Directed evolution approaches to generation of an industrially applicable biocatalyst

被称为细胞色素P450 （P450）的蛋白质在所有生命形式的生理中都是必不可少的。它们是血红素结合蛋白，与携带氧气的血液蛋白血红蛋白结合相同的血红素辅助因子。像血红蛋白一样，p450也能结合氧气。然而，与血红蛋白不同的是，它们通过从伙伴蛋白传递给血红素的电子来减少结合氧，并最终来源于细胞辅酶NADPH。这使得p450能够将氧分解成它的组成原子。两个原子中的一个形成水（H2O）；另一种用于氧化有机底物分子，该分子与靠近其血红素的P450结合。通常，羟基化（OH基团的引入）是催化的。在人类中，p450的活性是类固醇生产所必需的，也是体内信号传递所必需的许多脂质分子的产生（例如免疫系统激活）所必需的。人类有57个p450。它们最著名的作用是排毒和清除体内的药物。细菌p450在允许不寻常分子（例如樟脑）用于提供生长能量的途径中发挥重要作用，并且对抗生素（例如红霉素）的生产至关重要。p450在有机分子中特定位置引入氧原子的能力引起了工业/生物技术领域有机化学家的极大关注，他们正在寻找更清洁、更环保的途径来合成药物和其他重要分子。用“传统”化学方法将氧原子引入有机分子的精确位置是非常困难的。通常，会形成大量的产品混合物，然后必须对其进行分馏以分离出所需的产品。就废物而言，这一过程可能非常“肮脏”。p450在精细化学品和各种含氧中间体和药品的“更清洁”生产方面具有潜力。许多p450在被识别的分子和它们产生的产物方面都是高度特异性的。然而，人们普遍认识到，蛋白质工程（通过改变编码蛋白质的DNA序列可预测地改变蛋白质的结构）可以有效地用于改变酶（即P450）识别的分子（底物）类型和改变底物上氧原子引入的位置。因此，该方法可用于制造新型催化剂，以执行工业/药物化学所需的反应。蛋白质工程最近的进一步发展是使用“强制进化”；一种使用随机诱变使蛋白质结构发生多种变化的方法，并通过允许分离具有工业开发所需活性的变体的方法筛选具有改变特性的突变体。在这个项目中，我们将使用强制进化和大规模筛选（使用安装在曼彻斯特国家中心的新机器人设备）来识别和分离名为P450 BM3的P450酶的突变体。我们将通过一种涉及氧气消耗的新方法进行筛选；使我们能够更准确地定义（比其他小组先前的工作更准确）已经将特异性“切换”到所需底物的突变体。我们将切换活性(i)有利于在药物/制药生产所需化学物质合成中重要的化合物（从而节省大量成本），以及（ii）允许将氧引入另一类脂质分子，从而形成高价值的生理活性信号分子。与其他P450相比，P450 BM3具有独特的优势，它与伙伴酶的“融合”对驱动其功能至关重要。其他P450系统需要添加其他蛋白质成分，这些成分通常不溶于水。因此，我们将使用最合适的酶和新的筛选技术，以创建具有新活性的P450突变体文库，这些突变体可以直接被英国生物技术和工业部门利用。

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.