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，与携带氧气血蛋白血红蛋白相同的血红素辅因子结合。像血红蛋白一样，P450也结合氧（O2）。但是，与血红蛋白不同，它们用从伴侣蛋白传递给血红素的电子减少结合的氧，并最终源自细胞辅酶NADPH。这使P450可以将氧气分为其成分原子。两个原子之一形成水（H2O）；另一个用于用P450结合的有机底物分子氧合靠近其血红素。通常，羟基化（引入OH组的引入）是催化的。在人类中，P450的活性是类固醇产生所必需的，也需要创建许多对于体内信号传导所必需的脂质分子（例如免疫系统激活）。人类有57个P450。他们最著名的角色是从体内排毒和去除药物。细菌p450在允许异常分子（例如樟脑）提供生长的能量的途径中具有重要作用，对于抗生素的产生至关重要（例如红霉素）。 P450在有机分子中定义的位置引入氧原子的能力吸引了有机化学家在工业/生物技术领域的广泛关注，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。通过“传统”化学方法将氧原子引入有机分子的精确位置非常困难。通常，形成了大型产品混合物，然后必须将其分馏以隔离所需的产品。就浪费而言，这个过程可能非常“脏”。 P450具有大量“清洁”生产优质化学物质以及各种氧化中间体和药品的产生。许多P450在识别的分子和从中生产的产品方面都是高度特异性的。但是，可以有效地使用蛋白质工程（可以通过更改编码它的DNA的序列改变蛋白质的结构）有效地使用蛋白质工程来改变酶（即P450）识别的分子类型（即底物）（即P450）（即P450）并改变氧氧气植物位置的位置。因此，该方法可以通过创建新型催化剂来执行对工业/药物化学的反应。蛋白质工程的最新发展是使用“强迫进化”。一种使用随机诱变来对蛋白质结构进行多种变化的方法，并且具有改变特性的突变体是通过允许允许隔离变体具有具有行业利用活动的变体的方法来筛选的。在这个项目中，我们将使用强制进化和大规模筛查（使用安装在曼彻斯特国家中心的新机器人设施）来识别和隔离名为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。允许我们更准确地定义对所需底物的特异性“切换”特异性的突变体。我们将转换活性（i），而有利于对药物/药物生产必不可少的化学物质（可节省大量成本）重要的化合物，以及（ii）允许将氧气引入另一类的脂质分子，从而实现高价值生理活性活性信号分子的形成。 P450 BM3就其“融合”与伴侣酶的“融合”具有独特的优势，这对于推动其功能至关重要。其他P450系统需要添加其他蛋白质成分，这些蛋白质成分通常是不溶的。因此，我们将使用最合适的酶和新颖的筛查技术，以创建P450突变体的库，这些突变体具有由英国生物技术和工业部门直接利用的新活动。