Molecular mechanism and engineering of P450 peroxygenases for synthetic biology applications

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The proposed project will characterize an important new type of enzyme catalyst with uses in the production of biofuel molecules, as well as other chemicals with applications in industry. Two representatives of a new class of heme-containing enzymes will be produced and their structural and catalytic properties studied in detail. These enzymes are termed "peroxygenases" due to their ability to use hydrogen peroxide as a substrate; and the enzymes studied here (named P450 KR and P450 OleT) use peroxide to convert fatty acids into the valuable hydrocarbon molecules alkenes. The alkenes (of appropriate size) can be used in car engines as fuel, and have multiple other applications in the chemicals industry; e.g. in making plastics (and other polymers) and alcohols. In work underpinning this application, we have developed methods to produce the KR and OleT P450 enzymes (using genes cloned from different bacteria that naturally produce the enzymes) and for purifying the P450s. This has enabled us to establish that the ranges of lengths of fatty acids recognized by OleT and KR are different, such that the KR P450 produces a group of shorter chain alkenes than can OleT. These enzymes are thus complementary and together are able to produce a wide range of different alkenes using cheap fatty acids as substrates. In this project, we will analyse how these enzymes function to convert fatty acids into alkenes. This will be done using both computational/modelling procedures (to understand the chemistry involved and which parts of the enzymes are crucial for the alkene production process) and through a combination of structural, spectroscopic and fast reaction methods (to determine how the enzymes fold and bind their substrates, how fast the alkene production reaction occurs, and to understand the mechanism involved). These studies are essential to enable us to rationalize how this important biochemical transformation of fatty acids to alkenes occurs, and will also be crucial to allow protein engineering (i.e. mutating enzymes in a targeted way) to be done to improve binding of selected fatty acids (particularly short chain lipids that generate more volatile alkenes with better properties as biofuels) and to disfavour unwanted side reactions where a different product (hydroxylated fatty acid) is formed. Having engineered the KR and OleT enzymes to optimize their reactivity, different routes to driving their function will be explored - since another way of driving their reactions is by providing them with different proteins ("redox partners") that are used by other classes of P450 enzymes (e.g. those involved in human drug metabolism and steroid synthesis). Once the most efficient means of driving these enzymes is identified, work will be done to produce the desired short- to mid-chain alkenes using bacterial cells that make the OleT/KR P450s at high levels. Quantification of alkenes will be done to determine production levels and to establish the efficiency of generation of different chain length alkenes in an industrial-type fermentation process. In parallel studies, the ability of native and engineered forms of the OleT/KR P450s to produce alkene or hydroxylated products from different types of fatty acids (including polyunsaturated and branched chain lipids) will also be determined, in order to establish whether diverse types of lipids can be substrates for these enzymes, and to evaluate their potential to make distinct types of products with industrial applications. This project thus has both fundamental and applied aspects: first to enable a detailed understanding of the structure/mechanism of two members of a biotechnologically important class of enzyme catalyst (enabling us to engineer the OleT and KR enzymes rationally for improved performance), and second to demonstrate their versatility and uses in synthetic biology for industrial exploitation - most notably in generating alkenes for biofuel and chemical products applications.