipid bodies, when less nitrogen was added to the medium (C/N ratio increased from 12.6 to 126); 7) determine an elemental composition for the biomass of Y. lipolytica (CH1,98O0,58N0,13), in which the average carbon atom was more reduced with respect to the biomass of yeasts such as Dekkera bruxellensis and Saccharomyces cerevisiae. Bioreactor cultivations in batch mode (working volume 1 L, 28 oC, full aerobiosis and pH controlled at 5.0) were also carried out, which allowed us to: a) define a protocol for the cultivation of Y. lipolytica in this system (which involves mechanical agitation, aeration and the use of anti-foam, among other differences with respect to shake-flask cultivations); b) confirm the main physiological parameters presented by this yeast, previously obtained from shake-flask cultivations; c) confirm that the biomass yield on substrate (Yx/s) is higher on glycerol than on glucose (0.53 g/g and 0.48 g/g, respectively). Finally, N-limited chemostat cultivations with glycerol as the carbon and energy source and ammonium as the N-source were also performed (dilution rates of 0.25 h-1 and 0.15 h-1, C/N ratio in the medium of 126), allowing us to verify that the number of lipid particles per cell is around 2 under both conditions and that there was a decrease in the N content in the cells when the specific growth rate decreased from 0.25 to 0.15 h-1.

La compréhension des organismes vivant a été une quête pendant longtemps. Depuisles premiers progrès des derniers siècles, nous sommes arrivés jusqu’au point où desquantités massives de données et d’information sont constamment générées. Bien que,jusqu’au présent la plupart du travail a été concentré sur la génération d’un catalogued’éléments biologiques, ce n’est pas que récemment qu’un effort coordonné pour découvrirles réseaux de relations entre ces parties a été constaté. Nous nous sommes intéressésà comprendre non pas seulement ces réseaux, mais aussi la façon dont, à partir de sesconnexions, émergent des fonctions biologiques.Ce travail se concentre sur la modélisation et l’exploitation d’un de ces réseaux :le métabolisme. Un réseau métabolique est un ensemble des réactions biochimiquesinterconnectées qui se produisent à l’intérieur, ou dans les proximité d’une cellulevivante. Une nouvelle méthode de découverte, ou de reconstruction des réseaux métaboliquesest proposée dans ce travail, avec une emphase particulière sur les organismeseucaryotes.Cette nouvelle méthode est divisée en deux parties : une nouvelle approche pour lamodélisation de la reconstruction basée sur l’instanciation des éléments d’un modèlesquelette existant, et une nouvelle méthode de réécriture d’association des gènes. Cetteméthode en deux parties permet des reconstructions qui vont au-delà de la capacitédes méthodes de l’état de l’art, permettant la reconstruction de modèles métaboliquesdes organismes eucaryotes, et fournissant une relation détaillée entre ses réactions etses gènes, des connaissances cruciales pour des applications biotechnologiques.Les méthodes de reconstruction développées dans ce travail, ont été complétéespar un workflow itératif d’édition, de vérification et d’amélioration du modèle. Ceworkflow a été implémenté dans un logiciel, appelé Pathtastic.Comme une étude de cas de la méthode développée et implémentée dans le présenttravail, le réseau métabolique de la levure oléagineuse Yarrowia lipolytica, connucomme contaminant alimentaire et utilisé pour la biorestauration et comme usinecellulaire, a été reconstruit. Une version préliminaire du modèle a été générée avecPathtastic, laquelle a été améliorée par curation manuelle, à travers d’un travail avecdes spécialistes dans le domaine de cette espèce. Les données expérimentales, obtenuesà partir de la littérature, ont été utilisées pour évaluer la qualité du modèle produit.La méthode de reconstruction chez les eucaryotes, et le modèle reconstruit deY. lipolytica peuvent être utiles pour les communautés scientifiques respectives, lepremier comme un pas vers une meilleure reconstruction automatique des réseauxmétaboliques, et le deuxième comme un soutien à la recherche, un outil pour desapplications biotechnologiques et comme un étalon-or pour les reconstructions futures. / Understanding living organisms has been a quest for a long time. Since the advancesof the last centuries, we have arrived to a point where massive quantities of data andinformation are constantly generated. Even though most of the work so far has focusedon generating a parts catalog of biological elements, only recently have we seena coordinated effort to discover the networks of relationships between those parts. Notonly are we trying to understand these networks, but also the way in which, from theirconnections, emerge biological functions.This work focuses on the modeling and exploitation of one of those networks:metabolism. A metabolic network is a net of interconnected biochemical reactionsthat occur inside, or in the proximity of, a living cell. A new method of discovery, orreconstruction, of metabolic networks is proposed in this work, with special emphasison eukaryote organisms.This new method is divided in two parts: a novel approach to reconstruct metabolicmodels, based on instantiation of elements of an existing scaffold model, and a novelmethod of assigning gene associations to reactions. This two-parts method allows reconstructionsthat are beyond the capacity of the state-of-the-art methods, enablingthe reconstruction of metabolic models of eukaryotes, and providing a detailed relationshipbetween its reactions and genes, knowledge that is crucial for biotechnologicalapplications.The reconstruction methods developed for the present work were complementedwith an iterative workflow of model edition, verification and improvement. This workflowwas implemented as a software package, called Pathtastic.As a case study of the method developed and implemented in the present work,we reconstructed the metabolic network of the oleaginous yeast Yarrowia lipolytica,known as food contaminant and used for bioremediation and as a cell factory. A draftversion of the model was generated using Pathtastic, and further improved by manualcuration, working closely with specialists in that species. Experimental data, obtainedfrom the literature, were used to assess the quality of the produced model.Both, the method of reconstruction in eukaryotes, and the reconstructed model ofY. lipolytica can be useful for their respective research communities, the former as astep towards better automatic reconstructions of metabolic networks, and the latteras a support for research, a tool in biotechnological applications and a gold standardfor future reconstructions.

Das Interesse an biotechnologisch hergestellter Bernsteinsäure als ein potenzielles intermediäres Ausgangsmaterial und als Alternative zu petrochemischen Produktionsprozessen in der Industrie steigt stetig. Für industrielle Zwecke wird Succinat derzeit noch hauptsächlich petrochemisch aus Maleinsäureanhydrid ausgehend von Butan gewonnen. Aufgrund ihrer Struktur als lineare, gesättigte Dicarbonsäure hat Bernsteinsäure allerdings das Potenzial als sogenannte Building-Block-Chemikalie zu fungieren und das als Ausgangssubstanz für viele Prozesse dienende Maleinsäureanhydrid zu verdrängen. Wichtige Vorstufen für die chemische Synthese, Polyesterproduktion und andere Prozesse können aus Succinat gewonnen werden, eine preiswerte und zu Maleinsäureanhydrid günstigere Bereitstellung von Succinat vorausgesetzt. Aufgrund der zahlreichen Anwendungsmöglichkeiten und der mit der petrochemischen Herstellung verbundenen Nachteile, wie z. B. hohe Kosten und der Einsatz umweltschädlicher Substanzen, ist die Forschung an succinatproduzierenden Organismen durch den voraussichtlichen ökologischen Nutzen und der besseren Kosteneffizienz einer biobasierten Succinatproduktion motiviert. Bernsteinsäure wird natürlicherweise durch viele Mikroorganismen, Pflanzen und Tiere als Intermediat im zentralen Stoffwechsel oder als Stoffwechselendprodukt gebildet. Die Mehrheit der natürlichen und optimierten Produzenten stellen Bakterienstämme dar, die Succinat unter anaeroben Bedingungen akkumulieren. In den letzten Jahren rückten auch Hefen immer stärker in den Fokus der Untersuchungen zur Entwicklung von biotechnologischen Verfahren zur Succinatproduktion. Neben der konventionellen Hefe Saccharomyces cerevisiae, mit der bisher trotz gentechnischer Veränderungen nur maximal 6,3 g Succinat je Liter produziert werden konnte, ist besonders die als Säureproduzent bekannte Hefe Yarrowia lipolytica von Bedeutung. Yarrowia lipolytica ist einzigartig in ihrer Fähigkeit zur Produktion und Sekretion von großen Mengen verschiedener organischer Säuren, wie Citrat, Isocitrat, α-Ketoglutarat und Pyruvat. Aufgrund dieser hohen Sekretionskapazität für Metabolite und Proteine und einer effizienten Verwertung eines breiten Substratspektrums, aber auch wegen ihrer Apathogenität und der guten molekularbiologischen und verfahrenstechnischen Handhabbarkeit ist diese Hefe schon seit einigen Jahren von hohem industriellen Interesse. Neben den bereits genannten organischen Säuren ist Yarrowia lipolytica unter geeigneten Bedingungen auch zur Produktion und Sekretion von Succinat fähig. In der vorliegenden Arbeit sollte das Potenzial dieser Hefe als Succinatproduzent untersucht werden. Der Fokus der Untersuchungen lag dabei vor allem auf dem succinat-metabolisierenden Enzym Succinatdehydrogenase, das im Tricarbonsäurezyklus die Oxidation von Succinat zu Fumarat katalysiert. Zu Beginn der Arbeiten wurden die Auswirkungen einer Reduktion der Aktivität der Succinatdehydrogenase durch das Antibiotikum Carboxin untersucht. Carboxin bewirkt in verschiedenen Organismen eine Hemmung der Succinatdehydrogenase. Dabei bindet Carboxin wahrscheinlich an die Ubichinon-Bindestelle der Succinatdehydrogenase und verhindert so eine Reduktion von Ubichinon. Die Wirkung von Carboxin auf die Succinatdehydrogenase und die Succinatproduktion der Hefe Yarrowia lipolytica wurde bisher allerdings nicht untersucht. Im Rahmen dieser Arbeit wurde jedoch nachgewiesen, dass Carboxin nicht nur auf das Wachstum des Wildtypstamms Yarrowia lipolytica H222, sondern auch auf die spezifische Aktivität der Succinatdehydrogenase hemmend wirkte. Es wurde außerdem gezeigt, dass eine Reduktion der Succinatdehydrogenaseaktivität zu einer deutlichen Steigerung der maximalen Succinatmengen und der Produktbildungsraten führte. So wurden mit 150 μg L-1 Carboxin maximale Succinatmengen von 31,6 ± 5,4 g L-1 mit durchschnittlichen Produktivitäten von 65,5 ± 7,6 mg L-1 h-1 und 4,3 ± 1,7 μg OD-1 h-1 im Vergleich zur Kultivierung ohne Carboxin mit 2,0 ± 0,7 g L-1, 4,3 ± 0,9 mg L-1 h-1 und 0,2 ± 0,1 μg OD-1 h-1 nachgewiesen. Um auf das für großtechnische Verfahren ungeeignete Antibiotikum Carboxin zu verzichten, sollte die Aktivität der Succinatdehydrogenase anschließend durch gezielte gentechnische Veränderungen reduziert werden. Um dies zu erreichen, wurde das Gen SDH2, das für die Eisen-Schwefel-Untereinheit der Succinatdehydrogenase kodiert, unter die Kontrolle ausgewählter Promotoren (des Isocitratlyasegens ICL1 oder des 3-Oxo-Acyl-Thiolasegens POT1) gesetzt, die unter bestimmten Bedingungen, z. B. bei Einsatz von Glycerol als Kohlenstoffquelle nur schwach expremiert werden. Die spezifische Aktivität der Succinatdehydrogenase konnte dadurch um 40 (pICL1-SDH2) bzw. 64 % (pPOT1-SDH2) herabgesetzt werden. Die so konstruierten Yarrowia lipolytica Stämme H222-AZ1 (pICL1-SDH2) und H222-AZ2 (pPOT1-SDH2) produzierten maximal 15,3 ± 0,5 g L-1 bzw. 30,0 ± 3,7 g L-1 Succinat. Die Untersuchungen mit Carboxin und gentechnisch bedingter verringerter Expression zeigten, dass es offenbar einen Zusammenhang zwischen Reduktion der Aktivität der Succinatdehydrogenase und Steigerung der Succinatproduktion gibt. Weiterhin waren die Produktbildungsraten bei beiden Stämmen im Vergleich zum Wildtyp signifikant erhöht. Eine Reduktion der Aktivität der Succinatdehydrogenase – sei es durch Carboxin oder Promotoraustausch – bewirkte außerdem eine Reduktion der Malat- und Fumaratbildung und eine Erhöhung der detektierbaren α-Ketoglutaratgehalte. Auf Basis dieser Ergebnisse wurde geschlussfolgert, dass die Succinatdehydrogenase das Schlüsselenzym zur Beeinflussung der Succinatproduktion zu sein scheint. Diese Schlussfolgerung wurde außerdem durch die Arbeiten von Yuzbashev et al. (2010) unterstützt. Parallel zu diesen Arbeiten wurden außerdem die Effekte der Überexpression der α-Ketoglutaratdehydrogenase, der Isocitratlyase und der Pyruvatcarboxylase untersucht. Trotz einer signifikanten Steigerung der spezifischen Aktivitäten hatten weder eine Überexpression der α-Ketoglutaratdehydrogenase noch der Isocitratlyase eine Steigerung der Succinatproduktion zur Folge. Nur die erhöhte Kopienzahl des Pyruvatcarboxylase kodierenden Gens und eine damit einhergehende Steigerung der Aktivität resultierte in einer geringen Erhöhung der gebildeten Succinatgehalte im Vergleich zum Wildtyp. Bei gleichzeitiger Reduktion der Succinatdehydrogenaseaktivität durch Carboxin wurde diese Tendenz eindeutig bestätigt. Aufgrund dieses Befunds wurde ausgehend von H222-AZ2 ein Stamm konstruiert, in dem zusätzlich zum Austausch des DH2-Promotors mehrere Kopien des Pyruvatcarboxylase-kodierenden Gens PYC1 eingeführt wurden. Die Überexpression von PYC1 resultierte nicht nur in einer gesteigerten Aktivität der Pyruvatcarboxylase, sondern auch in einer weiteren Steigerung der Succinatproduktion im Vergleich zu H222-AZ2. So wurden maximale Succinatmengen von 43,9 ± 7,9 g L-1 mit durchschnittlichen Produktivitäten von 129,4 ± 13,0 mg L-1 h-1 und 8,2 ± 1,5 μg OD-1 h-1 nachgewiesen. Im Vergleich zum Wildtyp entspricht dies einer Steigerung der detektierbaren Succinatmengen um das 34fache. Die in der vorliegenden Arbeit erzielten Ergebnisse verdeutlichen das große Potenzial der Hefe Yarrowia lipolytica für die biotechnologische Herstellung von Succinat. Es wurde gezeigt, dass eine Reduktion der Succinatdehydrogenaseaktivität allein oder kombiniert mit der gesteigerten Pyruvatcarboxylaseaktivität zu einer drastischen Steigerung der Succinatproduktion sowie zu einer Reduktion der Nebenprodukte Fumarat und Malat führte. Es wird davon ausgegangen, dass eine weitere Steigerung durch eine Optimierung der Kultivierungsbedingungen bzw. durch zusätzliche gentechnische Veränderungen möglich ist. So sollte durch nachfolgende gentechnische Veränderungen, z. B. eine erhöhte Expression der Gene der am Glyoxylatzyklus beteiligten Enzyme Malatsynthase und Isocitratlyase, vor allem die weitere Reduktion der Nebenprodukte, besonders eine Reduktion der α-Ketoglutaratgehalte angestrebt werden. Des Weiteren sollten auch die Transportprozesse der organischen Säuren, insbesondere durch die Membranen, untersucht und gegebenenfalls zugunsten der Succinatproduktion manipuliert werden.

The retrotransposon Ylt1 was described previously from the genome of the dimorphic fungus Yarrowia lipolytica. Remarkably, Ylt1 is currently the largest LTR retrotransposon reported from fungal genomes. However, little was known about its biology and its interactions with host genome. So, the aim of this work was the characterization of properties of Ylt1.Analysis of proteins encoded by Ylt1 (Gag protein and integrase) was carried out during this work. To enable their detection, both proteins were tagged with HA epitopes. The sizes of Gag protein and putative precursors of Gag protein and integrase were estimated, and a model for the proteolytic processing of the polyprotein of Ylt1 was proposed. It was shown that Gag protein of Ylt1 is about 2-fold larger than Gag proteins of other studied yeast retrotransposons. An analysis of Ylt1 expression was also performed. Production of the Ylt1 Gag protein under different conditions was analyzed by Western blotting. Expression of Ylt1 occurred on all tested carbon sources. The amount of Ylt1 decreased rapidly upon transition to stationary growth phase, in the presence of copper sulfate and under heat shock conditions. It is suggested that Ylt1 is expressed in actively growing cells, whereas stress conditions have a negative impact on its expression. Such expression pattern was not previously reported for other yeast retrotransposons. Activity of Ylt1 in vivo was characterized using an Ylt1 elements tagged with SUC2 gene of Saccharomyces cerevisiae. Mobilization of the marked Ylt1 element and its transposition from autonomous plasmid into host genome was observed in performed experiments. Obtained results strongly support the idea that Ylt1 is transpositionally active. Formation of tandem repeats by newly inserted Ylt1 elements was observed in several cases. It is suggested that integrase function was affected in this case, and that the integration was mediated by homologous recombination instead. Analysis of the Ylt1 insertion specificity and of the Ylt1 distribution in the genome of Y. lipolytica E150 was done. The remarkable sequence specificity of Ylt1 insertions, which is unusual for LTR retrotransposons, was revealed during this analysis. Also, it was shown that Ylt1 insertions are found mainly in intergenic regions, often at a significant distance (>500 bp) from the next reading frame. No association of Ylt1 insertions with tRNA genes was observed. Searches for Ylt1-related elements in the Y. lipolytica genome database were performed. The novel Ty3/gypsy element Tyl6 was found in the genome of Y. lipolytica E150. The sequence analysis of this element was carried out. It was shown that structural properties of Tyl6 resemble the properties of the Ty3 element of S. cerevisiae. However, two reading frames of Tyl6 (gag and pol) are separated by -1 frame-shift, which was not previously reported for retrotransposons of hemiascomycetous yeasts. Phylogenetic analysis placed Tyl6 within chromoviruses, and the Tse3 element of S. exiguus was shown to be the closest relative of Tyl6. The distribution of Tyl6 among Y. lipolytica strains was analyzed. Interestingly, the novel element was found only in strains derived from the strain YB423-12. The strains of independent origin included in the analysis were shown to be Tyl6-free. The same distribution was previously reported for the retrotransposon Ylt1 and for the DNA transposon Mutyl. Two models of the evolution of transposable elements in Y. lipolytica genome were proposed based on these results.