Molecular mechanism and engineering of P450 peroxygenases for synthetic biology applications

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.

Few enzymes are known to produce alkenes/alkanes. A recently discovered class is the peroxygenase cytochrome P450 enzymes. In these P450s, hydrogen peroxide is used efficiently to generate a reactive iron-oxo species, leading to oxidative decarboxylation of a fatty acid substrate. A n-1 terminal alkene is the major product, with minor products hydroxylated at alpha- or beta-carbons also observed. In this project, we will characterize structural, spectroscopic and kinetic properties of two peroxygenase P450s - OleT from a Jeotgalicoccus sp. and KR from Kocuria rhizophila. In underpinning studies we have shown that these enzymes exhibit distinctive, but overlapping, fatty acid specificity profiles in terms of chain length selectivity. We have determined the OleT structure, and crystallized the KR P450. We will use computational approaches (DFT and QM/MM) to analyse reaction mechanism at the heme site and to establish the differing modes by which the P450s catalyse decarboxylation or hydroxylation reactions. We will use combined fast reaction and spectroscopic methods to identify transient intermediates in the peroxygenase catalytic cycle, and will determine the KR P450 structure in substrate-bound and -free forms. These data will inform a mutagenesis approach to promote OleT and KR P450 decarboxylation reactions at the expense of substrate hydroxylation, and to enhance binding of shorter chain lipids to produce hydrocarbons suitable for gasoline/diesel fuel, as well as other industrial applications. The efficiency of peroxide-driven substrate oxidation will be compared with that using a selection of NAD(P)H-dependent redox partners in both P450 systems, and we will also establish the productivity of alkenes by wild-type and engineered peroxygenase variants in bacterial fermentations. The project will thus produce both fundamental mechanistic understanding of an important enzyme class, as well as clear demonstrations of their applications in synthetic biology.

The proposed research impacts on the biofuels agenda and involves exploration of the mechanism of an important new class of enzymes that generate alkenes from fatty acids in a hydrogen peroxide-drive reaction. These microbial cytochrome P450 "peroxygenase" enzymes are rare examples of known enzymes that can produce alkenes/alkanes directly from lipid substrates. Moreover, they operate with a cheap chemical (H2O2) to drive these reactions efficiently. Our study will clarify the mechanistic features of two such peroxygenases (P450s KR and OleT), which exhibit differing fatty acid substrate chain length specificity ranges and thus have potential to generate a variety of different alkene products. Further, we will use computational and molecular modelling approaches to engineer these enzymes to (i) enable accommodation of shorter chain length lipids to expand their substrate specificity range, and (ii) perturb their regioselectivity of substrate oxidation to eradicate minor hydroxylated products and promote the near-exclusive formation of the desired alkenes. Work will also be done to optimize productivity of alkenes using diverse redox partner systems, and to facilitate isolation of alkene products from bacterial cell cultures as proof of concept for the industrial exploitation of peroxygenases in alkene production. Beneficiaries from this research includes researchers in academia and industry engaged in biofuel research and with interests in exploitation of new enzyme systems producing alkenes for applications as fuels and in other uses (e.g. synthetic processes). Through developing engineered peroxygenase systems with enhanced catalytic properties in alkene production, other researchers will be encouraged to develop these (and related) enzyme systems for biotechnological applications and chemicals manufacture. Prime candidates for exploitation of such technologies are petrochemical companies and other fine chemicals manufacturers. Benefits will come in the form of new routes to biofuel production and for generation of an array of terminal alkenes for synthetic applications. Detailed studies of the so-called "peroxide shunt" mechanism used by the peroxygenase P450s will also provide insights into how this cheap approach to driving P450 enzyme catalysis can be used more widely and effectively for monooxygenase applications in e.g. synthesis of steroids, drug metabolites and other important oxychemicals. In addition, our detailed research into biofuel enzymology will further consolidate this area and attract further attention to the field with long term benefits for more environmentally "friendly" routes to renewable generation of fuel molecules, as well as alkenes for use in diverse synthetic applications. Ultimately, this work will help contribute to a growing shift towards renewable energy and chemical synthesis applications, and will add to the White (Industrial) Biotechnology agenda by generation of new enzyme catalysts that are less environmentally damaging than existing processes for production of the same chemicals. The research will provide new means of accessing valuable alkenes for biofuels and other applications. Improvements of enzyme catalysts will be achieved and systems put in place for fermentation of alkenes from bacterial cells. Products generated will be appropriate for use in existing car engines. Impacts should thus come from provision of advanced technology for green chemicals manufacture, with potential timescales for benefits coming in the decade following completion of the study in which engineered peroxygenases are generated and demonstrated to be effective in alkene production from bacterial cells on a commercially viable scale. Staff working on the project will receive training in areas including enzymology, structural biology, bacterial fermentation and biofuel applications, enhancing their skills in a wide range of areas, particularly with respect to employment in the chemicals and energy sectors.