Succinat und ihre Derivate werden in vielfältiger Weise in den Bereichen Tenside, Lebensmittel, Pharmazeutika und Polymere angewendet. Aufgrund der derzeit kostenintensiven petrochemischen Synthese ist die aerobe nicht-konventionelle Hefe Yarrowia (Y.) lipolytica für die biotechnologische Succinatsynthese von großem Interesse. In der vorliegenden Arbeit wurde das Potential dieser Hefe für eine industrielle Succinatproduktion unter Betrachtung des Einflusses der enzymatischen Aktivitäten von Succinyl-CoA Synthetase und Pyruvat-Carboxylase auf die Succinatsynthese untersucht. Es wurde eine Steigerung der Succinatausbeute um 40 % durch die Erhöhung der Pyruvat-Carboxylase Aktivität um den Faktor 7-8 gemeinsam mit der Deletion des Gens der β Untereinheit der Succinyl-CoA Synthetase im genetisch veränderten Y. lipolytica Stamm H222-AK10 (mcPYC Δscs2) erzielt. Unter Verwendung von Glycerol als C-Quelle wurde eine Erhöhung der Succinatbildung der Transformande H222 AK10 im Vergleich zum Wildtyp von 5,1 ± 0,7 g/l auf 8,7 ± 1,6 g/l nachgewiesen. Die Raum-Zeit-Ausbeute dieses Hefestammes verdoppelte sich von 11,9 ± 1,3 mg/l*h auf 21,9 ± 2,5 mg/l*h. Eine Erhöhung der Sekretion organischer Säuren gelang hingegen nicht durch den alleinigen Verlust der Succinyl-CoA Synthetase Aktivität in den Stämmen H222-AK4 (scs1::URA3), H222-AK8 (scs2:.URA3) und H222-AK9 (scs1::URA3 Δscs2) oder durch die alleinige Aktivitätserhöhung der Pyruvat-Carboxylase in H222-AK1 (mcPYC). Des Weiteren wurde ein Y. lipolytica Stamm erzeugt, der durch die Überexpression der für die Succinyl-CoA Synthetase kodierenden Gene SCS1 und SCS2 charakterisiert ist. Die Transformande H222 AK2 (mcSCS1 mcSCS2) bildete unter den gleichen Kultivierungsbedingungen durchschnittlich 2 g/l weniger Succinat als der Wildtyp (5,1 ± 0,7 g/l). Auch die zusätzliche Erhöhung der Pyruvat-Carboxylase Aktivität um den Faktor 4 in der Transformande H222 AK3 (mcPYC mcSCS1 mcSCS2) konnte den negativen Effekt der erhöhten Gen-Dosen von SCS1 und SCS2 auf die Succinatsynthese nicht aufheben. Dementsprechend wurden für H222-AK3 eine Succinatausbeute von 3,1 ± 0,3 g/l bestimmt.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nos últimos anos o mercado de enzimas tem crescido muito devido a sua utilização em reações específicas importantes para diversos setores industriais. Os micro-organismos, em especial, as leveduras são capazes de produzir uma ampla variedade de enzimas, e assim, tornam-se cada vez mais interessantes para estudos da utilização dos seus compostos. Este trabalho teve como objetivo avaliar a capacidade de produção de enzimas hidrolíticas por leveduras isoladas de solos do Parque Nacional do Viruá e Campus Cauamé, em Roraima. O processo de isolamento permitiu quantificar 6,4x106 UFC/g, contabilizadas em 883 colônias, agrupadas em 11 morfotipos, e identificadas dentro de quatro gêneros: Candida, Cryptococcus, Rhodotorula e Yarrowia. Foram identificados Cryptococcus podzolicus, Candida pseudointermedia e Rhodotorula mucilaginosa; cinco isolados foram identificados como novas espécies, sendo quatro Yarrowia sp. e uma Candida sp., que estão em fase de descrição. Os isolados testados não produziram amilase e celulase pelos testes utilizados. A produção qualitativa de lipase foi observada em 36% dos isolados, representados por Yarrowia spp. (VR546, VR547, VR571 e VR572). A protease foi produzida por cerca de 90% dos isolados: Cryptococcus podzolicus (VR520, VR535 e VR558), Candida sp. (VR533), Candida pseudointermedia (VR544 e VR545) e Yarrowia sp. (VR546, VR547, VR571 e VR572). Três isolados de Cryptococcus podzolicus (VR520, VR535 e VR558) produziram xilanase e pectinase. O potencial multienzimático foi observado em 63% dos isolados, representados por Cryptococcus podzolicus (VR520, VR535 e VR558) e Yarrowia sp. (VR546, VR547, VR571 e VR572). Os resultados alcançados nesta pesquisa corroboram a importância das áreas protegidas como fontes de novas espécies, inclusive com especial interesse para a Biotecnologia. / In recent years the market for enzymes has grown a lot due to their use in specific reactions important for several industrial sectors. The micro-organisms, in particular, the yeasts are able to produce a wide variety of enzymes, and so, become ever more interesting for studies the use of your compounds. This work aimed to assess the capacity of production of hydrolytic enzymes by yeasts isolated from soils of the Viruá National Park and the Campus of Cauamé, in Roraima. The isolation process allowed to quantify 6,4x106 UFC/g, grouped into 11 morphtypes, and identified within four genuses: Candida, Cryptococcus, Rhodotorula and Yarrowia. Were identified Cryptococcus podzolicus, Candida pseudointermedia and Rhodotorula mucilaginosa; five isolates have been identified as new species, being four Yarrowia sp and one Candida sp, which are undergoing description. The isolates tested did not produce amylase and cellulase by tests used. Qualitative lipase production was observed in 36 % of the isolates, represented by Yarrowia spp. (VR546, VR547, VR571 e VR572). The protease was produced about 90% of the isolates: Cryptococcus podzolicus (VR520, VR535 and VR558), Candida sp. (VR533), Candida pseudointermedia (VR544 and VR545) and Yarrowia sp. (VR546, VR547, VR571 and VR572).Three isolates of Cryptococcus podzolicus (VR520, VR535 and VR558) produced xilanase e pectinase. The multienzymatic potential was observed in 63 % of the isolates, represented by Cryptococcus podzolicus (VR520, VR535 and VR558) and Yarrowia SP. (VR546, VR 547, VR571 and VR572). The results of this study confirm the importance of protected areas as sources of new species, including with special interest for biotechnology.

Os lipídios microbianos apresentam composição similar aos óleos vegetais, e tem despertado o interesse de muitos pesquisadores, uma vez que podem suprir a demanda por óleos e gorduras. Além disso, estudos demonstram que ao submeter um microrganismo a baixas doses de radiação gama, estímulos são promovidos, podendo aumentar a função, a resistência celular, e o crescimento. Objetivou-se com esse estudo: avaliar os efeitos de baixas doses de radiação gama no crescimento e na síntese lipídica de Yarrowia lipolytica. A levedura foi submetida às doses de: (controle) 0,3; 0,5; 0,75; 1,0; 1,25 e 2,5 kGy de radiação gama, sob taxa de dose de 0,259 kGy/h. Foi utilizada câmara de Neubauer para determinação da viabilidade celular da levedura. Uma curva de crescimento e outra de sobrevivência foi determinada por densidade óptica em um espectrofotômetro. Posteriormente, a levedura foi inoculada em meio composto por xarope de cana-de-açúcar e vinhaça, e incubada por 7, 14 e 21 dias para a obtenção da biomassa seca, por liofilização, e da síntese lipídica, por metodologia de extração. A irradiação não interferiu na produção de biomassa seca, no crescimento e na sobrevivência da levedura Y. lipolytica. O período de incubação afetou a produção final de biomassa, sendo a maior produção obtida no 14° dia (13,46 g/L). Para os resultados de síntese lipídica, observou-se interferência na produção final, durante 21 dias de incubação e dose de 0,5 kGy, com produção de 4,17 g/L (34,94%). Conclui-se que não foi observado o efeito hormético durante as análises de crescimento, sobrevivência e produção de biomassa, quando submetidas às doses de radiação. Este efeito foi observado apenas durante a síntese lipídica, quando avaliadas as junções entre dose de radiação e período de incubação / Microbial lipids present similar composition to vegetable oils and has aroused great interest among researchers. In addition, studies show that by submitting a microorganism to low doses of gamma radiation, stimuli are promoted, which could increase the function, cell resistance, and the growth. Therefore, this study aimed to evaluate the effects of low doses of gamma radiation on the growth and lipid synthesis of Yarrowia lipolytica. The yeast received irradiation doses of: (control); 0.3; 0.5; 0.75; 1.0; 1.25 and 2.5 kGy gamma radiation dose rate in 0.259 kGy/h. Neubauer Chamber was used to analyze cell viability of the yeast. A growth curve and survival curve was determined by spectrophotometry. Then, the yeast was inoculated in the medium composed of syrup sugarcane and vinasse and incubated for 7, 14 and 21 days for the obtaining of dry biomass, by lyophilization, and lipid synthesis, by extraction methodology. The low doses of irradiation had no effect on the growth, survival, and production of dry biomass of Y. lipolytica. For the incubation periods, there was interference in the final production; the highest production was obtained on day 14 (13.46 g/L). The results for lipid synthesis revealed interference with the final production of lipids, being better at a dose of 0.5 kGy to 21 days incubation with yield of 4.17 g/L (34.94%). In conclusion, the low doses of irradiation had no effect on the growth, survival and biomass production of Y. lipolytica. Hormetic effect was observed only in the lipid synthesis, when the radiation dose was associated with the incubation time

Les composés hydrophobes sont connus comme des sources de carbone qui peuvent être utilisées par les levures comme Yarrowia lipolytica pour de multiples applications. Ces composés causent parfois des perturbations aux levures mais sont aussi rapportés comme conférant aux cellules une certaine résistance contre les stress environnementaux. Dans le cadre de cette thèse, nous avons étudié le rôle de l'oléate de méthyle comme source de carbone sur la résistance de la levure Y. lipolytica en réponse au choc d’un composé amphiphile, la -dodécalactone, et au stress thermique. Les résultats obtenus montrent que les cellules ayant poussé sur oléate sont beaucoup plus résistantes au choc lactone ainsi qu’au stress thermique que les cellules ayant poussé sur glucose. L’action de la lactone se trouve au niveau de la membrane où elle cause une fluidification membranaire et une déplétion de stérols qui sont considérés comme la cause de la mort cellulaire. Ce travail met en évidence le rôle des corps lipidiques dans la réponse cellulaire qui se manifeste de différentes manières en réponse à ces stress. Une accumulation des corps lipidiques est importante pour la résistance de la cellule aux stress. Les cellules ayant poussé sur glucose transforment leur stérol libre en esters de stéryle pour former les corps lipidiques en réponse au choc lactone, ce qui augmente leur sensibilité. Tandis que les cellules ayant poussé sur oléate qui ont accumulé des corps lipidiques pendant leur croissance ont tendance à convertir leurs esters de stéryle en stérol libre pour compenser la déplétion de stérol membranaire causée par la lactone ce qui diminue leur sensibilité. L'homéostasie de l'ergostérol, liée à la présence de corps lipidiques, semble donc jouer un rôle clé dans la résistance cellulaire à ces stress. Ce travail relève aussi que la présence de lipides modifie le processus de mort cellulaire programmée de Y. lipolytica en réponse à un stress thermique. / Hydrophobe compounds are known as carbon source which could be used by yeast like Yarrowia lipolytica for multi purposes. These compounds may cause disturbance in yeast but also reported as confer some resistance to cells towards environmental stress. Here, we study the role of methyl oleate as carbon source on resistance of Y. lipolytica in response to stress of an amphiphilic compound, -dodecalactone, and to heat shock. Results show that cells grown in oleate are more resistant to these stresses than cells grown in glucose. This work reveals the role of the lipids bodies in cells response to stress and that cells manifest in different ways in response to these stresses. An accumulation of lipids bodies is required for the resistance of cells towards stress as glucose grown cells transform their free sterol into steryl esters to form the lipids bodies in response to lactone shock which increases their sensibility towards lactone. In the case of oleate grown cells which accumulated the lipids bodies during their growth, these cells have tendency to convert their steryl esters into free sterol in order to compensate sterol depletion causing by lactone shock and decrease their sensibility. Homeostasis of ergosterol, linked with presence of lipids bodies, seems to be the key for cellular resistance to stresses. This work reveals also that the presence of lipids bodies modifies the processes of programmed cells death in response to heat shock.

Os lipídios microbianos apresentam composição similar aos óleos vegetais, e tem despertado o interesse de muitos pesquisadores, uma vez que podem suprir a demanda por óleos e gorduras. Além disso, estudos demonstram que ao submeter um microrganismo a baixas doses de radiação gama, estímulos são promovidos, podendo aumentar a função, a resistência celular, e o crescimento. Objetivou-se com esse estudo: avaliar os efeitos de baixas doses de radiação gama no crescimento e na síntese lipídica de Yarrowia lipolytica. A levedura foi submetida às doses de: (controle) 0,3; 0,5; 0,75; 1,0; 1,25 e 2,5 kGy de radiação gama, sob taxa de dose de 0,259 kGy/h. Foi utilizada câmara de Neubauer para determinação da viabilidade celular da levedura. Uma curva de crescimento e outra de sobrevivência foi determinada por densidade óptica em um espectrofotômetro. Posteriormente, a levedura foi inoculada em meio composto por xarope de cana-de-açúcar e vinhaça, e incubada por 7, 14 e 21 dias para a obtenção da biomassa seca, por liofilização, e da síntese lipídica, por metodologia de extração. A irradiação não interferiu na produção de biomassa seca, no crescimento e na sobrevivência da levedura Y. lipolytica. O período de incubação afetou a produção final de biomassa, sendo a maior produção obtida no 14° dia (13,46 g/L). Para os resultados de síntese lipídica, observou-se interferência na produção final, durante 21 dias de incubação e dose de 0,5 kGy, com produção de 4,17 g/L (34,94%). Conclui-se que não foi observado o efeito hormético durante as análises de crescimento, sobrevivência e produção de biomassa, quando submetidas às doses de radiação. Este efeito foi observado apenas durante a síntese lipídica, quando avaliadas as junções entre dose de radiação e período de incubação / Microbial lipids present similar composition to vegetable oils and has aroused great interest among researchers. In addition, studies show that by submitting a microorganism to low doses of gamma radiation, stimuli are promoted, which could increase the function, cell resistance, and the growth. Therefore, this study aimed to evaluate the effects of low doses of gamma radiation on the growth and lipid synthesis of Yarrowia lipolytica. The yeast received irradiation doses of: (control); 0.3; 0.5; 0.75; 1.0; 1.25 and 2.5 kGy gamma radiation dose rate in 0.259 kGy/h. Neubauer Chamber was used to analyze cell viability of the yeast. A growth curve and survival curve was determined by spectrophotometry. Then, the yeast was inoculated in the medium composed of syrup sugarcane and vinasse and incubated for 7, 14 and 21 days for the obtaining of dry biomass, by lyophilization, and lipid synthesis, by extraction methodology. The low doses of irradiation had no effect on the growth, survival, and production of dry biomass of Y. lipolytica. For the incubation periods, there was interference in the final production; the highest production was obtained on day 14 (13.46 g/L). The results for lipid synthesis revealed interference with the final production of lipids, being better at a dose of 0.5 kGy to 21 days incubation with yield of 4.17 g/L (34.94%). In conclusion, the low doses of irradiation had no effect on the growth, survival and biomass production of Y. lipolytica. Hormetic effect was observed only in the lipid synthesis, when the radiation dose was associated with the incubation time

In der Hefe Yarrowia lipolytica führen Mutationen im GPR1-Gen zu Essigsäuresensitivität. Die Deletion dieses Genes hat demgegenüber keinen Effekt auf den Phänotyp. In dieser Arbeit wurde das Gpr1-Protein näher charakterisiert. Es zeigte sich, dass GPR1-Mutantenstämme wesentlich schneller Acetat akkumulierten als der Wildtyp. Außerdem konnte bestetigt werden, dass Gpr1p ein integrales Membranprotein ist. Mittels Ortspezifischer Analyse wurden verschiedene funktionelle Bereiche untersucht. Das Protein unterliegt zudem einer Phosphorylierung/Dephosphorylierung. Auf der Grundlage der dargelegten Ergebnisse wurde ein Funktionsmodell für Gpr1p erstellt.

Trans-dominante Mutationen im GPR1-Gen der Hefe Yarrowia lipolytica führen zur Sensitivität der Hefezellen gegenüber Essigsäure. Die Deletion dieses Genes hat dem gegenüber keinen Effekt auf den Phänotyp. In dieser Arbeit wurde das Gpr1-Protein aus Y. lipolytica und dessen Orthologe Ycr010cp, Ydr384cp und Ynr002cp von S. cerevisiae weiter charakterisiert. S. cerevisiae-Transformanden, welche die Mutantenallele GPR1-1 bzw. GPR1-2 exprimierten, zeigten bei gleichzeitiger Anwesenheit von Glucose eine erhöhte Sensitivität gegenüber Essigsäure. Mittels Ort-spezifischer und zufälliger Mutagenese konnten funktionell wichtige Bereiche in den Proteinen Ycr010cp und Ynr002cp identifiziert werden. Die GPR1-Orthologen in S. cerevisiae werden durch verschiedene C-Quellen und voneinander unabhängig reguliert. Die Expression von YCR010c und YDR384c wird weiterhin durch allgemeinen Stress induziert. Die Deletion von zwei oder allen drei Homologen hatte eine Verringerung der Ammoniumproduktion zur Folge. Aufgrund der geringen Ähnlichkeit der Gpr1p-Orthologen zu Ammoniumtransportern wird davon ausgegangen, daß sie selber keine Ammoniumtransporter darstellen. Es wird angenommen, dass die Gpr1p-orthologen Proteine eine regulatorische Funktion haben bzw. Bestandteil einer bisher nicht bekannten Signaltransduktionskette sind.

Trans-dominante Mutationen im GPR1-Gen der Hefe Yarrowia lipolytica führen zur Sensitivität der Hefezellen gegenüber Essigsäure. Die Deletion dieses Genes hat dem gegenüber keinen Effekt auf den Phänotyp. In dieser Arbeit wurde das Gpr1-Protein aus Y. lipolytica und dessen Orthologe Ycr010cp, Ydr384cp und Ynr002cp von S. cerevisiae weiter charakterisiert. S. cerevisiae-Transformanden, welche die Mutantenallele GPR1-1 bzw. GPR1-2 exprimierten, zeigten bei gleichzeitiger Anwesenheit von Glucose eine erhöhte Sensitivität gegenüber Essigsäure. Mittels Ort-spezifischer und zufälliger Mutagenese konnten funktionell wichtige Bereiche in den Proteinen Ycr010cp und Ynr002cp identifiziert werden. Die GPR1-Orthologen in S. cerevisiae werden durch verschiedene C-Quellen und voneinander unabhängig reguliert. Die Expression von YCR010c und YDR384c wird weiterhin durch allgemeinen Stress induziert. Die Deletion von zwei oder allen drei Homologen hatte eine Verringerung der Ammoniumproduktion zur Folge. Aufgrund der geringen Ähnlichkeit der Gpr1p-Orthologen zu Ammoniumtransportern wird davon ausgegangen, daß sie selber keine Ammoniumtransporter darstellen. Es wird angenommen, dass die Gpr1p-orthologen Proteine eine regulatorische Funktion haben bzw. Bestandteil einer bisher nicht bekannten Signaltransduktionskette sind.

The thesis is aimed at understanding of molecular mechanisms of autophagic degradation of peroxisomes (pexophagy) in the yeast Yarrowia lipolytica. This microorganism has been extensively used to explore peroxisome biogenesis (Titorenko and Rachubinski, 2000). Gunkel et al. (1999) intoduced Y. lypolitica into pexophagy studies. However, the field of pexophagic research on this yeast remains quite unexplored. This work involved following tasks: (1) the development and optimization of Y. lipolytica as a model system to study peroxisome degradation; (2) Y. lipolytica genes and proteins implicated in pexophagy should be found and characterized; (3) a proper easy-to-handle selection procedure to isolate novel peroxisome degradation-deficient(pdd) mutants of Y. lipolytica should be devised.

In der Hefe Yarrowia lipolytica führen Mutationen im GPR1-Gen zu Essigsäuresensitivität. Die Deletion dieses Genes hat demgegenüber keinen Effekt auf den Phänotyp. In dieser Arbeit wurde das Gpr1-Protein näher charakterisiert. Es zeigte sich, dass GPR1-Mutantenstämme wesentlich schneller Acetat akkumulierten als der Wildtyp. Außerdem konnte bestetigt werden, dass Gpr1p ein integrales Membranprotein ist. Mittels Ortspezifischer Analyse wurden verschiedene funktionelle Bereiche untersucht. Das Protein unterliegt zudem einer Phosphorylierung/Dephosphorylierung. Auf der Grundlage der dargelegten Ergebnisse wurde ein Funktionsmodell für Gpr1p erstellt.