Chemoenzymatic routes to rose oxide

The scent of the rose is one of the most sought after in the fragrance industry and for more general domestic as well as personal products. Eau de toilette contains the lighter, more volatile rose essential oil components such as citronellol and geraniol. Perfumes are longer lasting as they contain less volatile components such as the rose ketones damascone, damascenone, ionones and the like. Another component of rose essential oil is the small molecule rose oxide. Although present in tiny amounts in rose oil, it has an odour threshold of 0.5 parts-per-billion and contributes more to the rose note than all other compounds except for damascone. Rose oxide is described as having a floral, green note and so important that a stamp with its molecular structure printed on it was issued when the compound was first isolated from the oil of Bulgarian roses. Rose oxide is a high value fine chemical because it takes 3 tonnes of rose blossoms to generate 1 kg of rose oil which contains <0.1% rose oxide. Material isolated from natural sources is expensive ($7,000 per kg). Commercially, rose oxide is produced by chemical routes from citronellol; one route uses bromine while others require reagents that are polluting to produce and use. The aim of this project is to develop a biological process to rose oxide from citronellol, with the key step being the oxidation of a specific carbon-hydrogen bond in citronellol. These bonds are chemically inert, which leads to more complex routes or more polluting processes in order to use citronellol as feedstock. We have developed enzyme variants capable of carrying out this critical step in water at ambient temperature. The industry partner for the project is in the process of taking this to commercial scale rose oxide production by a sustainable and non-polluting biotechnological process. The purpose of this project is to develop more active enzymes and alternative feedstocks based on citronellol in order to make the rose oxide process even more efficient and productive. Advanced computational methods will also be applied to help guide enzyme design and process development.

This project seeks to develop an alternative substrate for a process to synthesise rose oxide synthesis via citronellol oxidation. The industry partner is developing such a process via engineering and evolution of a P450BM3 enzyme variant for citronellol oxidation. The proposed project will adopt two approaches that parallel this effort in enzyme and process refinement. Firstly, molecular dynamics simulations will be used to generate energy minimised structures of enzyme variants showing different levels of oxidation selectivity. The substrate will be docked into the active sites to provide information on binding orientations and residue interactions that alter/control product selectivity. These insights form the basis for more informed enzyme design and engineering. Secondly, ester and other derivatives of citronellol will be prepared and screened for activity and selectivity of oxidation by the extensive collection of enzyme variants in our laboratory to identify residues and amino acid substitutions that promote oxidation at the correct position. Further engineering will be undertaken to increase activity, product concentration and yield. Derivatisation alters the size and hydrogen bonding requirements for substrate binding, which could lead to enhanced activity and process efficiency. This approach may complement the effort on the unmodified substrate by the industry partner. Computational methods will also be applied to investigate residue-derivative interactions. We aim to develop an alternative process that is at least as, if not more, efficient than that based on the unmodified substrate. A significant feature of this project is the fact that the new variant/derivatised substrate combination can be introduced directly as replacements into the existing process with minimal effort.

In the broadest sense the results from this project will benefit the academic and industrial research community on publication of successes from the research approaches, namely computationally aided enzyme design and evolution and the use of alternative substrates. The economic performance and competitiveness of the UK will benefit directly from job creation and increased profits for the industry partner and in the longer term indirectly as the lessons learned from our work become enabling technology, leading to improved industrial processes. The academic community will gain new approaches to synthesis and process development. Researchers in biocatalysis and bioprocessing will be primary beneficiaries by being able to modify and redesign synthetic routes to take advantage of C-H functionalisation by new enzymes and new variants.

The industry partner will work in close collaboration with the research team. The University and industrial teams are motivated by the potential new insights into enzyme-substrate interactions and enzyme design and process strategies arising from the research. The project will have immediate impact on the industry team, with the output from computational approaches, based in part on their input, providing insights into substrate recognition in the enzyme active site. The slightly different approach to enzyme screening and engineering will also benefit these scientists.

The PDRA will benefit from the interdisciplinary nature of the research program, the methods and techniques. They will acquire knowledge and skills in computational approaches to enzyme-substrate interactions, enzyme engineering, biotransformation, process parameter selection and optimisation, and synthetic biology. They will derive invaluable benefits from the close collaboration with scientists at the industry partner, and develop key skills in setting objectives, interpreting results, data management and IP protection. This combination of academic and industrial research approaches to high priority areas will provide excellent opportunities for career development.