No description available.

No description available.

La levure Candida shehatae est le microorganisme modèle d’étude choisi. Cette levure peutconvertir le xylose et le glucose en éthanol, contrairement à Saccharomyces cerevisiae, levuretraditionnellement utilisée dans les procédés industriels, qui ne peut convertir le xylose.L’optimisation des performances de production d’éthanol à partir de xylose passe par unemaximisation des trois critères suivants : la productivité volumique, le titre final et lerendement éthanol/xylose. Pour diriger le flux de carbone vers la production d'éthanol defaçon optimale, le paramètre majeur qu’il faut contrôler est le degré de limitation en oxygène.Les cultures sont réalisées sur milieu minéral en mode fed-batch et conduites en deux phases :aérobie puis limitation en oxygène. Une valeur moyenne de la vitesse spécifique derespiration (qO2) de 1,19 mmolO2/gX/h permet de maximiser les trois critères deperformances sur xylose : le rendement en éthanol (0,327 gETOH/g-xylose), la productivitéspécifique maximale (0,22 gETOH/gX/h) et le titre en éthanol final (48,81 g/L). Pour lafermentation du glucose, le rendement en éthanol le plus élevé (0,411 gETOH/g-glucose) estobtenu lorsque qO2 est faible et a pour valeur moyenne 0,30 mmolO2/gX/h; tandis que laproductivité spécifique et le titre en éthanol final atteignent les valeurs maximales de 0,35gETOH/gX/h et 54,19 g/L pour respectivement qO2 de 1,7 et de 2,5 mmolO2/gX/h.Pour la consommation simultanée des deux substrats, un phénomène de répression du glucosesur le xylose est démontré par expérience en chemostat de pulse glucose en régime stabilisésur xylose. La simple présence intra-cellulaire des enzymes de la voie du xylose (XR andXDH) ne permet pas la co-consommation efficace des deux sucres et le glucose estpréférentiellement consommé.Afin de structurer la connaissance acquise sur le métabolisme de C. shehatae et pouvoiroptimiser par simulation les co-cultures C. shehatae / S. cerevisiae pour la productiond’éthanol à partir de mélanges xylose/glucose, un modèle cinétique de C. shehatae estconstruit. Ce modèle est validé avec des cultures sur substrats purs (xylose et glucoseséparés). Un modèle cinétique de co-culture est ensuite développé de manière à simulerdifférentes stratégies de fermentation pour l’optimisation de la production d’éthanol surmélange xylose/glucose de type hydrolysats de paille de blé / The yeast Candida shehatae was the model microorganism of the study. This yeast canconvert xylose and glucose into ethanol, unlike Saccharomyces cerevisiae traditionally usedin industrial processes, which cannot convert xylose. Performance optimization of ethanolproduction from xylose is performed through maximization of the following three criteria:volumetric productivity, final ethanol titer and yield of ethanol over xylose. To direct thecarbon flux towards ethanol production, the major parameter which must be controlled is thelevel of oxygen limitation. Cultures are carried out in fed-batch in mineral medium andperformed in two phases: the first one is not limited in oxygen and the second one is oxygenrestricted. A mean value of qO2 equal to 1.19 mmolO2/gX/h maximizes the three criteria ofperformance on xylose: ethanol yield (0.327 gETOH/g-xylose), the maximum specificproductivity (0.22 gETOH/gX/h) and the final ethanol titer (48.81 g/L). For glucosefermentation, ethanol yield is the highest (0.411 gETOH/g-glucose) when qO2 is low as anaverage value of 0.30 mmolO2/gX/h, while the specific productivity and the ethanol final titerreach maximum values of 0.35 gETOH/gX/h and 54.19 g/L for respectively qO2 of 1.7 and2.5 mmolO2/gX/h.For the simultaneous consumption of the two substrates, a phenomenon of glucose repressionover xylose is observed in chemostat experiment with glucose pulse on xylose steady state.The presence of intracellular enzymes of the xylose pathway (XR and XDH) is not sufficientfor efficient co-consumption of both sugars and glucose is preferentially consumed.In order to structure the knowledge obtained on the metabolism of C. shehatae and tooptimize by simulation the co-culture C. shehatae / S. cerevisiae to produce ethanol fromxylose/ glucose mixtures, a kinetic model of C. shehatae is built. This model is validated withpure substrate cultures (xylose and glucose separated). A kinetic model of co-culture is thenbuilt in order to simulate several fermentation strategies to optimize the ethanol productionfrom xylose/glucose mixture similar to wheat straw hydrolysates

A xilose é um dos principais componentes dos materiais lignocelulósicos, os quais são de grande interesse para produção de bioprodutos como etanol e polihidroxialcanoatos (PHA). Visando melhorar o consumo de xilose em Burkholderia sacchari, uma grande produtora de PHA, os seguintes genes codificadores de xilose isomerase foram nela inseridos e avaliados: xylABs, xylABc, xylAPl, xylABp e xylABx, respectivamente de B. sacchari, B. cenocepacia, Photorhabdus luminescens, B. phymatum e B. xenovorans. Foi ainda sintetizado o gene de B. sacchari (xylA*) no qual foram inseridas modificações descritas na literatura como capazes de aumentar o consumo de xilose em outros organismos. As linhagens recombinantes de B. sacchari abrigando os genes xylABs e xylA* tiveram um aumento de aproximadamente 30%, e aquelas abrigando os genes xylABp e xylABx de 23%, no consumo de xilose quando comparadas com a linhagem controle. Essas quatro linhagens recombinantes foram aquelas que conseguiram produzir maior quantidade de P3HB, aproximadamente 70% a mais do que linhagem controle. / Xylose is a major component of lignocellulosic materials, which are of great interest for the production of bio-products, such as ethanol and polyhydroxyalkanoates (PHA). To improve the consumption of xylose in Burkholderia sacchari, a major PHA producer, the following genes, encoding xylose isomerase, were introduced in these bacteria: xylABs, xylABc, xylAPl, xylABp and xylABx, respectively from B. sacchari, B. cenocepacia, Photorhabdus luminescens, B. phymatum e B. xenovorans. The gene of B. sacchari (xylA*) was also synthesized with several modifications described in the literature as able to increase the consumption of xylose in other organisms. Recombinant strains harboring B. sacchari xylABs and xylA* gene had an increase of approximately 30% in the xylose consumption compared to the control strain, and those harboring xylABx and xylABp gene an increase of 23%. These four recombinant strains were those that were able to produce more P3HB, approximately 70% more than the control strain.

A levedura Saccharomyces cerevisiae, amplamente utilizada na conversão de glicose e frutose a etanol, não é capaz de fermentar a xilose presente na biomassa lignocelulósica de resíduos agroindustriais. Apesar da introdução da via metabólica dessa pentose em linhagens de S. cerevisiae, a fermentação da xilose simultaneamente com outros açúcares ainda é pouco eficiente. A proposta deste trabalho foi aumentar a eficiência do consumo da xilose por linhagens de S. cerevisiae introduzindo genes de transportadores exógenos identificados em leveduras selvagens que naturalmente fermentam pentoses. A via do metabolismo da xilose foi integrada no genoma de uma linhagem industrial brasileira de S. cerevisiae usada na produção de etanol. A partir desta, linhagens isogênicas foram criadas e mostraram ser mais eficientes no metabolismo da xilose em meio sintético e capazes de co-fermentar glicose e xilose na presença de altas concentrações de inibidores resultantes da hidrólise da biomassa lignocelulósica. Os tranportadores identificados foram testados nas linhagens industriais geneticamente modificadas criadas neste estudo e em linhagens laboratoriais. Não foi possível confirmar a eficiência dos transportadores nas linhagens, embora os resultados mostraram diferenças nas curvas de crescimento das linhagens industriais expressando os transportadores. Este trabalho foi o início de um estudo dos fatores envolvidos no metabolismo da xilose e servirá como base para que futuros trabalhos sejam realizados na obtenção de uma linhagem mais eficiente para produção de etanol de segunda geração. / The yeast Saccharomyces cerevisiae, which efficiently ferments glucose and fructose to ethanol, is unable to ferment xylose present in lignocellulosic biomass of agroindustrial residues. Although the introduction of xylose metabolic pathways in S. cerevisiae strains has been described in the literature, the simultaneous fermentation of xylose and glucose in these modified strains is still very inefficient. The aim of this study was to increase the xylose consumption efficiency of S. cerevisiae by introduction of exogenous genes identified in wild yeast that naturally ferment pentose. The xylose metabolism pathway was integrated into the genome of a Brazilian industrial strain of S. cerevisiae used for the production of ethanol, which was then used to obtain isogenic modified strains. The isogenic strains showed to be more effective in xylose metabolism in synthetic medium and able to co-ferment glucose and xylose in the presence of high concentrations of inhibitors resulting hydrolysis of lignocellulosic biomass. The transporters identified were inserted into genetically modified industrial strains of S. cerevisiae created in this study and also in laboratory strains. It was not possible to confirm the transporters efficiency in laboratory strains but the results showed differences in the growth curves of the industrial strains expressing the transporters. This work was the beginning of a study of the factors involved in xylose metabolism and it will help to prepare future work to obtain an efficient strain for lignocellulosic ethanol production.

A levedura Saccharomyces cerevisiae, amplamente utilizada na conversão de glicose e frutose a etanol, não é capaz de fermentar a xilose presente na biomassa lignocelulósica de resíduos agroindustriais. Apesar da introdução da via metabólica dessa pentose em linhagens de S. cerevisiae, a fermentação da xilose simultaneamente com outros açúcares ainda é pouco eficiente. A proposta deste trabalho foi aumentar a eficiência do consumo da xilose por linhagens de S. cerevisiae introduzindo genes de transportadores exógenos identificados em leveduras selvagens que naturalmente fermentam pentoses. A via do metabolismo da xilose foi integrada no genoma de uma linhagem industrial brasileira de S. cerevisiae usada na produção de etanol. A partir desta, linhagens isogênicas foram criadas e mostraram ser mais eficientes no metabolismo da xilose em meio sintético e capazes de co-fermentar glicose e xilose na presença de altas concentrações de inibidores resultantes da hidrólise da biomassa lignocelulósica. Os tranportadores identificados foram testados nas linhagens industriais geneticamente modificadas criadas neste estudo e em linhagens laboratoriais. Não foi possível confirmar a eficiência dos transportadores nas linhagens, embora os resultados mostraram diferenças nas curvas de crescimento das linhagens industriais expressando os transportadores. Este trabalho foi o início de um estudo dos fatores envolvidos no metabolismo da xilose e servirá como base para que futuros trabalhos sejam realizados na obtenção de uma linhagem mais eficiente para produção de etanol de segunda geração. / The yeast Saccharomyces cerevisiae, which efficiently ferments glucose and fructose to ethanol, is unable to ferment xylose present in lignocellulosic biomass of agroindustrial residues. Although the introduction of xylose metabolic pathways in S. cerevisiae strains has been described in the literature, the simultaneous fermentation of xylose and glucose in these modified strains is still very inefficient. The aim of this study was to increase the xylose consumption efficiency of S. cerevisiae by introduction of exogenous genes identified in wild yeast that naturally ferment pentose. The xylose metabolism pathway was integrated into the genome of a Brazilian industrial strain of S. cerevisiae used for the production of ethanol, which was then used to obtain isogenic modified strains. The isogenic strains showed to be more effective in xylose metabolism in synthetic medium and able to co-ferment glucose and xylose in the presence of high concentrations of inhibitors resulting hydrolysis of lignocellulosic biomass. The transporters identified were inserted into genetically modified industrial strains of S. cerevisiae created in this study and also in laboratory strains. It was not possible to confirm the transporters efficiency in laboratory strains but the results showed differences in the growth curves of the industrial strains expressing the transporters. This work was the beginning of a study of the factors involved in xylose metabolism and it will help to prepare future work to obtain an efficient strain for lignocellulosic ethanol production.

A levedura Saccharomyces cerevisiae, amplamente utilizada na conversão de glicose e frutose a etanol, não é capaz de fermentar a xilose presente na biomassa lignocelulósica de resíduos agroindustriais. Apesar da introdução da via metabólica dessa pentose em linhagens de S. cerevisiae, a fermentação da xilose simultaneamente com outros açúcares ainda é pouco eficiente. A proposta deste trabalho foi aumentar a eficiência do consumo da xilose por linhagens de S. cerevisiae introduzindo genes de transportadores exógenos identificados em leveduras selvagens que naturalmente fermentam pentoses. A via do metabolismo da xilose foi integrada no genoma de uma linhagem industrial brasileira de S. cerevisiae usada na produção de etanol. A partir desta, linhagens isogênicas foram criadas e mostraram ser mais eficientes no metabolismo da xilose em meio sintético e capazes de co-fermentar glicose e xilose na presença de altas concentrações de inibidores resultantes da hidrólise da biomassa lignocelulósica. Os tranportadores identificados foram testados nas linhagens industriais geneticamente modificadas criadas neste estudo e em linhagens laboratoriais. Não foi possível confirmar a eficiência dos transportadores nas linhagens, embora os resultados mostraram diferenças nas curvas de crescimento das linhagens industriais expressando os transportadores. Este trabalho foi o início de um estudo dos fatores envolvidos no metabolismo da xilose e servirá como base para que futuros trabalhos sejam realizados na obtenção de uma linhagem mais eficiente para produção de etanol de segunda geração. / The yeast Saccharomyces cerevisiae, which efficiently ferments glucose and fructose to ethanol, is unable to ferment xylose present in lignocellulosic biomass of agroindustrial residues. Although the introduction of xylose metabolic pathways in S. cerevisiae strains has been described in the literature, the simultaneous fermentation of xylose and glucose in these modified strains is still very inefficient. The aim of this study was to increase the xylose consumption efficiency of S. cerevisiae by introduction of exogenous genes identified in wild yeast that naturally ferment pentose. The xylose metabolism pathway was integrated into the genome of a Brazilian industrial strain of S. cerevisiae used for the production of ethanol, which was then used to obtain isogenic modified strains. The isogenic strains showed to be more effective in xylose metabolism in synthetic medium and able to co-ferment glucose and xylose in the presence of high concentrations of inhibitors resulting hydrolysis of lignocellulosic biomass. The transporters identified were inserted into genetically modified industrial strains of S. cerevisiae created in this study and also in laboratory strains. It was not possible to confirm the transporters efficiency in laboratory strains but the results showed differences in the growth curves of the industrial strains expressing the transporters. This work was the beginning of a study of the factors involved in xylose metabolism and it will help to prepare future work to obtain an efficient strain for lignocellulosic ethanol production.

A xilose é um dos principais componentes dos materiais lignocelulósicos, os quais são de grande interesse para produção de bioprodutos como etanol e polihidroxialcanoatos (PHA). Visando melhorar o consumo de xilose em Burkholderia sacchari, uma grande produtora de PHA, os seguintes genes codificadores de xilose isomerase foram nela inseridos e avaliados: xylABs, xylABc, xylAPl, xylABp e xylABx, respectivamente de B. sacchari, B. cenocepacia, Photorhabdus luminescens, B. phymatum e B. xenovorans. Foi ainda sintetizado o gene de B. sacchari (xylA*) no qual foram inseridas modificações descritas na literatura como capazes de aumentar o consumo de xilose em outros organismos. As linhagens recombinantes de B. sacchari abrigando os genes xylABs e xylA* tiveram um aumento de aproximadamente 30%, e aquelas abrigando os genes xylABp e xylABx de 23%, no consumo de xilose quando comparadas com a linhagem controle. Essas quatro linhagens recombinantes foram aquelas que conseguiram produzir maior quantidade de P3HB, aproximadamente 70% a mais do que linhagem controle. / Xylose is a major component of lignocellulosic materials, which are of great interest for the production of bio-products, such as ethanol and polyhydroxyalkanoates (PHA). To improve the consumption of xylose in Burkholderia sacchari, a major PHA producer, the following genes, encoding xylose isomerase, were introduced in these bacteria: xylABs, xylABc, xylAPl, xylABp and xylABx, respectively from B. sacchari, B. cenocepacia, Photorhabdus luminescens, B. phymatum e B. xenovorans. The gene of B. sacchari (xylA*) was also synthesized with several modifications described in the literature as able to increase the consumption of xylose in other organisms. Recombinant strains harboring B. sacchari xylABs and xylA* gene had an increase of approximately 30% in the xylose consumption compared to the control strain, and those harboring xylABx and xylABp gene an increase of 23%. These four recombinant strains were those that were able to produce more P3HB, approximately 70% more than the control strain.

Résumé confidentiel / No abstract

Les ressources de carbone primaires doivent être progressivement remplacées par des ressources renouvelables plus complexes comme les matières lignocellulosiques, pour produire des biocarburants ou des synthons (bioraffineries de 2ième génération). Cette évolution nécessite de modifications importantes à différentes étapes du procédé, au niveau de la fermentation elle-même ou dans les étapes ultérieures nécessaire pour l'obtention du produit cible. Dans ce travail, nous avons étudié un procédé de production de succinate à partir du xylose. La fermentation a été réalisée en utilisant une souche d' Escherichia coli modifiée par ingénierie métabolique. La purification du succinate a été réalisée par nanofiltration. Des travaux précédents ont permis, par ingénierie métabolique, de mettre au point une souche E. coli KJ122 permettant de produire du succinate par fermentation anaérobie de glucose dans un milieu contenant des sels minéraux. Cette souche ne permet cependant pas une fermentation performante lorsque le xylose est utilisé comme substrat. Afin de lever cette limitation, E. coli KJ122 a été modifiée. Le transporteur ABC codant pour les gènes xylFGH a été inactivé par une technique de suppression de gènes. La souche ainsi obtenue, baptisée KJ12201 (E. coli KJ122 ?xylFGH) a permis d'atteindre des vitesses de croissances rapides, des consommations de xylose et une production de succinate améliorées par rapport à la souche parente. Après modification génétique, E. coli KJ12201-14T permet de produire en mode une concentration élevée de succinate de 84 g/L, la concentration d'acétate accumulée étant de 11 g/L, à partir d'un milieu de composition adaptée (AM1) contenant 10% de xylose. En fermentation fed-batch, E. coli KJ12201-14T permet de produire du succinate à une concentration de 84 g/L, avec un rendement de 0.85 g/g et une productivité de 2 g/L/h. Ces résultats démontrent les potentialités de cette souche pour produire du succinate à partir de xylose ou d'hydrolysats riches en xylose issus de matières lignocellulosiques. La nanofiltration a ensuite été considérée afin de purifier le succinate obtenu par fermentation. Les expériences ont été réalisées avec une membrane NF45 et des milieux de fermentation synthétiques contenant le succinate et différentes impuretés, sels minéraux, glucose ou autres sels d'acides organiques, acétate en particulier. L'influence des conditions opératoires (pH, pression) sur les performances de la NF a été évaluée. Les mécanismes gouvernant le transfert des espèces à travers la membrane ont été étudiés afin d'expliquer la variation des rétentions en fonction de la composition du milieu. En solution simple, les résultats ont montré que la rétention du succinate augmente avec la pression appliquée et avec le pH et diminue lorsque la concentration augmente. Pour des concentrations faibles, de l'ordre de 0.1M, les rétentions du succinate et de l'acétate en mélange sont différentes et identiques à celles en solution simples. Une bonne purification du succinate est ainsi possible. Au contraire, pour des concentrations plus élevées en succinate, la rétention diminue par suite de l'écrantage des effets de charge. Les rétentions étant trop proches, la séparation acétate/succinate devient impossible. Considérant les mécanismes ainsi mis évidence, une méthodologie a été proposée afin de réaliser la purification du succinate obtenu par fermentation. La séparation acétate/succinate est effectuée en deux étapes. Une diafiltration du jus de fermentation, préalablement dilué, est d'abord réalisée en utilisant la membrane NF45. Le rétentat purifié est ensuite concentré, en utilisant une membrane d'osmose inverse. Grace à ce procédé, il est possible d'augmenter la pureté du succinate de 85 à plus de 99.5% avec un rendement global supérieur à 92%. L'intérêt de la nanofiltration pour purifier le succinate produit par fermentation est ainsi démontrée. / Current trend is to move from primary carbohydrate resources to more complex ones like lignocellulosic materials as a bio-renewable feedstock, to produce biofuels or chemical building blocks. This evolution requires significant modifications at different stages in the bioprocess engineering, including fermentation and downstream processes. In this work, the succinate production by a newly metabolically engineered Escherichia coli from xylose, and its purification from fermentative broth by nanofiltration were studied. Escherichia coli KJ122 strain was previously engineered to produce high titers and yields of succinate in mineral salts medium containing glucose under simple-batch anaerobic conditions. However, this strain does not efficiently utilize xylose due to catabolic repression. To improve the xylose uptake and its utilization of E. coli KJ122, xylFGH genes were inactivated by the gene deletion technique. The mutant strain named KJ12201 (E. coli KJ122 ?xylFGH) exhibited high abilities in fast growth, xylose consumption and succinate production compared to those of the parental strains. After performing metabolic evolution, E. coli KJ12201-14T efficiently consumed 10% xylose to produce a high succinate concentration at 84 g/L with an accumulated acetate concentration at 11 g/L in mineral salts medium (AM1) under batch fermentation. During fed-batch fermentation, E. coli KJ12201-14T produced succinate at a concentration, yield, and overall productivity of 84 g/L, 0.85 g/g, and 1.0 g/L/h, respectively. These results demonstrated that E. coli KJ12201 would be a potential strain for the economic bio-based succinate production from xylose and other xylose-rich hydrolysates derived from lignocellulosic materials. The succinate purification from fermentation broth by nanofiltration (NF) was also investigated. The experiment was carried out with a NF45 membrane and various synthetic fermentation broths containing succinate salt and different impurities such as inorganic salts, glucose, and other organic acid salts including acetate. The influence of the operating conditions (pH, pressure) as well as the broth composition on the NF performances was evaluated. The mechanisms governing the transfer of the solutes through the membrane were studied in order to explain the different solute retentions observed according to the fermentation broth composition. In single-solute solutions, the succinate retention increases with increasing pressure and feed pH and decreases with increasing feed concentration. For instance, at a low salts concentration at 0.1 M, it was observed that the retentions of succinate and acetate in the mixture are identical to those in single solutions. Thus, a good purification of succinate can be obtained. On the contrary, with higher succinate concentrations, the retention was decreased due to the screening effect. Retentions of those solutes were then too close to achieve a separation. Based on abovementioned mechanisms observed, a methodology was proposed to perform the succinate purification from fermentation broth. The succinate/acetate separation was carried out in two steps. A diafiltration of the diluted fermentation broth was initially performed, and the concentration step followed. With this process, it was possible to increase the succinate purity from 85% to more than 99.5% while maintaining a total yield higher than 92%. From this work, it was shown that NF could be effectively used for the succinate purification from fermentation broth.