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Développement d’une nouvelle méthodologie pour la production de molécules par ingénierie métabolique en délocalisant tout ou partie des réactions enzymatiques sur la surface de S. cerevisiae / Development of a new methodology for molecular production via metabolic engineering by relocating all or part of the enzymatic reaction on the surface of S. cerevisiaePauthenier, Cyrille 07 November 2016 (has links)
L’ingénierie métabolique est une discipline qui vise à modifier artificiellement le métabolisme d’un organisme afin de lui faire produire un composé chimique d’intérêt. L’une des problématiques fréquemment rencontrées pour la production de nouvelles molécules est l’impossible diffusion du produit formé dans le cytoplasme vers l’extérieur de la cellule. Ce phénomène engendre une accumulation de ce dernier à l’intérieur du micro-organisme qui limite sa capacité de production pour des raisons de cinétiques chimiques et de toxicité pour l’hôte. Enfin, cela complique fortement la récupération et la purification de la molécule d’intérêt, ce qui peut réduire à néant le rendement économique du procédé industriel.Durant cette thèse, nous avons exploré la possibilité de réaliser les dernières étapes de synthèse de composés imperméables pour la membrane à l’extérieur de la cellule en utilisant des enzymes accrochées à la surface de la levure par la technique de “yeast surface display”.Dans une première partie, nous avons regardé l’intérêt industriel de la bioéconomie, puis nous nous sommes intéressés aux problématiques liées à la perméabilité des membranes plasmiques. Dans un second temps, nous avons évalué les méthodes de mesures de la perméabilité des membranes biologiques et exploré la possibilité de développer une méthode prédictive en utilisant une technique de relation structure-propriété. Dans un troisième temps, nous avons évalué les systèmes de “yeast surface display” disponibles et cherché à en découvrir de nouveaux, adaptés à nos problématiques. Dans l'objectif de construire ces bibliothèques nous avons aussi réalisé un outils informatique, permettant de calculer de grands nombres de primers. Enfin, nous avons réalisé différents circuits de production de molécules modèles pour évaluer la pertinence de l’approche pour la production de composés imperméables. / Sustainable chemical production is one of the endeavour of the post-oil era. Amongst the possible techniques, metabolic engineering which aims at producing novel compounds through genetic engineering of micro-organism is seen as one of the most promising techniques. One of the problem met by metabolic engineers is often the absence of diffusion or pumping mechanism expelling the compound of interest produced in the cell cytoplasm towards the outer environment, which reduces the process efficiency because of kinetic and toxicity concerns.During this PhD, we explored the possibility of producing impermeable compounds on the surface of a cell by anchoring the last reaction enzyme using « Yeast surface display » techniques.As PhD disputation we first looked at the industrial interest of metabolic engineering in the whole bioeconomy framework. We then looked at the membrane permeability issues met for the production of some compounds. We evaluated the different membrane permeability techniques and explored the possibility realizing a predictive technique using quantitative structure-property relationship (QSAR). We evaluated the different yeast-display systems available and paved the way for the discovery of new systems more suitable for metabolic engineenering. We developped a dedicated program tool for large PCR fragment library design. Finally we built several toy metabolic pathways in yeast in order to evaluate the interest of the technique.
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Flödescytometrisk undersökning av inbindning mellan designade topdomänen från transferrinreceptorn till virala glykoproteiner för potentiell användning inom läkemedelsframtagning / Flow cytometric investigation of binding between the designed top domain of the transferrin receptor to viral glycoproteins for potential use in drug developmentRydell, Emma January 2022 (has links)
Machupovirus är ett virus som kan orsaka hemorragisk feber hos människor. Efter utvärdering av bindning mellan designade proteiner och virala glykoproteiner skulle proteinerna potentiellt kunna användas vid framtagning av ett proteinbasserat läkemedel mot hemorragisk feber. Syftet med studien var att efter riktad evolution och framrening av optimerade varianter av proteinet AP01 undersöka inbindningen till virala glykoproteiner mellan designade AP01 proteiner och transferrinreceptorn med hjälp av flödescytometrisk undersökning. Den fysiologiska nivån av järn i kroppen upprätthålls av transferrin (Tf) och transferrinreceptorn (TfR), ett transmembranprotein bestående av tre domäner. TfR apikala domän används av glykoprotein 1 (MGP1) och Plasmodium vivax för att ta sig in i celler genom receptormedierad endocytos. Med rekombinant genteknik kan rekombinanta plasmider skapas där en gen av intresse ligeras in i en plasmid med hjälp av DNA-ligas. I studien skapades rekombinanta plasmider pET29b+/AP01 S2.1, S2.2, S2.3, S3.3, S3.4 och S3.6 som transformerades till E. coli. Erhållna resultat från sekvensering visade att samtliga sex AP01-gener hade ligerats i vektorn men sekvensering av rekombinanta plasmider visade att endast pET29b+/AP01 S2.1, S2.2, S2.3 och S3.6 hade nukleotidsekvens utan mutationer. Proteinuttryck inducerades innan proteiner renades fram med immobilized metal ion affinity chromatography (IMAC). Den uppskattade molekylvikten hos de framrenade proteinerna var 18 kDa som bestämdes med sodium dodecyl sulfate – polyacrylamid gel electrophoresis (SDS-PAGE) vilket överrenstämde med den teoretiska molekylvikten. Flödescytometri användes för att undersöka inbindningsförmågan mellan de uttryckta proteinerna och glykoprotein 1 (MGP1). Interaktionsbindningen mellan de designade proteinerna och MGP1 är bättre än interaktionen mellan originalgen AP01 och MGP1. De designade proteinerna visar på en svag effekt i den utförda ”competition assay” som gjorts vilket kan förklaras med en ej optimal struktur hos de designade proteinerna eller närvaro av BSA. / Machupovirus is a virus that can cause hemorragic fever in humans. After evaluating the binding between designed proteins and viral glycoproteins, the proteins could potentially be used in the development of a protein-based drug for hemorrhagic fever. The aim of the study was to investigate the binding to viral glycoproteins between designed AP01 proteins and the transferrin receptor after directed evolution and purification of optimized variants of the AP01 protein by means of flow cytometric examination. The physiological level of iron in the body is maintained by transferrin (Tf) and the transferrin receptor (TfR), a transmembrane protein consisting of three domains. The apical domain of TfR is used by glycoprotein 1 (MGP1) and Plasmodium vivax to enter cells through receptor mediated endocytosis. With recombinant DNA technology, recombinant plasmids can be created where a gene of interest is ligated into a plasmid using DNA ligase. In this study, recombinant plasmids pET29b+/AP01 S2.1, S2.2, S2.3, S3.3, S3.4 and S3.6 were created and transformed into E. coli. Sequencing results showed that all six AP01 genes had been ligated into the vector but sequencing of recombinant plasmids showed that only endast pET29b+/AP01 S2.1, S2.2, S2.3 and S3.6 had nucleotid sequence without mutations. Protein expression was induced before proteins were purified by immobilized metal ion affinity chromatography (IMAC). The estimated molecular weight of the purified proteins was 18 kDa as determined by sodium dodecyl sulfate – polyacrylamid gel electrophoresis (SDS-PAGE) which was consistent with the theoretical molecular weight. Flow cytometry was used to examine the binding ability between the expressed proteins and glycoprotein 1 (MGP1). The interaction binding between the designed proteins and MGP1 is better than the interaction between the original gene AP01 and MGP1. The designed proteins show a weak effect in the “competition assay” preformed, wich can be explained by a non-optimal structure how the designed proteins or the presence of BSA.
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Insights into the structure and functionality of feruloyl esterases for the efficient utilization of lignocellulosic biomass / リグノセルロース系バイオマスの有効活用に向けたフェルラ酸エステラーゼの構造と機能に関する研究Apisan, Phienluphon 25 March 2024 (has links)
京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第25398号 / エネ博第477号 / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 准教授 中田 栄司, 教授 菅瀬 謙治 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM
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