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PhD Defence Thibault Malfoy

Soutenance de thèse de doctorat le 4 décembre 2023 à 14h en salle 401.

The thesis jury is composed of Jean Marie François (Thesis Director), Stéphanie Heux (Examinator), Marie-Thérèse Giudici-Orticoni (Reporter) et Alain Perret (Reporter). Here is a short abstract of my work: Escherichia coli“.

Le jury sera composé de Jean Marie François (Directeur de thèse), Stéphanie Heux (Examinatrice), Marie-Thérèse Giudici-Orticoni (Rapporteure) et Alain Perret (Rapporteur). Vous trouverez ci-après un résumé de ma thèse :

Platform molecules are small molecules that act as an intermediate in the production of commodity products, between a carbon source (fossil or renewable) and these products. The production of these molecules in biotechnology is becoming increasingly significant as there is a wish to develop circular bioeconomy, utilising renewable carbon sources and generating environmentally friendlier molecules, especially within the field of plastics. 2,4-dihydroxybutyrate (DHB) is an example of this type of original platform molecule, because on one hand, it is not naturally produced by microorganisms, and on the other, it can be used to produce molecules for the fine chemicals and commodity sectors (1,3-propanediol and hydroxypropionic acid, and precursors for new biopolymers), pharmaceuticals (2-butyrolactone, butanetriol) and animal nutrition (methionine derivative). However, its production needs to be industrially attractive, which has been the subject of several projects carried out prior to my thesis and still ongoing today.
DHB is not a naturally occurring molecule in its production host organism Escherichia coli, This means that, aside from a few instances where a few substrate promiscuities were used for DHB production or enhancing strains that produce DHB, there is no detailed information available about how DHB interacts with the physiology of E. coli. My thesis work therefore contributed to the annotation of the metabolic pathways enabling DHB assimilation. By adding a racemic of DHB to the medium, it was found that DHB can be oxidised by threeE. colioxidases, and produce 2-oxo-4-hydroxybutyrate (OHB), which turns out to be the precursor of DHB in some of the pathways designed for its biosynthesis. OHB can then be cleaved into formaldehyde and pyruvate by a number of specific pyruvate aldolases, dissimilated into formate and pyruvate respectively, which is then redirected to the cells' central carbon metabolism. This work also enabled identification of potential DHB importers.E. coli, et produire le 2-oxo-4-hydroxybutyrate (OHB), qui s’avère être le précurseur de DHB dans certaines voies conçues pour sa biosynthèse. L’OHB peut ensuite être clivé en formaldéhyde et en pyruvate par plusieurs aldolases pyruvate spécifiques, respectivement dissimilé en formate et pyruvate, lequel peut se voir redirigé vers le métabolisme carboné central des cellules. Ce projet a également permis de mettre en évidence de potentiels importeurs de DHB.
In parallel, the construction of a DHB biosensor was carried out, and its use in various applications was demonstrated. Finally, the possibility of using DHB as a monomer for the biosynthesis of innovative polymers was also investigated in this work. Before addressing this aspect, the requirement for the enzyme catalysing polymerisation to use the (D)-enantiomer of the substrate made it necessary to identify an enzyme capable to specifically reduce OHB to D-DHB.While other DHB uptake pathways have yet to be identified, and polymer synthesis still requires additional research, all these findings support the significance of substrate promiscuity in E. coli proteins (enzymes, transcription factor, transporter). Enzyme promiscuity simplifies the creation of new synthetic pathways for molecule production. However, the production of molecules, especially unnatural ones, demands special attention to prevent unwanted interactions that could be harmful or toxic to the cells. These interactions could severely hinder the production within the resulting constructed cellular factory.

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