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Final Report Summary - P4FIFTY (Development of Cytochrome P450 Enzymes for the Chemical Manufacturing Industries)

1. Publishable Summary
P4FIFTY Final Report 01/01/2012 to 31/12/2015
P4FIFTY – FP7 PEOPLE ITN 2011-289217
Development of Cytochrome P450 Enzymes for the Chemical Manufacturing Industries

Professor Neil Bruce, P4FIFTY Project Coordinator, CNAP, Department of Biology, University of York, Wentworth Way, York YO10 5DD, UK. Tel +44 (0)1904 328777, Email: neil.bruce@york.ac.uk Website: www.p4fifty.eu

1.1 Summary of Project Objectives
P4FIFTY is an EU-funded (FP7) European Marie Curie Initial Training Network of academic and industrial researchers, working to develop enzymatic methods for green oxidation chemistry, through the isolation, redesign and application of cytochrome P450 enzymes.
Cytochromes P450 (P450s or CYPs) are heme-containing oxygenase enzymes that catalyse crucial reactions in physiology, biosynthesis and biodegradation. P450s have the potential to be harnessed as catalysts for cleaner and greener synthesis of important intermediates in the bulk chemical, pharmaceutical and agrochemical industries. However, the application of P450s has hitherto been thwarted by inherent limitations of the enzymes, such as low activity, insoluble expression and the dependence on auxiliary electron transport proteins for full efficacy. These factors have created barriers to the scale-up of P450 reactions.
As part of the P4FIFTY network, research teams at 8 European institutions, supported by 2 industrial partners, have worked in collaboration to address some of these issues.
The project was coordinated by Prof. N. Bruce, at the University of York, UK. The other groups were led by Prof. G. Grogan (University of York), Prof. N. Turner & Prof. S. Flitsch, (CoEBio3, University of Manchester, UK); Prof. B. Hauer & Prof. J. Pleiss, (University of Stuttgart, Germany); Prof. D. Janssen & Dr. A-M. Thunnissen, (University of Groningen, Netherlands); Prof. D. Werck, (CNRS, University of Strasbourg, France); Prof. B. L. Møller & Prof. Søren Bak, (University of Copenhagen, Denmark); Prof. J. Woodley, (Technical University of Denmark, Lyngby, Denmark), and Prof. R. Bernhardt, (University of Saarland, Germany), with two industrial partners; Dr. J. Riegler, (Lonza, Switzerland) and Dr. M. Hayes, (Astra Zeneca, Sweden).
As an ITN project, a key objective of P4FIFTY was to train junior scientists in the technologies involved in the industrial application of P450s. The overall purpose of this has been to deliver a trans-European network of industrially oriented biotechnologists, whose expertise will help to advance the state-of-the-art of chemical manufacturing and pharmaceutical industries in Europe.
1.2 Work Performed since the Beginning of the Project
Work performed throughout the project has focussed on the following areas: gene discovery for new P450 activity in plants and microbes; enabling P450 application through fusion protein technology; protein engineering, using strategies informed by X-ray structure and bioinformatics; process technology for the scale-up of P450-catalysed reactions.
1.3 Main Results Achieved (Final Results)
Libraries of new P450s were identified in Arabidopsis, grapevine and the fungus Beauveria bassiana. Annotation of the grapevine genome identified target P450s involved in the biosynthesis of oxidised monoterpenols such as wine lactone; a key determinant of wine flavour. P450s involved in the cyanogenic glucoside synthesis pathway were identified in barley, Lotus japonicas and eucalyptus. A homolog of CYP79A1 was identified in sorghum. A P450 from Arabidopsis (CYP81D11), which was upregulated when exposed to the phytotoxic xenobiotic TNT (trinitrotoluene), was shown to have a role in TNT tolerance in plants and therefore has potential for use in phytoremediation of contaminated soil.
High Throughput Screens (HTS) were developed by several partners (including RUG, AZ, and UNIMAN) to facilitate P450 activity screening. These included fluorescent screens, UPLC/MS methods and novel and highly selective colorimetric assays.
Mutant libraries of CYP106A1 and CYP106A2 were created and screened for steroid hydroxylation potential. New substrates were identified for both enzymes, and unprecedented 11-oxidase activity was also discovered. A library of mutants of another potential target (CYP102A1) was created and screened, revealing mutants with new activities and selectivities. A 100% regioselective mutant was identified for this target reaction (ring hydroxylation of 4-ethylphenol to 4-ethylcatechol).
Protein fusion technology was exploited to develop libraries of P450-reductase fusions, which were made available to the consortium for screening for improved activity, for example in alkane hydroxylation reactions. A P450BM3-phosphite dehydrogenase fusion protein, able to use (cheap) phosphite as an electron donor, was characterized, and mutants with improved activity constructed. An active fusion was created by combining a truncated and codon-optimised Arabidopsis cinnamate hydroxylase (CYP73A5) with ATR2, an Arabidopsis NADPH-cytochrome P450 reductase. Enzyme engineering was used to improve the activity and selectivity of a chimeric protein (CYP153AMaq-CPRBM3) for the terminal hydroxylation of fatty acids.
Two bacterial P450s (CYP109E1, CYP109A2) were expressed, purified and crystallized for the first time, providing insights into steroid binding. The crystal structure of CYP153A (from Marinobacter) was solved.
The P450 engineering database (CYPED v6.0) was updated and used for bioinformatic improvement of P450s for synthesis. This approach was used to understand CYP101A1 selectivity towards methylated ethylbenzene substrates, which were subsequently characterized experimentally. Bioinformatics was also used to design thermostable mutants of adrenodoxin reductase and to model the conformational space of CYP109E1, as well as to design mutants for the conversion of industrially relevant compounds catalysed by CYP102A1.
In addition, new insights were obtained into biochemical pathways. Comparative metabolic profiling was carried out in 3 cyanogenic plant species and a novel endogenous turnover pathway for cyanogenic glycosides was proposed, involving their conversion into non-cyanogenic compounds without the release of hydrogen cyanide. CYP79A1 and CYP71E1, components of the Dhurrin metabolon in sorghum, were inserted into nanodiscs and studied using cyclic voltammetry, to reveal the electrochemical properties of the proteins.
Promising biotransformations from screens described above were scaled-up, and process bottlenecks identified. The economics of the processes were evaluated, using modelling software.
1.3.1 Training of Fellows
The researchers were supervised and mentored by internationally recognized experts and had access to state-of-the-art equipment. Hands-on project training was supplemented with formal training courses in relevant and related fields, with further complementary training provided by the host institutions. Integral to the research program was a strong industrial training element for the fellows, whereby ERs (Experienced Researchers) in industry underwent high level training in the application of enzymes for industrial processes and passed on their experience to the ESRs (Early Stage Researchers).
The researchers have presented their work at P4FIFTY project review meetings, held at 6 month intervals over the course of the project. Attendance at these meetings has encouraged the fellows to develop strong network connections. The researchers have also had opportunities to participate in ‘masterclass’ workshops on various topics, including Bioprocess Engineering (May 2013), Protein Engineering of P450s (September 2013), Assembly of Nanodiscs and SMALPs (April 2014), Proposal writing (October 2014) and Training in Scientific Commercialization (October 2014).
In addition, fellows took full advantage of opportunities for dissemination of the project, taking poster presentations to conferences such as the 18th International Cytochrome P450 Meeting in Seattle, USA (June 2013) and the 12th International Symposium on Cytochrome P450 in Kyoto, Japan (September 2014). Fellows also engaged in a range of outreach activities, such as Manchester Science and Engineering weeks (March 2014 and 2015).
1.4 Potential Impact of Results
As described above, the results of P4FIFTY have included the discovery of new enzymes and improvements in enzyme activity and selectivity, coupled with the development of novel screening techniques and advances in bioinformatics and process engineering. These achievements have brought the P450 community several strides closer to their goal of creating an ‘off the shelf’ toolbox of P450 biocatalysts for industrially relevant biotransformations. Significant progress has been made in overcoming some of the hurdles that have historically hampered the use of P450s in industrial processes. Through P4FIFTY, the wider community will reap the benefits of improved efficiency and reduced environmental impacts. As well as this, the project has delivered high level training of a cohort of junior experts equipped with the skills needed to continue to advance P450 science and maintain Europe’s place at the forefront of this cutting edge technology.

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UNIVERSITY OF YORK
United Kingdom
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