PhD Oral Exam - Sara Ouadhi, Chemistry

When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.

Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.

Abstract

Glycosylated natural products, or GNPs, are omnipresent molecules in nature. They are also very well represented in the pharmaceutical and food production industries, among others. Therefore, their manufacturing is a crucial endeavour towards compound discovery. These molecules are synthesized through the glycosylation reaction between an NDP-activated sugar donor and a non-sugar molecule, or aglycone. This reaction is catalyzed by enzymes named glycosyltransferases (GTs). Using GTs, new-to-nature GNPs can be produced by mixing and matching NDP-sugars and aglycones. Producing these GNPs is however set back by two obstacles: the first one concerns the NDP-sugars. These molecules have long and complex natural pathways towards their production, making their availability limited and their synthesis in a laboratory setting challenging. The second obstacle comes from wild type GTs: these enzymes are promiscuous, meaning they can accept a range of substrates and therefore cannot produce specific GNPs. Furthermore, their activity at large scale is subpar. In this work, we have established an in vitro enzymatic pathway towards the synthesis of dTDP-3-amino-2,3,6-trideoxy-D-threo-hexopyranos-4-ulose, a key intermediate for the production of multiple important dTDP-sugars, as well as designed pathways towards the production of multiple rare and new-to-nature dTDP-sugars. Furthermore, we have developed in silico GT engineering tools. Using GT AknS and GT helper protein AknT as templates for our pipeline, we have developed homology models for the two proteins and performed molecular dynamics simulations and molecular docking experiments to elucidate the mechanism of action of the proteins and pinpoint the regions to engineer towards increased selectivity for new substrates. Through this, we will design in vitro tools for the glycorandomization of GNPs with robust GTs and varied dTDP sugars.