Characterisation and Engineering of Alkene Producing P450 Peroxygenases for Bioenergy Applications

  • Sarah Matthews

Student thesis: Phd


OleTJE (CYP152L1) is a P450 peroxygenase that was first isolated from Jeotgalicoccus sp. 8456 in 2011. OleTJE is primarily a fatty acid decarboxylase, converting mid-chain fatty acids (C10:0 to C22:0) to terminal alkenes, which are industrially useful petrochemicals. Terminal alkenes are hydrophobic with high energy density, and are compatible with existing transportation infrastructure. Thus OleTJE has attracted considerable interest due to potential applications for generating "drop-in" biofuels. As a P450 peroxygenase, OleTJE is able to utilise H2O2 as a sole oxygen and hydrogen donor. This is atypical of P450s, which usually require electron transfer from redox partners to perform substrate oxidation. Other P450 peroxygenases have previously been characterised, including fatty acid hydroxylases P450 Spalpha (CYP152B1) from Sphingomonas paucimobilis and P450 BSβ (CYP152A1) from Bacillus subtilis. In addition to decarboxylation, OleTJE also hydroxylates fatty acids, generating 2-OH and 3-OH fatty acids as minor products. P450 BSβ has also been reported to perform low levels of decarboxylation. However, OleTJE has superior decarboxylase activity, posing questions about the mechanism of OleTJE. This thesis describes initial structural and biochemical characterisation of OleTJE. These data highlighted three amino acid residues thought to be key for effective catalysis: His85, Phe79 and Arg245. We hypothesised that the active site His85 could act as a proton donor to thereactive ferryl-oxo species compound I, allowing homolytic scission of the substrate C-Calpha bond to form the alkene product. Phe79 sandwiches His85 between the heme, and Arg245 co-ordinates the fatty acid carboxylate moiety. I performed mutagenesis studies to probe the roles of these residues, creating H85Q, F79A, F79W, F79Y, R245L and R245E OleTJE mutants, and characterised them by a combination of spectroscopic, analytical and structural methods. I also developed a novel system, where OleTJE was fused to alditol oxidase (AldO) from Streptomyces coelicolor, creating a fusion protein where addition of glycerol drives hydrogen peroxide production and the decarboxylation of fatty acids. Finally, studies showed that OleTJE is capable of performing secondary oxidation of hydroxylated products, which has expanded our knowledge of OleTJE's catalytic repertoire. This thesis also describes the initial characterisation of the OleTJE orthologue P450 KR from Kocuria rhizophila, which is also a terminal alkene-forming fatty acid decarboxylase. The crystal structure of P450 KR revealed an unusual dimeric state, with structural interactions unprecedented for a P450 enzyme. These data thus provide characterisation of two P450 peroxygenases involved in the production of terminal alkenes and which are of great interest as tools for the development of alternative sources of advanced biofuels.
Date of Award1 Aug 2017
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorAndrew Munro (Supervisor), David Leys (Supervisor) & Stephen Rigby (Supervisor)


  • Biofuels
  • OleT
  • Cytochrome P450

Cite this