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Polycétide synthases : étude de l’influence de l’organisation macromoléculaire sur la fonction par ingénierie génétique

Abstract : The polyketides, medically valuable bacterial secondary metabolites, are synthesized by multimodular mega-enzymes called polyketide synthases (PKSs). The goal of this work has been to exploit genetic engineering tools including CRISPR/Cas9 to evaluate the effect of macromolecular context on the function of PKSs at two molecular levels. In order to assess the influence of the modular context on the stereocontrol exerted by certain domains, the module exchanges were carried out within our model PKS system. These modules were introduced by a homologous recombination system in yeast following several module exchange strategies. The expression of these hybrid proteins was then performed in Saccharopolyspora erythraea, followed by analysis of the products by gas chromatography coupled with mass spectrometry. Our results suggest that whole-module-to-whole-module exchange would be more efficient because it preserves contacts between the KS and ACP domains of the same module, to maintain an efficient decarboxylative condensation reaction. In addition, the module exchange internal to a subunit is more efficient, because the KS domain of this type of module has a greater substrate specificity. The modules which showed the most significant results were then used to modify an entire PKS to attempt for the first time to modify the stereochemistry of the erythromycin intermediate. Regarding the macromolecular organization of PKS, it has been described that the trans-AT-type PKS responsible for the synthesis of bacillaene in Bacillus subtilis assemble to form a mega-complex, and that this mega-complex is localized close to the inner membrane. These findings should be generalized to the second major class of PKS, that of cis-AT-type PKS. To be able to locate the cis-AT-type PKS responsible for the synthesis of coelimycin (Cpk) within its native host Streptomyces coelicolor, we fused the first and / or the last subunit of Cpk to a fluorescent protein. Creation of the protein fusions was performed with the CRISPR / Cas9 genetic engineering tool. Subsequently, we obtained images of the localization of the fusion proteins by confocal microscopy. We show for the first time that the subunits of a cis-AT-like PKS also localize in macrocomplexes but present a different macromolecular organization. We also show the colocalization of one of the Cpk subunits with the associated CpkF transporter, to assess the formation of an authentic biosynthetic factory.
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Submitted on : Wednesday, June 30, 2021 - 5:26:57 PM
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  • HAL Id : tel-03275171, version 1

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Jean-Malo Massicard. Polycétide synthases : étude de l’influence de l’organisation macromoléculaire sur la fonction par ingénierie génétique. Sciences du Vivant [q-bio]. Université de Lorraine, 2020. Français. ⟨NNT : 2020LORR0278⟩. ⟨tel-03275171⟩

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