Adaptation of heterologous cytochrome P450s to plant expression and membrane environment for improvement of biocatalytic function

• Applicant: Iurii Sheludko, Ph.D.
• Subject Area: Plant Biochemistry and Biophysics ; Plant Physiology ; Structural Biology ; Plant Cell and Developmental Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 516587177

Our project is focused on the optimization of conditions for the efficient expression of heterologous membrane-bound cytochrome P450 monooxygenases (CYPs) in plant cells and studying the molecular and structural mechanisms of their function in different plant membrane surroundings and scaffold structures. This will allow for developing a robust and sustainable platform for highly efficient production of functionally active CYPs for targeted modulation of metabolic pathways, rapid testing of substrates, increasing the biodiversity of natural products and preparative scale biotransformation of valuable prodrug molecules of synthetic and natural origin. We plan to increase the accumulation of foreign CYPs in transient nuclear and constitutive plastome expression systems using genetic cassettes providing elevated transcript levels and enhanced translational capacity. To improve the function of heterologous CYPs in a foreign membrane environment, we will study and optimize the molecular structures of the native CYP transmembrane domain and heterologous transport signals to adapt them to the corresponding subcellular compartment. We will consider the advantages of CYP scaffolding or clusterization for enzyme activity, stability and product yield. The most efficient expression system will be validated by performing preparative biotransformation of pharmaceutically valuable prodrug molecules and scalable synthesis of new-to-nature indican derivatives. Implementation of the project will contribute to understanding chloroplast protein transport mechanisms and CYP functionality in foreign membrane environments and scaffold clusters, and provide a possibility for scaled biocatalytic oxidation of compounds for the pharmaceutical or chemical industry. As a result, we expect to achieve the productivity of the plant CYP expression system surpassing that of yeast or animal cells. Development of the plant-based platform for enzymatic synthesis of chemicals, which are used daily by millions of people worldwide, brings overall economic, social and environmental benefits. The new processes can substitute the traditionally used chemical techniques resulting in less energy expenditure and independence from fossil raw materials. The utilization of water-based enzyme catalytic systems mitigates the effects of conventional solvents, toxic intermediates and inorganic catalysts and reduces the need for their exploitation, containment and disposal.

• DFG Programme: Research Grants