Author: Robert Osborne, Ph.D., Scientist II Technical Lead
Cytochrome P450 monooxygenases (P450s) have been the subject of extensive research for decades. These enzymes are involved in numerous metabolic and synthetic processes, and are best known for their role in clearing toxic chemicals including pharmaceuticals. This in turn makes them highly significant for the pharmaceutical industry, not only for their potential to influence drug interactions and drug metabolism, but also as therapeutic targets. P450s continue to be a valuable research tool at the small scale due to their ability to catalyze the incorporation of an oxygen atom into unactivated C-H bonds. However, the incorporation of a P450 enzyme by process chemists has come up against hurdles that to date have prevented these powerful enzymes from being used at manufacturing scale. Here we consider some of those limitations along with recent developments in biocatalysis that may overcome these challenges, allowing pharma and other manufacturing industries to gain from the significant cost advantages of incorporating P450-regulated chemistries into their processes.
P450 enzymes are of great interest for synthetic chemistry and biocatalysis due to their ability to catalyze oxidative reactions including C-H hydroxylation and heteroatom oxidations under mild reaction conditions and often at positions that are challenging to target using traditional chemistry. (reviewed in reference 1). Furthermore, P450s can provide stereo- and regioselectivity advantages that are superior to chemical approaches.
The potential of exploiting P450 chemistries for basic research and development (e.g. metabolite production), industrial syntheses, and even manufacturing in the pharmaceutical, agrochemical, fine chemical and flavors and fragrances industries is undeniable.
A classic example for the hydroxylation of an unactivated C-H bond was the demonstration by Upjohn (1952) using a whole cell bioreactor to convert progesterone to 11-a hydroxyprogestrone en route to the production of cortisone; it is well-established that a P450 enzyme is responsible for the biocatalytic transformation.
Safety considerations and a general chemical paradigm continue to limit the frequency of incorporating oxidative chemistries at manufacturing scale. Many organic substrates require a solubilizing and often flammable solvent, and the reaction conditions for traditional, oxidative reactions are often harsh (e.g. require heat heavy metals, etc.). These factors combined with oxygen present a combustion risk. However, this risk can often be mitigated if enzymes are used to catalyze the desired oxidative transformation. Due to the safety considerations described, production of chiral alcohols is most often achieved by chemical or enzymatic reduction of a ketone. Concerns about safety and a chemical paradigm shift should be achieved as more examples of enzyme-catalyzed oxidative transformations at manufacturing scale emerge (e.g. BVMO for esomeprazole production).
Overcoming P450 limitations with directed evolution
Solvent tolerance and the need for expensive cofactors such as NADPH, FMN, and FAD have often been cited as limitations for using a P450 for industrial processes.1 However, solvent tolerance is a selection pressure routinely used for identifying beneficial diversity to generate robust enzyme variants. By increasing solvent concentration as a selection pressure over time, it is possible to evolve an enzyme to tolerate very high fractions of solvent (50-70%). Similarly, scientists at Codexis and elsewhere have used directed evolution to overcome the requirement for cofactors. For example, Joo et al.2 engineered P450 mutants that could hydroxylate naphthalene without the need for cofactors, through the peroxide shunt pathway. Alternatively, cofactor recycling systems are routinely incorporated, and the efficiency of the recycling enzyme has been improved via directed evolution, further reducing the cost associated with and the supply requirement of cofactors.
Currently, bacterial P450s are most widely used for small-scale metabolite production offering a cost-effective, quick and easy alternative to whole cell liver microsomes. Similarly, P450s are being applied in medicinal chemistry for drug lead optimization. The primary application of P450s as a small scale R&D research tool is in part due to a complex mechanism in which substrate/product inhibition and coupling efficiency contribute to generally low turnover rates that manifest in low volumetric productivities that are cost-prohibitive.
Optimal substrate loading for P450s is typically in the range of <1.0-3.0 g/L due to the steep decrease in the rate of product formation associated with increasing amounts of starting material, and this phenomenon is observed for multiple substrates. Simply, substrate loads that achieve maximal velocity are typically at least one order of magnitude (10X) less than what is generally required (50-100 g/L) for a cost-competitive, manufacturing scale biocatalytic process.
Coupling efficiency is simply described as a productive turnover. For a single turnover, P450s require two separate long-range electron transfers which are coupled to the activation of oxygen and subsequent hydroxylation of a C-H bond. Several intermediates formed during this process can lead to the disruption of or a reduction in coupling efficiency typically referred to as uncoupling. The consequence of uncoupling is enzyme inactivation and low volumetric productivities.
Directed evolution can overcome the limitations imposed by inhibition and coupling efficiency. One notable example is that of P450PMO (propane monooxygenase).3P450PMOwas engineered in Frances Arnold’s laboratory at the California Institute of Technology (CalTech) using directed evolution. The final variant hydroxylates propane to propanol with >35,000 total turnovers.
The use of directed evolution is the best approach to overcome these challenges. Directed evolution enables rapid screening and ranking of diversity from screening thousands of non-native enzymes. Success will be dependent on designing screens that implement the appropriate selection pressure for identifying beneficial diversity for overcoming inhibition and improving coupling efficiency.
During the last couple of years, scientists have discovered and characterized single domain fungal peroxygenase enzymes that catalyze P450-like chemistries. These enzymes are potentially easier to understand, manipulate, and evolve for successful incorporation into large-scale biocatalytic manufacturing processes. Fungal peroxygenases use hydrogen peroxide as the oxidant to catalyze chemical transformations analogous to those catalyzed by P450s using molecular oxygen. Use of a simpler, single domain enzyme could be much more amenable to optimizing an oxidative enzyme for large-scale manufacturing.
The immense benefits of P450s and P450-like enzymes have yet to be fully achieved, but their unique chemistry capabilities combined with the great economic benefits of biocatalysis and cleaner chemistry will bring significant advantages to those manufacturing companies that can incorporate them into their processes.
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