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

Descripción del proyecto

Un método de ingeniería genética para obtener enzimas sostenibles y más asequibles

Los procesos de fabricación insostenibles convierten a la industria química en uno de los principales responsables de la contaminación mundial. Con el apoyo de las acciones Marie Skłodowska-Curie, el proyecto UNSHACKLED contribuirá al desarrollo de reacciones ecológicas impulsadas por enzimas que se ajusten al Pacto Verde Europeo. La investigación se centrará en una clase de enzimas muy poco estudiada que podría generar valiosos componentes químicos básicos, pero que actualmente resulta costosa para las fábricas, ya que depende de una proteína asociada de alto coste. Los investigadores utilizarán simulaciones de dinámica molecular e ingeniería genética para estudiar estas proteínas a nivel molecular y rediseñarlas de modo que funcionen de forma totalmente independiente. Estas enzimas modificadas se someterán a pruebas en experimentos de laboratorio. Los resultados del proyecto podrían ayudar a crear herramientas asequibles y sostenibles que abran nuevas posibilidades químicas para aplicaciones industriales.

Objetivo

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.