Submitted in fulfilment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2001. / Kluyveromyces sp. have adapted to existence in milk due to the evolution of permeabilisation and hydrolytic systems that allow the utilisation of lactose, the sugar most abundant in milk. Lactose hydrolysis, to equimolar units of glucose and galactose, is facilitated by a glycoside hydrolase, i.e., β-galactosidase (EC 3.2.1.23). The versatility of this enzyme allows its application in numerous industrial processes, amongst the most significant of which, is its role in the alleviation of lactose intolerance, one of the most prevalent digestive ailments, globally. In this study, β-galactosidase production by Kluyveromyces lactis UOFS y-0939 was initially optimised in shake flask culture with lactose as the sole carbon source, and thereafter, production was scaled up to batch, fedbatch and continuous culture. Shake flask studies revealed optimum conditions of 30°C, pH 7 and a 10% inoculum ratio, to be most favourable for β-galactosidase synthesis, producing a maximum of 0.35 ± 0.05 U.ml-1 when cell lysates were prepared by ultrasonication with glass beads. Batch cultivation in 28.2 and 40 g.L-1 lactose revealed that elevated levels of the carbon source was not inhibitory to β-galactosidase production, as maximum enzyme activities of 1.58 and 4.08 U.ml-1, respectively, were achieved. Cell lysates prepared by ultrasonication and homogenisation were compared and homogenised cell lysates were more than 3.5 fold higher that those prepared by ultrasonication, proving homogenisation to be the superior method for cell disruption. The lactose feed rate of 4 g.L-1.h-1 in fed-batch culture operated at ± 20.4% DO, appeared to be inhibitory to biomass production, as indicated by the lower biomass productivity in fed-batch (0.82 g.L-1.h-1) than batch culture (1.27 g.L-1.h-1). Enzyme titres, however, were favoured by the low DO levels as a maximum of 8.7 U.ml-1, 5.5 fold more than that obtained in batch culture, was achieved, and would be expected to increase proportionally with the biomass. Continuous culture operated at a dilution rate of 0.2 h-1, under strictly aerobic conditions, revealed these conditions to be inhibitory to the lactose consumption rate, however, the non-limiting lactose and high DO environment was favourable for β-galactosidase synthesis, achieving an average of 8 ± 0.9 U.ml-1 in steady state.

Halle, Wittenberg, Universiẗat, Diss., 2005.

Halle, Wittenberg, University, Diss., 2002.

Düsseldorf, Universiẗat, Diss., 2003.

Les fromages au lait cru présentent un écosystème microbien complexe où chaque acteur interagit avec ses voisins et avec le milieu. Parmi ces interactions, l’une d’elle est particulièrement intéressante, l’antagonisme, autrement dit la capacité d’un individu à inhiber la croissance d’un autre. Ces interactions antagonistes sont particulièrement étudiées dans les écosystèmes bactériens fromagers et ont conduit à l’identification de bactéries lactiques productrices de bactériocines. Parmi elles, certaines souches ont révélé des propriétés probiotiques et présentent ainsi un fort potentiel dans la lutte contre les résistances aux antibiotiques. D’un autre côté, l’écosystème fongique des fromages est encore peu étudié. Ce dernier est cependant prometteur, notamment par la possibilité d’abriter des levures productrices de mycocines et/ou possédant des propriétés probiotiques. Dans ce cadre, les présents travaux ont porté sur l’étude d’un fromage du terroir des Hauts-de-France, la Tomme d’Orchies. Ils ont permis la mise en évidence de deux levures antagonistes non-Saccharomyces, présentant un potentiel probiotique. L’écosystème microbien de la Tomme d’Orchies sera décrit et la recherche et la caractérisation de levures antagonistes seront effectuées au sein de son écosystème fongique. Deux souches de Kluyveromyces (K. marxianus S-2-05 et K. lactis S-3-05) seront mises en évidence. Ces dernières présentant un pouvoir antagoniste large, contre des bactéries à Gram positifs et négatifs et contre une levure pathogène. De plus, un haut potentiel d’utilisation comme agents probiotiques a été observé, complété par une activité antioxydante d’intérêt médical, observé chez K. marxianus. / Raw milk cheeses show a complex microbial ecosystem, where each actor interacting with its neighbor and with its environment. Among these interactions, one is particularly interesting, the antagonism, in other words the capacity of an individual to inhibit the growth of another. Antagonistic interactions are massively studied in bacterial cheese ecosystems and lead to the identification of bacteriocin-producing lactic acid bacteria. Among these strains, some revealed probiotics properties and a high potential in the fight against antibiotics resistances. Additionally, cheese fungal ecosystems were poorly studied. However, this ecosystem is promising as it might contain mycocin-producing yeasts and/or showing probiotic properties. In this frame, our work focused on a local French cheese, the “Tomme d’Orchies”. Two non Saccharomyces antagonistic yeasts, with a probiotic potential, were identified in this cheese. Microbial ecosystems of the “Tomme d’Orchies” will be described before presenting the research and the characterization of antagonistic yeast in the fungal ecosystem. Two strains of Kluyveromyces (K. marxianus S-2-05 and K. lactis S-3-05) will be highlighted, with broad antagonistic properties, against Gram positive and negative bacteria, but also against pathogenic yeast. A high potential to be used as probiotic agents could be observed, in addition to antioxidative properties for K. marxianus, with medical relevance.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-07-17T16:04:41Z No. of bitstreams: 1 texto completo.PDF: 305457 bytes, checksum: 62f65ddfb7b0cad1f80dadfaef449290 (MD5) / Made available in DSpace on 2017-07-17T16:04:41Z (GMT). No. of bitstreams: 1 texto completo.PDF: 305457 bytes, checksum: 62f65ddfb7b0cad1f80dadfaef449290 (MD5) Previous issue date: 2001-09-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Foi isolado um mutante auxotrófico para uracila de Kluyveromyces lactis com potencial para ser hospedeiro em sistemas de expressão heteróloga de proteínas recombinantes. Uma população selvagem de K. lactis foi irradiada com luz ultravioleta e selecionada na presença de ácido 5 fluoro orótico (5FOA). Dez colônias auxotróficas para uracila e resistentes a 5FOA foram selecionadas. Todas cresceram em meio utilizando lactose como única fonte de carbono e exibiram atividade de β galactosidase normal em relação à célula selvagem. Os mutantes apresentaram velocidades específicas de crescimento inferiores a da célula selvagem em soro de queijo ultrafiltrado (SUF), o que indicou ser o soro de queijo um meio limitante em uracila, afetando o crescimento dos mutantes. O mutante M7 foi selecionado por apresentar uma freqüência de reversão baixa (1,7 x 10 -10 ). A complementação da auxotrofia na linhagem M7 mediante transformação com o plasmídeo PTG802 contendo o gene URA3 de S. cerevisiae foi analisada. Melhor eficiência de transformação de K. lactis foi obtida com o método de choque térmico com acetato de lítio, polietilenoglicol e DNA carreador fita simples. Um transformante mostrou viiiintegração do plasmídeo em um fragmento de 6,0 Kb. A velocidade de crescimento da célula mutante (0,04 h -1 ) em SUF foi inferior a da célula selvagem (0,26 h -1 ). Estes resultados sugerem que o SUF é limitante em uracila. Conseqüentemente a transformação dos mutantes, com o gene URA3, fisiologicamente causou aumento na velocidade especifica de crescimento (0,22-0,25h -1 ) quando cultivadas em SUF, atingindo os níveis da célula selvagem e evidenciando a complementação da auxotrofia. / A Kluyveromyces lactis uracil auxotrophic mutant was isolated with potential as host for heterologous recombinant protein expression systems. A wild-type K. lactis population was irradiated with ultraviolet light and mutants selected in the presence of 5-fluoroorotic acid (5FOA). Ten uracil auxotrophic colonies resistant to 5FOA were selected. All grew in medium with lactose as sole carbon source and exhibited β-galactosidase activity comparable to that of the wild-type cell. The mutants presented specific growth rates lower than the wild-type cell when grown in ultrafiltered cheese whey (SUF), indicating that cheese whey is uracil limited, thereby affecting mutant growth. Mutant M7 was selected since it presented a low reversion frequency (1.7 x 10 -10 ). Auxotrophic complementation of the M7 lineage, through transformation with the PTG802 plasmid containing the URA3 gene from S. cerevisiae was analyzed. Better K. lactis transformation efficiency was obtained with the thermal shock method with lithium acetate, polyethylene glycol and single stranded DNA carrier. One transformed cell presented a 6.0 kb fragment integrated into the plasmid. Transformed cells grown in SUF presented specific growth rates (0,22-0,25 h -1 ) similar to that of the wild-type cell. (0,26 h -1 ) and superior to that of the untransformed mutant cell (0,04 h -1 ). Consistent with this observation, transformation of the mutants with the URA3 gene physiologically causes an increase in specific growth, when grown in SUF, reaching levels of the wild-type cell and exhibiting auxotrophic complementation. / Dissertação importada do Alexandria

Submitted in fulfilment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2001. / Kluyveromyces sp. have adapted to existence in milk due to the evolution of permeabilisation and hydrolytic systems that allow the utilisation of lactose, the sugar most abundant in milk. Lactose hydrolysis, to equimolar units of glucose and galactose, is facilitated by a glycoside hydrolase, i.e., β-galactosidase (EC 3.2.1.23). The versatility of this enzyme allows its application in numerous industrial processes, amongst the most significant of which, is its role in the alleviation of lactose intolerance, one of the most prevalent digestive ailments, globally. In this study, β-galactosidase production by Kluyveromyces lactis UOFS y-0939 was initially optimised in shake flask culture with lactose as the sole carbon source, and thereafter, production was scaled up to batch, fedbatch and continuous culture. Shake flask studies revealed optimum conditions of 30°C, pH 7 and a 10% inoculum ratio, to be most favourable for β-galactosidase synthesis, producing a maximum of 0.35 ± 0.05 U.ml-1 when cell lysates were prepared by ultrasonication with glass beads. Batch cultivation in 28.2 and 40 g.L-1 lactose revealed that elevated levels of the carbon source was not inhibitory to β-galactosidase production, as maximum enzyme activities of 1.58 and 4.08 U.ml-1, respectively, were achieved. Cell lysates prepared by ultrasonication and homogenisation were compared and homogenised cell lysates were more than 3.5 fold higher that those prepared by ultrasonication, proving homogenisation to be the superior method for cell disruption. The lactose feed rate of 4 g.L-1.h-1 in fed-batch culture operated at ± 20.4% DO, appeared to be inhibitory to biomass production, as indicated by the lower biomass productivity in fed-batch (0.82 g.L-1.h-1) than batch culture (1.27 g.L-1.h-1). Enzyme titres, however, were favoured by the low DO levels as a maximum of 8.7 U.ml-1, 5.5 fold more than that obtained in batch culture, was achieved, and would be expected to increase proportionally with the biomass. Continuous culture operated at a dilution rate of 0.2 h-1, under strictly aerobic conditions, revealed these conditions to be inhibitory to the lactose consumption rate, however, the non-limiting lactose and high DO environment was favourable for β-galactosidase synthesis, achieving an average of 8 ± 0.9 U.ml-1 in steady state.

A levedura Kluyveromyces marxianus, apesar de apresentar propriedades fisiológicas vantajosas para a produção heteróloga de proteínas, foi utilizada apenas poucas vezes como hospedeira na síntese dessa classe de moléculas. Em contrapartida, a sua congênere Kluyveromyces lactis possui mais de 40 sistemas de expressão desenvolvidos, inclusive comerciais. Além disso, não há literatura disponível sobre glicosilação de proteínas em K. marxianus. Levando-se isso em consideração, este trabalho visou a desenvolver sistemas para a expressão heteróloga da enzima glicose oxidase (GOX) de Aspergillus niger e de uma esterase termófila (EST) de Thermus thermophilus em K. marxianus. A linhagem K. lactis CBS 2359 foi utilizada como parâmetro de comparação em todos os sistemas de expressão construídos. Primeiramente, foi realizado um estudo fisiológico com a finalidade de selecionar, dentre três linhagens de K. marxianus pré-selecionadas a partir de informação da literatura, a que apresentasse as melhores características fisiológicas para se tornar uma hospedeira de expressão heteróloga. A linhagem selecionada foi a CBS 6556, baseando-se numa combinação das seguintes características: velocidade específica de crescimento, formação de metabólitos, rendimento de substrato em biomassa e secreção da enzima homóloga inulinase. Após, foram construídos dois sistemas de expressão epissomais. No primeiro, o gene era expresso sob controle do promotor PGK de S. cerevisiae e no segundo, sob controle de INU1 de K. marxianus. Um sistema integrativo foi utilizado, no qual a expressão era dirigida pelo promotor INU1. Estudos bioquímicos e de glicosilação foram realizados nas enzimas produzidas. Em relação aos sistemas para expressão de GOX, foram alcançados níveis de produção de 1722 U/gMS (unidades por grama de biomassa seca) em K. marxianus transformado com o sistema epissomal no qual a expressão era controlada pelo promotor INU1. As caracterizações bioquímicas da enzima mostraram que a molécula produzida apresentava propriedades semelhantes à enzima homóloga de A. niger. Além disso, os estudos de glicosilação mostraram uma menor tendência de hiperglicosilação de K. marxianus quando comparada com K. lactis. Já em relação à esterase, K. lactis apresentou maiores níveis de expressão (294 U/gMS), porém a enzima produzida em K. marxianus apresentou temperatura ótima de atividade (50 °C) ligeiramente superior à enzima produzida por sua congênere (45 °C), temperaturas abaixo da qual ocorre maior atividade da enzima homóloga (65 °C). Isso pode ser explicado pela glicosilação exercida por ambas espécies de leveduras sobre a proteína, ao contrário da homóloga, não glicosilada. Além disso, os produtos das leveduras apresentaram três padrões de glicosilação. Dessa forma, o trabalho desenvolvido alcançou seu objetivo de desenvolver esses sistemas de expressão, bem como de avaliar a síntese heteróloga de proteínas nessa levedura de destacado potencial. Os resultados obtidos devem servir à comunidade científica, no sentido de estimular e orientar futuros trabalhos que objetivem a síntese heteróloga de proteínas em microrganismos. / In spite of the advantageous physiological properties of the yeast Kluyveromyces marxianus to produce heterologous proteins, this species has not been widely explored for the synthesis of these biomolecules. On the other hand, more than 40 heterologous expression systems, including commercial ones, were developed for Kluyveromyces lactis. Moreover, there is no available literature concerning heterologous protein glycosylation in K. marxianus. Taking these facts into account, this work aimed at developing systems for the heterologous production of Aspergillus niger glucose oxidase (GOX) and of a thermophilic esterase (EST) from Thermus thermophilus in K. marxianus. The strain K. lactis CBS 2359 was utilized as a reference throughout the whole work. First, a physiological study was carried out in order to select one K. marxianus strain, out of three which had been chosen based on literature information, that exhibited the best physiological traits to be a heterologous expression host. The chosen strain was CBS 6556, based on a combination of the following properties: specific growth rate, metabolites formation, biomass yield on substrate, and secretion of the homologous enzyme inulinase. Subsequently, two episomal systems were constructed. In one of them, the heterologous gene was expressed under control of the S. cerevisiae PGK promoter, whereas in the other system, heterologous gene expression occurred under control of the K. marxianus INU1 promoter. An integrative expression system was also constructed, in which the KmINU1 promoter drove foreign gene expression. Both heterologous enzymes were characterized biochemically and also with respect to their glycosylation. The results attained with GOX led to an expression level of 1722 U/g DW (unit per gram of dry cell weight) in K. marxianus transformed with the episomal INU1-based system. The biochemical studies showed that the enzyme was very similar to the A. niger GOX. Furthermore, analysis of the glycosylation pattern showed a lower tendency of K. marxianus to hypermannosylate proteins, when compared to K. lactis. Higher levels of esterase (294 U/gDW) were obtained in K. lactis than in K. marxianus. However, the enzyme produced in the latter host presented a higher temperature for maximal activity ((50 °C), when compared to the former organism (45 °C). Both values are lower than the temperature for maximal activity of the homologous enzyme (65 °C), which can be explained by the glycans added by both yeast species to the peptide, resulting in a glycosylated protein, in contrast to the homologous esterase. Moreover, the yeast products presented three glycosylation patterns. In conclusion, the work presented in this thesis reached its aims, which were to develop these expression systems and to characterize biochemically the heterologous enzymes expressed, which included an analysis of the glycosylation pattern. The results presented here will certainly be of interest and aid the scientific community working on the expression of heterologous proteins in microorganisms.

Thèse (M.Sc.)--Université Laval, 2003. / Titre de l'écran-titre (visionné le 29 novembre 2004). Bibliogr.

A levedura Kluyveromyces marxianus, apesar de apresentar propriedades fisiológicas vantajosas para a produção heteróloga de proteínas, foi utilizada apenas poucas vezes como hospedeira na síntese dessa classe de moléculas. Em contrapartida, a sua congênere Kluyveromyces lactis possui mais de 40 sistemas de expressão desenvolvidos, inclusive comerciais. Além disso, não há literatura disponível sobre glicosilação de proteínas em K. marxianus. Levando-se isso em consideração, este trabalho visou a desenvolver sistemas para a expressão heteróloga da enzima glicose oxidase (GOX) de Aspergillus niger e de uma esterase termófila (EST) de Thermus thermophilus em K. marxianus. A linhagem K. lactis CBS 2359 foi utilizada como parâmetro de comparação em todos os sistemas de expressão construídos. Primeiramente, foi realizado um estudo fisiológico com a finalidade de selecionar, dentre três linhagens de K. marxianus pré-selecionadas a partir de informação da literatura, a que apresentasse as melhores características fisiológicas para se tornar uma hospedeira de expressão heteróloga. A linhagem selecionada foi a CBS 6556, baseando-se numa combinação das seguintes características: velocidade específica de crescimento, formação de metabólitos, rendimento de substrato em biomassa e secreção da enzima homóloga inulinase. Após, foram construídos dois sistemas de expressão epissomais. No primeiro, o gene era expresso sob controle do promotor PGK de S. cerevisiae e no segundo, sob controle de INU1 de K. marxianus. Um sistema integrativo foi utilizado, no qual a expressão era dirigida pelo promotor INU1. Estudos bioquímicos e de glicosilação foram realizados nas enzimas produzidas. Em relação aos sistemas para expressão de GOX, foram alcançados níveis de produção de 1722 U/gMS (unidades por grama de biomassa seca) em K. marxianus transformado com o sistema epissomal no qual a expressão era controlada pelo promotor INU1. As caracterizações bioquímicas da enzima mostraram que a molécula produzida apresentava propriedades semelhantes à enzima homóloga de A. niger. Além disso, os estudos de glicosilação mostraram uma menor tendência de hiperglicosilação de K. marxianus quando comparada com K. lactis. Já em relação à esterase, K. lactis apresentou maiores níveis de expressão (294 U/gMS), porém a enzima produzida em K. marxianus apresentou temperatura ótima de atividade (50 °C) ligeiramente superior à enzima produzida por sua congênere (45 °C), temperaturas abaixo da qual ocorre maior atividade da enzima homóloga (65 °C). Isso pode ser explicado pela glicosilação exercida por ambas espécies de leveduras sobre a proteína, ao contrário da homóloga, não glicosilada. Além disso, os produtos das leveduras apresentaram três padrões de glicosilação. Dessa forma, o trabalho desenvolvido alcançou seu objetivo de desenvolver esses sistemas de expressão, bem como de avaliar a síntese heteróloga de proteínas nessa levedura de destacado potencial. Os resultados obtidos devem servir à comunidade científica, no sentido de estimular e orientar futuros trabalhos que objetivem a síntese heteróloga de proteínas em microrganismos. / In spite of the advantageous physiological properties of the yeast Kluyveromyces marxianus to produce heterologous proteins, this species has not been widely explored for the synthesis of these biomolecules. On the other hand, more than 40 heterologous expression systems, including commercial ones, were developed for Kluyveromyces lactis. Moreover, there is no available literature concerning heterologous protein glycosylation in K. marxianus. Taking these facts into account, this work aimed at developing systems for the heterologous production of Aspergillus niger glucose oxidase (GOX) and of a thermophilic esterase (EST) from Thermus thermophilus in K. marxianus. The strain K. lactis CBS 2359 was utilized as a reference throughout the whole work. First, a physiological study was carried out in order to select one K. marxianus strain, out of three which had been chosen based on literature information, that exhibited the best physiological traits to be a heterologous expression host. The chosen strain was CBS 6556, based on a combination of the following properties: specific growth rate, metabolites formation, biomass yield on substrate, and secretion of the homologous enzyme inulinase. Subsequently, two episomal systems were constructed. In one of them, the heterologous gene was expressed under control of the S. cerevisiae PGK promoter, whereas in the other system, heterologous gene expression occurred under control of the K. marxianus INU1 promoter. An integrative expression system was also constructed, in which the KmINU1 promoter drove foreign gene expression. Both heterologous enzymes were characterized biochemically and also with respect to their glycosylation. The results attained with GOX led to an expression level of 1722 U/g DW (unit per gram of dry cell weight) in K. marxianus transformed with the episomal INU1-based system. The biochemical studies showed that the enzyme was very similar to the A. niger GOX. Furthermore, analysis of the glycosylation pattern showed a lower tendency of K. marxianus to hypermannosylate proteins, when compared to K. lactis. Higher levels of esterase (294 U/gDW) were obtained in K. lactis than in K. marxianus. However, the enzyme produced in the latter host presented a higher temperature for maximal activity ((50 °C), when compared to the former organism (45 °C). Both values are lower than the temperature for maximal activity of the homologous enzyme (65 °C), which can be explained by the glycans added by both yeast species to the peptide, resulting in a glycosylated protein, in contrast to the homologous esterase. Moreover, the yeast products presented three glycosylation patterns. In conclusion, the work presented in this thesis reached its aims, which were to develop these expression systems and to characterize biochemically the heterologous enzymes expressed, which included an analysis of the glycosylation pattern. The results presented here will certainly be of interest and aid the scientific community working on the expression of heterologous proteins in microorganisms.