Biocatalysis using P450 Monooxygenases for the Synthesis of Novel Biodegradable Fragrances

Flavour and fragrance (F&F) oils are widely used in the food, pharmaceutical, perfume, cosmetic and detergent industries. However, many F&F compounds that are widely used across many industries are not readily biodegraded in the environment and hence accumulate over time. The poor biodegradation of such materials will make them untenable F&F products in the future; and certainly, from the perspective of Unilever, the industrial partner of this project, will not be used in any products by 2030. Further, materials used as ingredients in perfumes, cosmetics and household products, and flavourings in oral care products must be registered under the European REACH regulation, and so information on biodegradation and bioaccumulation is required. F&F molecules with high performance in many home and personal care products can bioaccumulate because of their hydrophobicity and chemical inertness. The aim of this project is to insert oxygen in the carbon skeletons of problematic F&F molecules that, upon oxidation, could afford F&F molecules for the future. The application of designed cytochrome P450 monooxygenases may render the new molecules more water soluble, bioavailable and biodegradable compared to the parent molecule. The approach has good precedence from nature as metabolically diverse microorganisms contain oxidative enzymes such as P450s to catalyse C-H bond oxidation to activate chemically inert compounds in metabolic and catabolic processes. The project involves screening of F&F molecules with an existing library of P450 enzyme variants known to oxidise a wide range of organic compounds to diverse products, characterisation of products isolated from preparative scale (0.1g-1g) reactions, followed by the design of new variant enzymes via docking-guided mutagenesis of the most selective variant to increase substrate conversion, scalability and selectivity for target products. In collaboration with Unilever scientists, the oxidation products will be studied for skin sensitisation and by odorant-binding assays for receptor activation to demonstrate that they have the potential to provide a sensory modality. Their biodegradability will be assessed using BioWin biodegradation and QSAR methods and experimental biodegradation assays. Current biodegradation models can provide poor fits due to having no data on a particular structure; hence, the project will use bioinformatics techniques to establish structure-function relations for the new molecules designed with respect to their bioavailability and biodegradation within an aqueous media. The project aligns perfectly with the BBSRC Strategic Priority Area 2: Tackling Strategic Challenges - Bioscience for Renewable Resources and Clean Growth. Specifically, the application of enzymatic processes to create new high value industrially relevant materials, developing higher performing bio-based processes, and the use of Techno Economic Analysis to provide an impact analysis will be at the heart of this project. The Clean Growth potential is realised by the application of biotechnology to tackle the most difficult step in creating new materials, that of C-H bond oxidation, which would otherwise require high energy and polluting chemical processes. The converted products include those from renewable sources, components of which will be converted to more degradable products with desirable properties. This positive downstream impact will be delivered by enzyme engineering and evolution methodology that is adaptable to generate the product with the most desirable combination of properties. The combination of library screening and computationally-aided design is transformative because it delivers initial diversity for discovery purposes followed by selectivity enhancements to produce the desired new entries.