Thèse Ingénierie Avancée d'Enzymes Actives sur les Sucres pour la Conception à Façon de Glycoconjugués d'Intérêt pour la Santé H/F - Doctorat.Gouv.Fr

Établissement : Institut National des Sciences Appliquées de Toulouse École doctorale : SEVAB - Sciences Ecologiques, Vétérinaires, Agronomiques et Bioingenieries Laboratoire de recherche : TBI - Toulouse Biotechnology Institute, Bio & Chemical Engineering Direction de la thèse : Claire MOULIS ORCID 0000000219379052 Début de la thèse : 2026-10-01 Date limite de candidature : 2026-06-01T23:59:59 La thèse porte sur la découverte et l'ingénierie d'enzymes guidée par des approches computationnelles avancées pour la production biosourcée de glycoconjugués bioactifs destinés à des utilisations en santé humaine. A TBI, dans l'axe EnzyOne de l'équipe EAD1-CIMES, nous disposons d'une collection de transglycosylases qui peuvent être de puissants outils de glycosylation à partir de substrats naturels et abondants. L'objectif majeur sera d'investiguer en détail les relations existant entre la structure et la fonction d'enzymes candidates pour entreprendre un remodelage ciblé et conséquent de leur site actif afin de forcer la reconnaissance de molécules naturellement peu -voire- non reconnues et d'optimiser les rendements de glycosylation.
Pour cela, des méthodologies avancées d'ingénierie des protéines couplées à des approches informatiques développées dans notre équipe (modélisation moléculaire et intelligence artificielle) seront utilisées pour adapter ces enzymes aux différents substrats envisagés.Glycosylation is a major reaction in many biological processes. The glycosylated molecules can be involved in structural roles, constitute energy reserves or play a part in cell communication, signaling and protection. In cellulo, the transfer of a glycosyl residue from a donor substrate to an acceptor molecule is almost exclusively catalyzed by Leloir-type glycosyltransferases, which use activated sugars (nucleotide-sugars) as substrates1. Yet demand for oligosaccharides, polysaccharides and glyco-conjugates has been growing steadily for several decades. Well-defined structures are needed to study the role of glycosylation in biological phenomena. In addition, there is growing interest in the application of bioactive oligosaccharides and glycoconjugates in the food, cosmetics and pharmaceutical industries (such as nutraceutical ingredients, prebiotics, antigenic motives for vaccines, anti-oxidant, anti-inflammatory and anti-cancer molecules, etc.). In particular, intense research are dedicated to the glycosylation of therapeutic molecules with the aim to tune their solubility, prevent their toxicity, modify their mechanism of action, improve the target recognition or acting as pro-drugs2.

Identifying and engineering carbohydrate acting enzymes that can efficiently graft sugars on aglycone molecules is a real challenge, both to obtain new structures with new properties, and to move towards greener and more sustainable processes. However, Leloir-type glycosyltransferases are not ideal candidates for large-scale in vitro synthesis of glycosylated products, as the nucleotide sugars used are still very expensive and not widely available. An alternative is to use glycoside hydrolases acting on more available and lower cost substrates than nucleotide activated sugars.

In this context, the doctoral project will aim to take advantage of the latest advances in the analysis, discrimination and grouping of enzyme sequences in order to select the best enzymes of interest. Once selected, one or two enzyme scaffolds will be adapted to recognize aglycone acceptors. Multiple approaches including 3D structure determination of enzymes in complex with such ligands, molecular modelling and the latest AI-based digital tools for protein design will be used to identify structural determinants important for glycosylation. On this basis, libraries of enzyme variants of different sizes will be generated and screened using high-throughput technologies if necessary. Fundamental data will be acquired on the molecular determinants to be modified to control the architecture of the active site.
The main objective is to discover or engineer enzymes enabling to explore new routes for the enzymatic production of bioactives molecules. To this end, transglycosylases of interest will be selected and adapted to the reaction of interest via rational and/or semi-rational engineering including artificial intelligence-based approaches for protein design. The PhD project will comprise four main work-packages

WP1- Sequence-based identification of new Glycoside-Hydrolases
a. Data mining of sequences available in public databases and clustering through sequences similarity networks. Selection of a set of sequences (max10 sequences).
b. Design of ancestral sequences (max 10 sequences) using AI-based computational tools
c. Cloning of synthetic genes corresponding to the selected sequences. Expression in E. coli for enzyme characterization and screening.
d. If interest, biochemical characterization of the most relevant enzymes (nature of products, kinetic parameters)

WP2- Acquisition of structural data
The 3D structure of the best performing enzymes (max two enzymes) in complex with the aglycone ligands (2 to 4 moelcules) and/or glycosyl donors will be solved using X-ray crystallography or CryoEM experiments in order to identify the determinants involved in enzyme specificity and target of mutagenesis sites.

WP3 - Enzyme engineering to adapt them to acceptor molecules
a. In parallel to structural data acquisition, the molecules of interest (2 to 4) will be docked in the enzyme active sites to identify the amino acids to mutate
b. AI-based construction of small-size and structure-guided libraries of mutants
c. Variant library construction and screening to determine the variant product profiles and further guide iterative round of engineering.
Protein fitness models based on machine learning from (i) mutant sequences with assay-labeled data obtained from screening and from (ii) evolutionary information extracted from sequence data, will be used in combination with protein design methods to guide the next mutagenesis iteration.

WP4 - Characterization of glycoconjugates
a. Biochemical characterization of the best performing variants selected in WP1 and WP3
b. Glycoconjugates of interest will be produced at 50-mg scale, purified and their structure determined by NMR and Mass spectrometry at TBI.
c. Biological properties will be assessed in collaboration with partners

Solides connaissances en biochimie, biologie moléculaire et méthodes d'analyse. Expérience en ingénierie enzymatique et analyse bioinformatique des séquences protéiques. De bonnes connaissances en biologie structurale et/ou computationnelle serait un atout supplémentaire.