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Directed evolution approaches to generation of an industrially applicable biocatalyst

生成工业适用生物催化剂的定向进化方法

基本信息

批准号：
BB/F00883X/1
负责人：
Andrew Munro
金额：
$ 74.89万
依托单位：
University of Manchester
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2008
资助国家：
英国
起止时间：
2008 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FF00883X%2F1
关键词：
Directed evolution approaches generation industrially

项目摘要

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.

被称为细胞色素P450(P450)的蛋白质在所有生命形式的生理学中都是必不可少的。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因子。像血红蛋白一样，P450也与氧(O2)结合。然而，与血红蛋白不同的是，它们通过伙伴蛋白传递到血红素的电子来减少结合氧，最终来自细胞辅酶NADPH。这使得P450能够将氧分解成其组成原子。两个原子中的一个形成水(H2O)；另一个用来氧化与其血红素接近的P450结合的有机底物分子。通常，羟化反应(引入羟基)是催化的。在人类中，P450的活性是类固醇产生所必需的，也是产生体内信号(例如免疫系统激活)所必需的许多脂质分子所必需的。人类有57个P450。它们最著名的作用是解毒和清除体内的药物。细菌P450在允许不寻常的分子(例如樟脑)被用于为生长提供能量的途径中具有重要作用，并且对于生产抗生素(例如红霉素)是必不可少的。P450在有机分子中的特定位置引入氧原子的能力引起了工业和生物技术领域的有机化学家的极大关注，他们正在寻找更清洁、更环保的合成药物和其他重要分子的途径。用传统的化学方法很难将氧原子引入有机分子中的精确位置。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。从浪费的角度来看，这个过程可能是非常“肮脏”的。P450具有更清洁地生产精细化学品以及各种含氧中间体和药物的潜力。许多P450在识别的分子和它们生产的产品方面是高度特定的。然而，众所周知，蛋白质工程(通过改变编码蛋白质的DNA序列来改变蛋白质的结构)可以有效地改变酶识别的分子(底物)的类型(即P450)，并改变底物上引入氧原子的位置。因此，该方法可用于创建执行工业/药物化学所需的反应的新型催化剂。蛋白质工程的另一个最新发展是使用“强制进化”；通过这种方法，随机突变被用来对蛋白质结构进行多重改变，并通过允许分离具有工业开发所需活性的突变体的方法来筛选具有改变性质的突变体。在这个项目中，我们将使用强制进化和大规模筛选(使用作为曼彻斯特国家中心安装的新机器人设施)来鉴定和分离名为P450 BM3的P450酶的突变。我们将通过一种涉及耗氧量的新方法进行筛选；使我们能够更准确地定义(比其他小组之前的工作)已将特异性‘切换’到所需底物的突变体。我们将把活性(I)转向对合成药物/药品生产所必需的化学物质(实现大量成本节约)非常重要的化合物，以及(Ii)允许将氧气引入另一类脂分子，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，因为它与驱动其功能所必需的一种辅助酶“融合”。其他P450系统需要添加其他蛋白质成分，这些成分通常是不溶于水的。因此，我们将使用最合适的酶和新的筛选技术来创建具有新活性的P450突变体文库，这些突变体库可直接被英国生物技术和工业部门利用。