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(BBSRC DTP) Characterization of P450 peroxygenases and their applications in alkene/biofuel production


Project Description

The project involves characterization of P450 peroxygenase enzymes, a class of the P450 enzymes which naturally use hydrogen peroxide rather than redox partner systems to drive catalysis. These peroxygenases have important biotechnological applications in production of terminal alkenes as biofuel components and fine chemicals. The project will exploit bioinformatics and molecular modelling approaches to identify new, more robust peroxygenases with improved activities conducive to biotechnological applications. Proteins identified will be purified and crystallized using high-throughput crystallographic methods, with data collection at the Diamond Synchrotron and structural determination done in the MIB. Computational analysis will be used to predict mutations conducive to enhanced catalytic activity. High throughput analysis of products formed using novel peroxygenases identified from bioinformatics screens will be done using LC-/GC-MS to characterize alkenes and other products. Evolved catalysts will be interrogated using kinetic/spectroscopic approaches with state-of-the-art equipment available at the MIB through the SYNBIOCHEM Synthetic Biology Research Centre.

Dwindling oil resources have stimulated researchers to identify novel routes to the production of molecules such as alkenes and alkanes from microbial and other sources. Among the most efficient enzymes identified in this way are the P450 peroxygenases, which have different active site architecture to typical P450 monooxygenases, and bind mid- to long-chain fatty acids which interact through their carboxylate group to a conserved arginine residue. The P450 peroxygenases then use hydrogen peroxide to form a reactive iron-oxo species on the P450’s heme group, leading to the oxidative attack on the bound lipid substrate and to the production of either an alkene product, or a 2- or 3-hydroxylated form of the fatty acid. The alkene is the desired product in the context of biofuel, but at present there is a limited number of P450 peroxygenases characterized, some of which make little or no alkenes. However, we have identified P450 peroxygenases that make substantial amounts of alkenes, with the OleT enzyme from a Jeotgalicoccus species producing an excess of alkene product over hydroxylated fatty acids. The project aim is to expand the available repertoire of these enzymes in order to identify and characterize P450 peroxygenases with greater thermal stability and activity than the present set of enzymes. To achieve this, we will use bioinformatics approaches to locate novel P450 peroxygenases in thermophiles and other microbes living in challenging conditions. The genes for such enzymes will be synthesised or cloned, and expressed in E. coli or other appropriate hosts. The new peroxygenases will be expressed and purified for catalytic and structural analysis, using protein crystallography and X-ray diffraction methods to determine novel P450 peroxygenase structures and LC-/GC-MS methods to explore product (alkene and hydroxylated fatty acid) formation. Novel peroxygenases will be assessed for lipid binding affinity using UV-visible binding assays based on peroxygenase heme perturbation. Other work will focus on a novel type of P450 peroxygenase in which the enzyme dimerizes through interlocking interactions made by the N-terminal helices of each monomer, and will involve protein engineering studies to enhance its stability. The project offers training in areas such as enzymology, analytical chemistry, protein expression and engineering, structural biology and bioinformatics, all of which are key tools in protein science. The appointee will join a vibrant and supportive group with expertise in these areas and will participate in an exciting research programme involving the characterization of a biotechnologically important enzyme class.

Contact for further information:

https://www.research.manchester.ac.uk/portal/Andrew.Munro.html
https://www.research.manchester.ac.uk/portal/David.Leys.html

Entry Requirements:
Applications are invited from UK/EU nationals only. Applicants must have obtained, or be about to obtain, at least an upper second class honours degree (or equivalent) in a relevant subject.

Funding Notes

This project is to be funded under the BBSRC Doctoral Training Programme. If you are interested in this project, please make direct contact with the Principal Supervisor to arrange to discuss the project further as soon as possible. You MUST also submit an online application form - full details on how to apply can be found on the BBSRC DTP website View Website

As an equal opportunities institution we welcome applicants from all sections of the community regardless of gender, ethnicity, disability, sexual orientation and transgender status. All appointments are made on merit.

References

1) Girvan HM, Poddar H, McLean KJ, Nelson DR, Hollywood KA, Levy CW, Leys D, Munro AW. Structural and catalytic properties of the peroxygenase P450 enzyme CYP152K6 from Bacillus methanolicus. J. Inorg. Biochem. 188:18-28 (2018). 2) Munro AW, McLean KJ, Grant JL, Makris TM. Structure and function of the cytochrome P450 peroxygenase enzymes. Biochem. Soc. Trans. 46:183-196 (2018). 3) Matthews S, Belcher D, Tee KL, Girvan HM, McLean KJ, Rigby SE, Levy CW, Leys D, Parker DA, Blankley RT, Munro AW. Catalytic determinants of alkene production by the cytochrome P450 peroxygenase OleTJE. J Biol Chem 292:5128-5143 (2017). 4) Belcher J, McLean KJ, Matthews S, Woodward LS, Fisher K, Rigby SE, Nelson DR, Potts D, Baynham MT, Parker DA, Leys D, Munro AW. Structure and biochemical properties of the alkene producing cytochrome P450 OleTJE (CYP152L1) from the Jeotgalicoccus sp. 8456 bacterium. J Biol Chem 289, 6535-6550 (2014). 5) Grant JL, Mitchell ME, Makris TM. Catalytic strategy for carbon-carbon bond scission by the cytochrome P450 OleT. Proc. Natl. Acad. Sci. U. S. A. 113, 10049-10054.

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