Directed evolution approaches to generation of an industrially applicable biocatalyst

Proteins known as cytochromes P450 (P450s) are essential in physiology of all life forms. They are heme-binding proteins, binding the same heme cofactor as the oxygen-carrying blood protein hemoglobin. Like hemoglobin, P450s also bind oxygen (O2). However, unlike hemoglobin they reduce bound oxygen with electrons delivered to the heme from partner proteins, and ultimately derived from the cell coenzyme NADPH. This enables P450s to split oxygen into its component atoms. One of the two atoms forms water (H2O); the other is used to oxygenate an organic substrate molecule bound by the P450 close to its heme. Frequently, hydroxylation (introduction of an OH group) is catalysed. In humans, activity of P450s is required for steroid production, and also for creation of many lipid molecules essential for signalling in the body (e.g. immune system activation). Humans have 57 P450s. Their most famous roles are in detoxification and removal of drugs from the body. Bacterial P450s have important roles in pathways that allow unusual molecules (e.g. camphor) to be used to provide energy for growth, and are essential for production of antibiotics (e.g. erythromycin). The ability of P450s to introduce oxygen atoms at defined positions in organic molecules has attracted much attention from organic chemists in industrial/ biotechnology sectors, who are looking for cleaner, more environmentally friendly routes to synthesis of drugs and other important molecules. It is very difficult to introduce oxygen atoms into precise positions in organic molecules by 'traditional' chemistry approaches. Frequently, large mixtures of products are formed, which then must be fractionated to isolate the desired one. This process can be very 'dirty' in terms of waste. P450s have potential for much 'cleaner' production of fine chemicals and of various oxygenated intermediates and pharmaceuticals. Many P450s are highly specific in terms of molecules recognised and products they produce from them. However, it is well recognised that protein engineering (changing the structure of a protein predictably by altering the sequence of the DNA that encodes it) can be used effectively to change both the types of molecules (substrates) recognised by the enzyme (i.e. P450) and to alter the position on the substrate at which oxygen atoms are introduced. This method can thus by used to create novel catalysts that perform reactions desirable for industrial/pharmaceutical chemistry. A further recent development of protein engineering is the use of 'forced evolution'; a method by which random mutagenesis is used to make multiple changes in protein structure, and mutants with altered properties are screened by methods that allow isolation of variants with the activity desired for exploitation in industry. In this project we will use forced evolution and mass screening (using new robotics facilities installed as a national centre at Manchester) to identify and isolate mutants of a P450 enzyme named P450 BM3. We will screen by a novel method involving oxygen consumption; allowing us to define more accurately (than in previous work by other groups) mutants that have 'switched' specificity towards the desired substrates. We will switch activity (i) in favour of compounds that are important in synthesis of chemicals essential for drug/pharmaceutical production (enabling large cost savings), and (ii) to allow introduction of oxygen into another class of lipid molecules, enabling formation of high value physiologically active signalling molecules. P450 BM3 has unique advantages over other P450s in terms of its 'fusion' to a partner enzyme that is essential for driving its function. Other P450 systems need addition of other protein components, which are often water-insoluble. Thus, we will use the most appropriate enzyme and novel screening technologies in order to create libraries of P450 mutants that have new activities directly exploitable by the UK biotech and industrial sectors.

The stereo and regioselective oxidation of organic molecules is difficult to achieve by standard synthetic chemistry approaches. There are generally several products formed (requiring fractionation) and processes can be dirty and inefficient. The use of enzyme chemistry offers several advantages with respect to cleaner, more effective production of oxychemicals, but demands that efficient biocatalysts are available for specific reactions. The cytochrome P450 monooxygenases catalyse several specific oxidation reactions, but are frequently components of multiprotein redox chains and/or membrane proteins. Both of these issues present problems with respect to cost effective exploitation in the biotechnology sector. In this application, we will use a single component P450 system (P450 BM3 from B. megaterium) and rationally and irrationally engineer the enzyme for specific oxidation reactions that are of immediate exploitability in synthesis of fine oxychemicals as key intermediates (synthons) in pathways of synthesis of key pharmaceutical compounds, or as fine chemicals in their own right. BM3 is a structurally well characterized oxygenase with an efficient, self-contained electron transport system - making it ideal for these studies. We will employ novel screening strategies based on oxygen consumption and evasion of enzyme inactivation to generate mutants that have specific characteristics required. We will determine atomic structures of BM3 in presence of the required substrates to help refine mutagenesis strategies and optimise collection of variants with desired catalytic properties. We will systematically characterize structurally, kinetically, thermodynamically and spectroscopically the evolved mutant enzymes we generrate, to obtain a detailed understanding of the mechanisms by which their substrate recognition has been altered. Collectively, these studies will provide for a series of robust oxygenase catalysts tailored to biotechnologically important roles.