Manipulation of partner protein expression levels in Cytochrome P450 systems by molecular and fermentation-based manipulations

Context
Cytochrome P450 monooxygenases (CYPs) are heme-containing enzymes involved in the oxidation of steroids, fatty acids, and xenobiotic compounds and play a major role in human Phase I metabolism. Microbial cytochrome P450s mimic human cytochrome P450 activity and are used in industrial biotechnology for the production of hydroxylated derivatives and generation of standards for metabolite identification and/or toxicology studies (See FDA's MIST guidelines) [1].
Hypha Discovery together with John Ward's group in Innovate UK projects, developed recombinant microbial cytochrome P450s mined from some of Hypha's microorganisms. These recombinant cytochrome P450 systems were scaled to bioreactor format for the generation of enzyme materials and led to the successful launch of the PolyCYPs(r) kit in January 2019.
Despite the potential of microbial cytochrome P450s, they often exhibit poor catalytic activity and rates of hydroxylation due to their requirements for a compatible electron transport system.

Aims and Objectives
This PhD project will investigate the molar ratios of the Class I P450 systems consisting of a cytochrome P450 and two redox partners, a FAD-containing ferredoxin reductase and an iron-sulfur containing ferredoxin (Fd). The redox partners deliver two electrons from NAD(P)H to the cytochrome P450's heme and catalyze the reduction of molecular oxygen where one oxygen atom is introduced into the substrate and the second atom is reduced to water [2].
Altering the relative ratio of each protein component can lead to more efficient reactions per mole of P450 and a ratio of P450 to Fd to ferredoxin reductase (approximately 1P450:24Fdx:6FdR) is best. The current PolyCYPs(r) materials are expressed in a single polycistronic operon developed at UCL encoding the three component proteins in the order of P450, ferredoxin and ferredoxin reductase [3]. By manipulating the expression of the redox partners, the system will be improved to enhance the biocatalytic efficiency of the P450. The aim is to provide a catalytic system surpassing the current operons enabling the use of these P450 enzymes for industrial applications beyond research-scale applications.

Methodology
Yr1, the current ratio of the protein components will be measured, this will generate the baseline for the performance of the current Hypha strains. The molar ratios of the three component system will be confirmed with purified tagged protein components and to establish whether the relationship at other CYP concentrations is linear or non-linear relationship.
2nd and and 3rd years, molecular biology approaches will be used to modify the current cytochrome P450 system to alter the expression levels of the partner proteins including changing the position of protein components, altering protein expression levels by modifying ribosome binding sites and the use of multiple individual transcriptional units; results from each optimization will be combined to generate a superior single cytochrome P450 cell factory. Once established in shake-flask format, the robustness of the expression system will be scaled-up in 5L fed-batch bioreactors.

Alignment to EPSRC objectives
This project aligns to the EPSRC objectives of Catalysing growth 3.1.1, Transforming Healthcare 3.1.3. Also all of the Objective 2 Aims: Realising the potential of engineering and physical sciences research and Accessing talent through equality, diversity and inclusion (EDI) (3.3.3)

Collaborating company
Hypha Discovery Ltd.

References
[1] Girvan, H. M. & Munrow, A. W. (2016). Curr. Opin. Chem. Biol. 31: 136-45.
[2] Hubbard, P. A et al. (2001). J. Biol. Chem. 276: 29163-70.
[3] Hussain, H.A. & Ward, J.M. (2003). Appl Environ Microbiol. 69 (1): 373-82.

The CDT has a proven track record of delivering impact from its research and training activities and this will continue in the new Centre. The main types of impact relate to: (i) provision of highly skilled EngD and sPhD graduates; (ii) generation of intellectual property (IP) in support of collaborating companies or for spin-out company creation; (iii) knowledge exchange to the wider bioprocess-using industries; (iv) benefits to patients in terms of new and more cost effective medicines, and (v) benefits to the wider society via involvement in public engagement activities and impacts on policy.

With regard to training, provision of future bioindustry leaders is the primary output of the CDT and some 96% of previous EngD graduates have progressed to relevant bioindustry careers. These highly skilled individuals help catalyse private sector innovation and biomanufacturing activity. This is of enormous importance to capitalise on emerging markets, such as Advanced Therapy Medicinal Products (ATMPs), and to create new jobs and a skilled labour force to underpin economic growth. The CDT will deliver new, flexible on-line training modules on complex biological products manufacture that will be made available to the wider bioprocessing community. It will also provide researchers with opportunities for international company placements and cross-cohort training between UCL and SSPC via a new annual Summer School and Conference.

In terms of IP generation, each industry-collaborative EngD project will have direct impact on the industry sponsor in terms of new technology generation and improvements to existing processes or procedures. Where substantial IP is generated in EngD or sPhD programmes, this has the potential to lead to spin-out company creation and job creation with wider economic benefit. CDT research has already led to creation of a number of successful spin-out companies and licensing agreements. Once arising IP is protected the existing UCL and NIBRT post-experience training programmes provide opportunities for wider industrial dissemination and impact of CDT research and training materials.

CDT projects will address production of new ATMPs or improvements to the manufacture of the next generation of complex biological products that will directly benefit healthcare providers and patients. Examples arising from previous EngD projects have included engineered enzymes for greener pharmaceutical synthesis, novel bioprocess operations to reduce biopharmaceutical manufacturing costs and the translation of early stem cell therapies into clinical trials. In each case the individual researchers have been important champions of knowledge exchange to their collaborating companies.

Finally, in terms of wider public engagement and society, the CDT has achieved substantial impact via involvement of staff and researchers in activities with schools (e.g. STEMnet), presentations at science fairs (Big Bang, Cheltenham), delivery of high profile public lectures (Wellcome Trust, Royal Institution) as well as TV and radio presentations. The next generation of CDT researchers will receive new training on the principles of Responsible Innovation (RI) that will be embedded in their research and help inform their public engagement activities and impact on policy.