Relieving Fe/αKG-dependent Halogenases from their Carrier-dependence

Description du projet

Une approche de génie génétique pour des enzymes durables et plus abordables

Les procédés de fabrication non durables font de l’industrie chimique l’un des principaux contributeurs à la pollution mondiale. Soutenu par le programme Actions Marie Skłodowska-Curie, le projet UNSHACKLED contribuera au développement de réactions respectueuses de l’environnement fondées sur des enzymes et conformes au pacte vert pour l’Europe. Les travaux porteront sur une catégorie d’enzymes très peu étudiée, qui pourrait produire des composants chimiques de valeur, mais dont l’utilisation industrielle reste actuellement coûteuse en raison de sa dépendance à une protéine partenaire onéreuse. Les chercheurs utiliseront des simulations de dynamique moléculaire et le génie génétique pour étudier ces protéines à l’échelle moléculaire et les remanier afin qu’elles fonctionnent de manière totalement autonome. Ces enzymes modifiées seront testées dans le cadre d’expériences en laboratoire. Les résultats du projet pourraient contribuer à créer des outils abordables et durables, ouvrant de nouvelles possibilités chimiques pour les applications industrielles.

Objectif

The chemical industry contributes majorly to environmental pollution due to unsustainable synthetic processes. Thus, green chemistry has been coined, defining principles to achieve environmental benign development.
Within the scope of this project we will pave the way for enzyme-catalyzed aliphatic halogenation. This approach will provide valuable building blocks to the chemical industry in compliance with the UN Sustainable Development Goals and the European Green Deal. We will focus our studies on iron/alpha-ketoglutarate-dependent halogenases (αKGH) which are a heavily understudied enzyme class. Especially, members that depend on an acyl-carrier protein, to deliver the substrate, are too costly for industrial implementation. While current research focuses on the discovery of carrier-free αKGHs, we will relieve carrier-dependent αKGHs from their protein partner. This will make those enzymes, and their vastly untapped substrate scope, more accessible to industrial application.
We will tackle this shortcoming through advanced enzyme design. The application of molecular dynamics simulation will generate insight about the interaction between acyl-carrier protein and αKGH. This will be used to expand the understanding of their relationship on a molecular level. The obtained findings will then provide the foundation for rational enzyme engineering to rid αKGHs from their carrier-dependence. In the following, rational design rules will be devised generally applicable to any carrier-dependent αKGH. Finally, in vitro experiments will be employed to validate our predictions, designing the first αKGH relieved from their carrier protein. By approaching this project I anticipate to expand my skill set with computational methods to guide rational enzyme design. The results of this project will not only provide a new understanding about the interaction between acyl-carrier protein and αKGHs but also make their valuable substrate scope accessible to industrial application.