Integrative approach to promote hydroxylations with novel P450 enzymes for industrial processes

Cytochrome P450s are an interesting superfamily of haemoprotein monoooxygenases, which have been studied for over 50 years due to their vast reaction ability. However, previous studies on P450 enzymes have demonstrated how exploiting this reaction ability can be a challenging task. In some cases P450s enzymes are membrane bound and they require complex electron transport systems. For example isolated enzymes require efficient NAD(P)H regeneration systems. Using whole cells can overcome these issues but other physiological problems concerning substrate uptake, product/substrate toxicity, product degradation and poor substrate solubility may also arise. Mammalian cytochrome P450s can metabolise drugs, steroids and herbicides, however, their expression yields can be limited. Consequently, the discovery of novel P450s which can be cloned into E. coli and other host expression systems makes them attractive for industrial use.
For the aforementioned reasons the number of industrial processes utilising P450s is currently limited. Traditionally, P450 monooxygenases have been utilised in toxicity determination or to elucidate the effects of xenobiotic compounds in vivo. Commercial examples include the biotransformation of the steroid hydrocortisone produced at 100 ton/yr by Bayer Pharmaceuticals using P450 monooxygenases to conduct hydroxylation, the production of cortisone from progesterone by Pfizer and Bristol-Myers Squibb Company markets Pravastatin by microbial oxidation of compactin. Future applications could include antibiotic synthesis, anticancer drug synthesis, bioremediation, polymer and flavour production. Strategies aimed at optimising product yields of P450 catalysed reactions are needed for industrial processes to consider using this technology.
The ability to rapidly generate data on whole bioprocesses using automated microscale technologies has been previously demonstrated. In particular, accurate quantification of gas liquid mass transfer rates for a range of process conditions provided crucial insight into the occurrence of oxygen limitations and their effect on enzyme expression, activity and process yield. The application of this approach to challenging P450 catalysed bioconversions would allow quicker identification of optimised process conditions, making them more amenable to large scale development studies and industrial uptake. The proposal aim will be to establish automated microscale methodologies for the whole process evaluation of P450-based novel processes. In this project previous studies conducted at UCL will be extended by the introduction of microscale methods able to evaluate a large number of variables in parallel that have the potential to overcome the limitations of current P450s. An increase in throughput is considered as mandatory to identify P450s with activities suitable for commercial application as is the ability to look for synergistic improvements in both choice of biocatalyst and the processes by which it is produced and applied.

The functionalization of non-activated C-H bonds is one of the major challenges in chemistry. Methods are very rare and selectivity is even more challenging. At the same time, this reaction is highly demanded by both academic and industrial chemists to get a first activation of simple starting molecules. The direct hydroxylation of C-H bonds leads very often to specific compounds vastly needed in the specialty chemistry as well as in the development of active pharmaceutical ingredients. Although chemists have recently made progress towards the hydroxylation of inactivated C-H compounds, enzymes such as cytochrome P450 remain unsurpassed in their targeted specificity and scope. Consequently, the application of P450 enzymes in synthetic organic chemistry is considered "potentially the most useful of biotransformations". However, despite this favourable situation the synthetic application of P450 reactions in industry has been hampered by the limited access to suitable biocatalysts and inefficient biotransformation processes. Within this project we are aiming to solve this bottleneck by the development of a platform for P450 synthesis using automated microscale technologies, a whole process approach and appropriately developed scale-up parameters. The application of this approach to challenging P450 catalysed bioconversions would allow quicker identification of optimised process conditions, making them more amenable to large scale development studies and industrial uptake.

The proposal includes elements of microscale and process automation technologies applied to P450 bioconversions. The proposal fits within the BBSRC Bioenergy and Industrial Biotechnology strategic research priority area and in particular it addresses the "New strategic approaches to industrial biotechnology' within the overall Knowledge Bio-Based Economy (KBBE) strategic theme, through the development and utilisation of whole cells for the generation of high value chemical products. The proposed microscale approach utilises advanced robotics technology, facilitating accurate and quantitative data collection so as to speed up bioprocess optimisation and also significantly enhance translation to manufacturing scale. Research on oxidative biocatalysts and on the scale translation of these is an emerging field with a direct impact on the chemical and pharmaceutical industry sector in the UK and worldwide. Findings from this project will facilitate ways in which such enzymes can be produced in larger quantities and with a higher level of activity in reduced timescales, and will therefore have a direct impact on the chemical sector in terms of improvement of process yield and decrease of development times. The project is intended to provide a significant contribution to maintain and improve the competitiveness of the European chemical and pharmaceutical industry in an international context by providing new innovative technologies to effectively address new chemical products. The P450 enzyme platform will expand biotechnology companies' collection of in-house enzymes and will strengthen their position in the development of customized processes for the industry. The concept of having high quality in-house enzyme collections has proven to be a key asset for fulfilling customer demands in a time and cost efficient manner for the biotech industry. As currently such a diverse and efficient enzymatic hydroxylation platform with successful application in pilot scale is simply not available, the successful completion of the project will place biotech companies collaborating within this project in a premium position in that field. The proposal aims at providing faster and more cost-effective processes by investigating the causal relationship between process conditions and resulting biocatalytic activity using high throughput approaches. Such knowledge will benefit technology and equipment companies as it will provide them with information useful to improve the design of the next generation of enzyme host and genes. Technology and knowledge transfer will also be achieved by the definition of EngD collaborations with existing and new industrial partners within our Industrial Doctorate Training Centre.Results and findings from this work will be disseminated by publishing them in peer-reviewed journals. Given the multidisciplinary nature of the proposal three sets of journals can be approached, depending on whether the publication has a biological focus (Protein Science), a prevailing processing element (Biotechnology Progress, Biotechnology&Bioengineering) or demonstrates the integration of the two aspects for the development of a currently unavailable chemical process (Organic Process Research and Development). The participation to national and international conferences (BIOTRANS 2013; Enzyme Engineering 2014) will be employed as an effective way of dissemination of the results to the academic and industry community alike. The findings will also be introduced and disseminated via the Industrial Biotechnology MBI post-experience training course at UCL attended by international industrialists. The results will be made available to the European scientific community and ease their exploitation in a broader sense. During the execution of the project it is intended to generate new processes for chemical products which already have a market potential, like biopolymers, flavour compounds, and steroids.