Synthèse chimioenzymatique d'analogues de monosaccharides utilisés comme sondes pour le développement d'un test de sélection de la transcétolase de Saccharomyces cerevisae basé sur l'auxotrophie

Abstract : Transketolase (TK) catalyses the D-threo ketose synthesis by stereospecific formation of a C-C bond. The objective of this work consisted in modifying the substrate specificity of TK from Saccharomyces cerevisiae by mutagenesis to extend the synthetic potential to D-threo aldoses and Lerythro ketoses. Our strategy was based on the creation of TK libraries from TKL1 gene short sequences (identified from 3D structure) using cassette mutagenesis techniques. For identifying desired TK, we developed an in vivo selection test based on amino acid auxotrophy. Consequently, we synthesised corresponding probes bearing a moiety recognised by wild type TK (D-threo ketose) or mutant TK (L-erythro ketose or D-threo aldose) and the side chain of an amino acid (alanine, valine, leucine, methionine, threonine). We investigated this selection test in the presence of D-threo ketoses and wild type TK. In vitro we showed that these compounds were substrates for TK. Concerning the in vivo test, we chose leucine and methionine precursors due to the tightness and stability of E. coli auxotrophs. The higher growth rates were obtained with D-threo ketose probe, precursor of methionine.
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Grégory Simon. Synthèse chimioenzymatique d'analogues de monosaccharides utilisés comme sondes pour le développement d'un test de sélection de la transcétolase de Saccharomyces cerevisae basé sur l'auxotrophie. Chimie organique. Université Blaise Pascal - Clermont-Ferrand II, 2009. Français. ⟨NNT : 2009CLF22005⟩. ⟨tel-00724437⟩

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