Thèse Étude de l'Architecture Atypique des Nrps Impliquées dans la Biosynthèse des Pyrrolamides en Vue d'Une Optimisation de la Production de Peptides Non Ribosomaux H/F - 75

Établissement : Université Paris-Saclay GS Life Sciences and Health
École doctorale : Structure et Dynamique des Systèmes Vivants
Laboratoire de recherche : I2BC - Institut de Biologie Intégrative de la Cellule
Direction de la thèse : Sylvie LAUTRU ORCID 000000027935704X
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-03-23T23:59:59

Contexte :
Les métabolites spécialisés, produits par des micro-organismes ou des plantes, jouent des rôles écologiques majeurs (compétition, communication, signalisation) et présentent un intérêt médical (antibiotiques, anticancéreux, etc.) ou agricole (antifongique...) . Parmi eux, les pyrrolamides (ex. : congocidine, distamycine) sont synthétisés par des NRPS (Non-Ribosomal Peptide Synthetases), des enzymes modulaires responsables de l'assemblage de peptides complexes. Contrairement à la plupart des NRPS, organisées en modules fusionnés (architecture canonique), les NRPS des pyrrolamides sont composées de domaines ou modules discrets, une organisation rare et mal comprise.
Problématique :
Une étude récente sur les PKS (polycétide synthases) suggère qu'une structure en petites sous-unités favorise la production de métabolites. Cette découverte soulève une question : l'architecture atypique des NRPS des pyrrolamides optimise-t-elle leur biosynthèse ? Comprendre les mécanismes moléculaires régissant les interactions entre leurs sous-unités pourrait alors ouvrir des perspectives pour la biologie de synthèse des NRPS.
Objectifs :
1. Reconstituer une architecture canonique de la NRPS produisant la congocidine, en fusionnant progressivement les domaines/modules discrets la constituant et en évaluant l'impact de ces fusions sur la production de congocidine dans un hôte hétérologue.
2. Identifier les régions d'interaction entre sous-unités de la NRPS produisant la congocidine, par une combinaison d'approches in vivo (biosynthèse combinatoire), in vitro (empreinte protéique par spectrométrie de masse) et in silico (modèles structuraux, co-évolution). Les résidus identifiés comme cruciaux pour les interactions seront validés par des mutagénèses ciblées.
3. Analyser la localisation cellulaire de NRPS de la congocidine dans les hyphes de Streptomyces ambofaciens via imagerie de fluorescence (protéines de fusion fluorescentes ou marquage HaloTag).

Enjeux :
Ce projet vise à élucider les liens entre architecture enzymatique, interactions protéine-protéine et efficacité biosynthétique, avec des applications potentielles pour l'ingénierie des NRPS et la production optimisée de composés bioactifs.

Specialised metabolites are small molecules with often complex and highly diverse structures (peptides, polyketides, terpenes, aminoglycosides, or alkaloids, for example) synthesised by microorganisms and plants. These metabolites (also referred to as secondary metabolites or natural products) are not essential for the growth and survival of the producing organism under laboratory conditions. Nevertheless, they are generally thought to confer a selective advantage to the organism in its ecological niche, where they play roles in competition, communication and signaling.
Specialised metabolites frequently exhibit biological activities of interest to humans. Microbial specialised metabolites are used in agriculture (as pesticides or herbicides), in biological research (as tools for selection, inhibition, or imaging), and in medicine. In particular, they are used as anticancer agents, immunosuppressants, and antimicrobials: about two-thirds of clinically used antibiotics are specialised metabolites or derivatives thereof.
Nonribosomal peptide synthetases (NRPS) are large, multimodular enzymes organised into subunits (synthetases) that are themselves composed of modules. Each module is responsible for the incorporation of one amino acid into the growing peptide chain. Each module is further subdivided into structural and functional domains. There are four types of core domains. The adenylation (A) domain is responsible for the recognition of the substrate amino acid, its activation, and its transfer to the peptidyl carrier protein (PCP). The condensation domain (C) catalyses peptide bond formation and the elongation of the nascent peptide, which remains covalently tethered to the enzyme at the PCP domain throughout synthesis. Finally, the thioesterase domain (TE) allows the release of the peptide into solution, through hydrolysis or cyclization. In addition to these core domains, NRPSs often contain accessory domains such as the epimerization domains that further expand the chemical diversity of the synthesised peptides, a diversity also enhanced by the incorporation of non-proteinogenic monomers.
Most NRPS follow the co-linearity rule, i.e. the sequence of the synthesised peptide corresponds to the order of the modules within the NRPS. However, NRPS are frequently composed of multiple subunits, and correct peptide synthesis therefore requires tightly controlled interactions between these subunits. These inter-subunit interactions are ensured by pairs of small COM domains located at the N-terminal and C-terminal extremities of the NRPS subunits.
Pyrrolamides constitute a family of metabolites whose biosynthesis involves NRPS. Most of these compounds bind non-covalently to the minor groove of DNA, targeting sequences rich in A/T bases (four consecutive bases). This interaction confers various biological properties on them, including antibacterial, antiviral, or antitumor activities. However, their cytotoxicity limits their clinical use. Our team has elucidated the biosynthetic pathways of several pyrrolamides (congocidine, distamycin, anthelvencin) and has undertaken the characterisation of several others (pyrronamycins, amidinomycin, and kikumycins). Our work has revealed that these metabolites are assembled from a limited set of precursors, which are combined in various ways to generate structural diversity.
Pyrrolamides are synthesised by non-canonical NRPS composed of discrete domains or modules. This structural organisation is highly atypical. Indeed, the vast majority of NRPS adopt the classical architecture. In the rare instances where biosynthetic pathways involve discrete NRPS domains or modules, these generally participate in the synthesis of precursors that are subsequently assembled by canonical NRPS.
The unconventional architecture of pyrrolamide NRPS provides a simplified framework to investigate fundamental aspects of NRPS function, such as protein-protein interactions and the intracellular spatial organisation, features that are likely to be essential for the development of the synthetic biology of nonribosomal peptides. A recent study on the engineering of polyketide synthases (PKS), enzymes of specialised metabolism sharing a domain and module architectural organisation similar to that of NRPS, suggests that a small subunit structure (i.e., with a low number of modules) could enhance the polyketide production yields. This finding raises questions regarding the influence of NRPS architecture on nonribosomal peptide biosynthesis. In particular, could the unusual architecture of the NRPS involved in pyrrolamide biosynthesis also favour their production? If so, deciphering the molecular mechanisms governing interactions between the subunits of these atypical NRPS could prove valuable for the synthetic biology of NRPS. Indeed, no COM domains have been identified in these systems so far and very little is known about the interactions between the different subunits of these atypical NRPSs.

The project aims to achieve three main objectives:
1. Reconstitute a canonical architecture for the NRPS responsible for congocidine biosynthesis
2. Identify regions mediating interactions between the subunits of the congocidine NRPS by combining invivo, in vitro and in silico approaches
3. Analyse the cellular localization of the congocidine NRPS using fluorescence imaging.